CN101130081A - Use of PNAS-4 gene in preparing antineoplastic and antineoplastic auxiliary medicament - Google Patents

Abstract

The invention relates to a use of PNAS-4 gene in aspects of preparing antineoplastic in the tumour gene treatment field. The invention with PNAS-4 recombinant vector can limit the growth and the migration of the endocytosis effectively, which can limit the growth of various tumors and extend the survive time of mouse. The antineoplastic of the invention with PNAS-4 recombinant vector liposome and chemotherapy drug cisplatin and magnolol as the active component has better effect than the single application and can reduce the usage of both parties. The product has the appreciable effect, the little toxic and side effect and the easy preparing method, which compensates the defect of protein infusion drug, increases the time interval of medicine, reduces the usage, reduces the economic burden of the patient, improves the life quality of patient and has the wide market prospect.

Description

The purposes of PNAS-4 gene in preparation antitumor and antitumour auxiliary drug

Technical field

The invention belongs to field of tumor gene therapy, be specifically related to the purposes of PNAS-4 gene aspect preparation antitumor drug and antitumour auxiliary drug.

Background technology

Tumor cell is the cell mass that propagation is out of control, express original traits, and the generation of tumor is unbalance relevant with " propagation-apoptosis " of tumor cell.Generation, the development of apoptosis (apoptosis) and tumor, and substantial connection is arranged with cell death in the oncotherapy.Apoptosis is the important ingredient of cell monitoring system.When cellular genome sustained damage, cell at first started repair mechanism, in case damage is repaired fully, cell then reenters the normal growth state; If repairing failure, Apoptosis Mechanism will be activated, and the cell of damage enters apoptosis and is eliminated, thereby avoid the genome damage to entail daughter cell, eliminate tumorigenic potential possibility.Apoptosis has been the hot subject that becomes current life science research.The pair cell apoptosis is unbalance intervenes and is expected to bring new Therapeutic Method for tumor, and cell death inducing has also become the fresh target of oncotherapy.Therefore, be the main policies that the theory and technology of target has become the treatment malignant tumor with inducing apoptosis of tumour cell optionally.Wherein the most tempting strategy is to be the gene therapy of target with the cell death inducing, just in tumor cell, import the suppressor gene of apoptosis-induced activating gene or deactivation apoptosis, remove tumor cell by cell death inducing by molecular biological approach.

Although global scientist has made the contribution of many brilliances in field of tumor gene therapy, make genetic treatment of tumor once obtain vigorously development fast, up to now, still there is the problem of many needs solutions.And wherein urgent problem is: the gene that is used for therapy of tumor at present very little, the gene that can suppress tumor growth is few in number, is badly in need of providing more for the genes that utilize.The selection that focuses on genes of interest or its target spot of gene therapy, excavation also identifies that it is the focus and the advanced subject of therapy of tumor that the gene of therapeutic value is arranged clinically all the time.Along with finishing of human genome order-checking plan, the Human Genome Project has entered functional genome's epoch, and its purpose is clone new gene and illustrates the function and the intergenic interaction relationship thereof of these genes.Identify that for comprising the vertical many treatment of diseases of tumor more treatment target spot or therapeutic gene provide condition extremely easily.

On the other hand, the present radiotherapy that adopts clinically, most of chemotherapeutics and biopharmaceuticals are mainly all removed tumor cell by cell death inducing.Traditional chemotherapy and radiation curative effect is limited, the apoptosis of tumor cells opposing is an one of the main reasons, apoptosis opposing Study on Molecular Mechanism has obtained progress in recent years, and for oncotherapy provides the theory and practice basis, the new therapy that overcomes the tumor death resistance has entered clinical trial.Because the heterogeneity of tumor cell, different tumor cells obtain different apoptosis resistance mechanisms, and may have multiple apoptosis resistance mechanisms at same tumor cell, thus various medicine and method be combined with may but will inevitably not produce better therapeutic.Simultaneously, traditional chemotherapy and radiation also can cause negative effect to patient's immune system, can damage body health, reduces quality of life, therefore, seek used as adjuvant drug for antitumor especially the chemicotherapy adjuvant drug also be a research focus in current oncotherapy field.

People's PNAS-4 (h PNAS-4) gene is one of the new gene identified in the human genome large scale sequencing (Strausberg, RL, Feingold, EA, Grouse, LH, et al.Generation and initial analysis ofmore than 15,000 full-length human and mouse cDNA sequences.Proc.Natl.Acad.Sci.U.S.A.2002.99 (26): 16899-16903), login is in the data base of America NI H, and its homogenic searching and functional study are not yet carried out comprehensively.

Summary of the invention

First technical problem to be solved by this invention provides the new purposes of PNAS-4 gene aspect preparation antitumor drug or antitumour auxiliary drug.

Wherein, at least a in the above-mentioned behaviour of PNAS-4 gene, mice or the Xenopus laevis PNAS-4 gene.

Wherein, the sequence of above-mentioned PNAS-4 gene is SEQ ID NO.1, SEQ ID NO.3 or SEQ IDNO.5.

Second technical problem to be solved by this invention provides the purposes of PNAS-4 albumen in preparation antitumor drug or antitumour auxiliary drug.

Wherein, at least a in the above-mentioned behaviour of PNAS-4 albumen, mice or the Xenopus laevis PNAS-4 albumen.

Wherein, the above-mentioned proteic sequence of PNAS-4 is respectively SEQ ID NO.2, SEQ ID NO.4 or SEQ IDNO.6.

The 3rd technical problem to be solved by this invention provides a kind of recombinant vector.This recombinant vector contains the PNAS-4 gene.

Wherein, above-mentioned carrier is a carrier for expression of eukaryon.

Further, above-mentioned carrier for expression of eukaryon is plasmid, adenovirus.

Preferably, above-mentioned adenovirus is a replication-defective adenoviral.

The 4th technical problem to be solved by this invention provides the host cell that contains above-mentioned described recombinant vector.Further, described host cell is an eukaryotic cell.

The 5th technical problem to be solved by this invention provides the purposes of above-mentioned recombinant vector in preparation antineoplastic pharmaceutical compositions or antitumour auxiliary drug compositions.

The 6th technical problem to be solved by this invention provides a kind of antineoplastic pharmaceutical compositions or antitumour auxiliary drug compositions.This antineoplastic pharmaceutical compositions or antitumour auxiliary drug compositions are that the albumen by PNAS-4 gene or PNAS-4 gene code adds pharmaceutically as main active that the acceptable adjuvant is prepared from.

The 7th technical problem to be solved by this invention provides a kind of antineoplastic pharmaceutical compositions or antitumour auxiliary drug compositions.This antineoplastic pharmaceutical compositions or antitumour auxiliary drug compositions are to add pharmaceutically by above-mentioned recombinant vector as main active that the acceptable adjuvant is prepared from.

Wherein, the active component of above-mentioned antineoplastic pharmaceutical compositions or antitumour auxiliary drug compositions is by quilt that liposome wraps.

Wherein, the dosage form of above-mentioned antineoplastic pharmaceutical compositions or antitumour auxiliary drug compositions is an injection.

Above-mentioned injection can be prepared as the injection that pH value is 7.5-8.5.

Further, also contain tumor chemotherapeutic drug in the above-mentioned antineoplastic pharmaceutical compositions as active component.

Preferably, above-mentioned tumor chemotherapeutic drug is cisplatin or honokiol.

The 8th technical problem to be solved by this invention provides a kind of method for preparing above-mentioned recombinant vector.

This method may further comprise the steps:

A, go into the recombinant vector that carrier obtains containing PNAS-4 with PNAS-4 is gene constructed;

B, the recombinant vector that a step gained is contained PNAS-4 change host cell over to, amplification preparation recombinant vector.

Wherein, employed carrier can be eukaryon expression plasmid.Be preferably pcDNA3.1, its concrete preparation method can be:

A, the gene constructed pcDNA3.1 of going into of PNAS-4 is obtained pcDNA3.1-PNAS-4;

B, change a step gained pcDNA3.1-PNAS-4 over to host cell, amplification preparation recombiant plasmid.

Wherein, employed carrier can be adenovirus vector.Be preferably pAd/PL-DEST, its concrete preparation method can be:

A, the gene constructed pAd/PL-DEST of going into of PNAS-4 is obtained pAd-DEST-PNAS-4;

B, with a step gained pAd-DEST-PNAS-4 rotaring redyeing 293 cell, amplification preparation recombinant adenovirus.

The 9th technical problem to be solved by this invention provides above-mentioned antineoplastic pharmaceutical compositions of a kind of preparation or antitumour auxiliary drug method for compositions, it is characterized in that may further comprise the steps:

A, under aseptic condition, get water for injection, add raw material by following proportioning again: reorganization PNAS-4 carrier, 5% glucose solution;

B, under aseptic condition with step a product mix homogeneously, regulate pH value to 7.5-8.5 with buffer;

C, will be distributed into injection after the aseptic filtration of step b product, or be made as lyophilized formulations, cryopreservation.

According to above-mentioned various aspects of the present invention as can be known: for the function of research PNAS-4 and explore its application on therapy of tumor, the present invention clone has obtained Xenopus laevis, mice and people's PNAS-4 coding gene sequence; On cellular level and Xenopus laevis embryo, found PNAS-4 energy inducing apoptosis of tumour cell; PNAS-4 plasmid that has prepared liposome mice or people etc. has the carrier for expression of eukaryon of medical value; Then, PNAS-4 plasmid by liposome mice or people is treated tumor-bearing mices such as pulmonary carcinoma, colon cancer and ovarian cancer, all obtained good effect, found that PNAS-4 can become a new therapy of tumor gene, genetic treatment of tumor provides a new target spot.In addition, the present invention is also by finding in mice CT26 colon cancer and the IL/2 lung cancer model body that mPNAS-4 gene and chemotherapeutics cisplatin and honokiol have the synergistic antitumor effect, can or be prepared into combination dosage forms with chemotherapeutics as used as adjuvant drug for antitumor and play a role; Simultaneously, also investigate PNAS-4 and whether had toxic and side effects, found that it has slight side effect, can improve patient's quality of life well.

Need to prove that especially the concrete routine techniques method and apparatus of more than producing and operate recombination disclosed by the invention, recombinant vector and antitumor injection is well known by persons skilled in the art, and can finish according to the technology of having described.

Beneficial effect of the present invention is: evidence PNAS-4 of the present invention crosses the propagation that expression can suppress mouse tumor cell, shows that PNAS-4 has the antineoplastic potentiality.The present invention contains the recombinant vector of PNAS-4 can be can be in the external growth and the migration that can suppress endotheliocyte effectively; But intravital tumor experiment also shows, in the tumor-bearing mice tumor or tail vein injection liposome pcDNA3.1-mPNAS-4 provided by the invention or pcDNA3.1-hPNAS-4 injection can express PNAS-4 albumen, with the growth of remarkable inhibition kinds of tumors, prolong the life cycle of tumor-bearing mice.The present invention with liposome contain the recombinant vector of PNAS-4 and chemotherapeutics cisplatin or honokiol jointly as the antineoplastic pharmaceutical compositions of active component than separately separately use obviously have more excellent effect, can reduce both use amounts.Product efficacy of the present invention is obvious, toxic and side effects is little, preparation method is simple, can overcome that toxic and side is big, the difficult defective such as lasting of curative effect, can increase administration time the interval, significantly reduce dosage, alleviate patient economy burden and improve quality of life of patient; And the antitumor injection of recombinant vector of the present invention preparation can also remedy, and medicine costliness, body internal stability that the present invention uses PNAS-4 albumen infusion techniques scheme to have separately are poor, infusion waits defective often, have good market prospect.

Description of drawings:

Fig. 1 is pcDNA3.1 (+) carrier figure spectrogram.

Fig. 2 is the collection of illustrative plates of plasmid pVAX1.

Fig. 3 is the adenovirus vector collection of illustrative plates.

Fig. 4 is the painted nucleus outward appearance of the PI under the fluorescence microscope. (A) negative control group, (B) independent liposome group, (C) pcDNA3.1 group, (D) pcDNA3.1-mPNAS-4 group.As can be seen cell rupture, cell volume diminish, the feature of natural death of cerebral cells such as fragment and apoptotic body.

Fig. 5 represents inhibition and the elimination result of pcDNA3.1-mPNAS-4 to C57BL/6 mice LL2 mice model of lung cancer and BALB/c mouse C26 model of colon cancer tumor, and it is 100ul that each group is injected total amount respectively.Wherein the compound pcDNA3.1-mPNAS-4 plasmid of liposome group with * expression; PcDNA3.1 plasmid group with ▲ expression); Liposome is represented with ■.With the mice that began to inoculate 100 μ 1NS on the 10th day is contrast (representing with ◇ among the figure).Fig. 5-A represents C57BL/6 mice LL2 mice model of lung cancer, and each is subjected to tumor size between the examination group, Fig. 5-C represents BALB/c mouse C26 model of colon cancer, and each is subjected to tumor size between the examination group, and the result shows between pcDNA3.1-mPNAS-4 processed group and other processed group evident difference.Fig. 5-B represent C57BL/6 mice LL2 mice model of lung cancer each be subjected to tumour inhibiting rate, Fig. 5-D of examination group to represent BALB/c mouse C26 model of colon cancer each is subjected to the tumour inhibiting rate of examination group, the result shows between pcDNA3.1-mPNAS-4 processed group and other processed group evident difference.

Fig. 6 represents the histochemical stain analysis result of tumor.Whether because the apoptosis of tumor cell has caused result shown in Figure 5, utilization original position tunel detection kit detects by TUNEL has carried out histology to tumor cell in order to estimate.These paraffin sections respectively embedding be: E is the mouse tumor matched group that PBS handles; F is the matched group of liposome-treated; G is the matched group that liposome and empty carrier are handled; H is the treatment group (* 200) that liposome and pcDNA3.1-mPNAS-4 handle; The apoptotic body of visible green under fluorescence microscope is compared at interior matched group with PBS, liposome, liposome vectors, can observe more obvious apoptotic cell in the tumors remaining cell of the processed group of the compound pcDNA3.1-mPNAS-4 of liposome.

Fig. 7 represents flow cytometry transfection apoptosis situation after 48 hours, wherein: A is that serum-free medium, B are that liposome, C are that pcDNA3.1+ liposome, D are the hPNAS-4-pcDNA3.1+ liposome, the signal peak at arrow indication place is represented the signal of apoptotic cell among the D, and signal is strong more or the big more expression apoptotic cells of peak area is many more.

Fig. 8 represents after the transfection that Hoechst33258 dyeing in 48 hours shows apoptosis situation (fluorescence microscope, 20 *), wherein:

A. be the serum-free medium group; B is the liposome group; C is a pcDNA3.1+ liposome group; D is a hPNAS-4-pcDNA3.1+ liposome group.

Fig. 9 represents the contrast of nude mice oophoroma tumor outward appearance volume.

Figure 10 represents nude mice oophoroma tumor volume-time graph.Wherein: 1 is the PBS group; 2 are the Liposome group; 3 are the pAd-DEST-Null+liposome group; 4 are the pAd-DEST-hPNAS-4+liposome group.

Figure 11 apoptosis PI experiment of dyeing.

Figure 12 represents the inhibiting result of mouse tumor.

Figure 13 represents the mice survival rate.

Figure 14 represent external mPNAS-4 with and cisplatin (cisplatin) result of the test Figure 14-A of suppressing tumor cell jointly be the result of processing CT26 cell; Figure 14-B is for handling the result of IL/2 cell.

Figure 15 represents mouse tumor inhibitory action and the contrast of mice survival rate.

Figure 16 tumor tissues TUNEL testing result (A) matched group; (B) pcDNA3.1 group; (C) cisplatin group; (D) mPNAS-4 group; (E) mPNAS-4 adds the cisplatin group.

Figure 17 represents that external mPNAS-4 and honokiol (honokiol) suppress the result of the test Figure 17-A of tumor cell jointly for handling the result of CT26 cell; Figure 17-B is for handling the result of IL/2 cell.

Figure 18 represents mouse tumor inhibitory action and survival rate contrast.

Figure 19 represents tumor tissues TUNEL testing result: wherein (A) is matched group; (B) pcDNA3.1 group; (C) honokiol group; (D) mPNAS-4 group; (E) mPNAS-4 adds the honokiol group.

The invention will be further described below in conjunction with the description of accompanying drawing by the specific embodiment, but this is not a limitation of the present invention, those skilled in the art are according to basic thought of the present invention, can make various modification or improvement, only otherwise break away from basic thought of the present invention, all within the scope of the present invention.

The specific embodiment

Obtaining of embodiment one PNAS-4 gene

1, the clone of people PNAS-4 (hPNAS-4) gene cDNA

According to the people PNAS-4 full-length gene order of submitting among the NCBI (NM-016076), design primer amplification PNAS-4 gene coded sequence.In order further to be cloned into the pGEX-6p-1 carrier, in the primer of coded sequence upstream and downstream, introduce BamH I, Xho I restriction enzyme site respectively.

Forward primer (SEQ ID NO.7):

5′-GC GGATCCAAGATGGCAGATTTTTTGAAAGG-3′；

Downstream primer (SEQ ID NO.8):

5 '-CGC GATATCTTAAGTGTCCTC GTGCATGTCTG-3 ' (underscore shows restriction enzyme site).

The pcr amplification primer is synthetic by Shanghai Bo Ya company.For strengthening PNAS-4 expression of gene level, when making up the pcDNA3.1-hPNAS-4 recombiant plasmid, adopt following primer:

Forward primer (SEQ ID NO.9): 5 '-GC

GGATCCAAGATGGCA

GATTTTTTGAAAGG-3 ' (box indicating Kozak sequence, underscore is represented restriction enzyme site);

Downstream primer is the same.

From the HEK293 cell strain, extract the capable RT-PCR of total RNA with amplification hPNAS-4 coding region cDNA.The RT-PCR reaction amplifies about 600bp fragment, and is consistent with the theoretical prediction value.

After above-mentioned PCR product and pcDNA3.1 (+) used restriction endonuclease BamH I, Hind III double digestion respectively, through connection, conversion, screening, enzyme action identify, dna sequencing identifies, obtains eukaryotic expression recombiant plasmid pcDNA3.1-hPNAS-4.

Enzyme action identifies that correct recombiant plasmid delivers to the order-checking of Shanghai Bo Ya company, and the result shows that the sequence of the hPNAS-4 gene base sequence that inserts among the recombiant plasmid hPNAS-4 and GenBank is in full accord.The sequence of people PNAS-4 (seeing Table 1, table 2):

The base sequence of table 1 people PNAS-4cDNA (SEQ ID NO.1):

atgggg gctaaccagt?tagtggtgct?caacgtgtac?gacatgtatt?ggatgaacga

atatacctca?tccattggaa?ttggagtttt?tcattcagga?attgaagtct?atggcagaga

atttgcttat?ggtggccatc?cttacccctt?ttctggaata?tttgaaattt?ccccaggaaa

tgcttctgaa?ctaggagaaa?catttaaatt?taaagaagct?gttgttttag?ggagcacgga

cttcctagaa?gatgatatag?aaaaaattgt?agaagaactg?ggaaaagaat?acaaaggcaa

tgcttatcat?ttaatgcata?aaaactgcaa?tcatttttct?tcagctttat?cagagattct

ttgtgggaaa?gagattcctc?gctggatcaa?tcgacttgcc?tacttcagct?cctgtatacc

ctttctacag?agttgcctcc?cgaaggagtg?gctcacgccc?gcagccctgc?agtctagtgt

cagccaagaa?ctccaggatg?aactggagga?agcagaggat?gctgccgcat?ccgcttccgt

ggcaagcact?gcagcaggct?ccagacccgg?gcgccacact?aaactataa

Table 2 people PNAS-4 protein sequence (SEQ ID NO.2):

Met?Gly?Ala?Asn?Gln?Leu?Val?Val?Leu?Asn?Val?Tyr?Asp?Met?Tyr?Trp

Met?Asn?Glu?Tyr?Thr?Ser?Ser?Ile?Gly?Ile?Gly?Val?Phe?His?Ser?Gly

Ile?Glu?Val?Tyr?Gly?Arg?Glu?Phe?Ala?Tyr?Gly?Gly?His?Pro?Tyr?Pro

Phe?Ser?Gly?Ile?Phe?Glu?Ile?Ser?Pro?Gly?Asn?Ala?Ser?Glu?Leu?Gly

Glu?Thr?Phe?Lys?Phe?Lys?Glu?Ala?Val?Val?Leu?Gly?Ser?Thr?Asp?Phe

Leu?Glu?Asp?Asp?Ile?Glu?Lys?Ile?Val?Glu?Glu?Leu?Gly?Lys?Glu?Tyr

Lys?Gly?Asn?Ala?Tyr?His?Leu?Met?His?Lys?Asn?Cys?Asn?His?Phe?Ser

Ser?Ala?Leu?Ser?Glu?Ile?Leu?Cys?Gly?Lys?Glu?Ile?Pro?Arg?Trp?Ile

Asn?Arg?Leu?Ala?Tyr?Phe?Ser?Ser?Cys?Ile?Pro?Phe?Leu?Gln?Ser?Cys

Leu?Pro?Lys?Glu?Trp?Leu?Thr?Pro?Ala?Ala?Leu?Gln?Ser?Ser?Val?Ser

Gln?Glu?Leu?Gln?Asp?Glu?Leu?Glu?Glu?Ala?Glu?Asp?Ala?Ala?Ala?Ser

Ala?Ser?Val?Ala?Ser?Thr?Ala?Ala?Gly?Ser?Arg?Pro?Gly?Arg?His?Thr

Lys?Leu

2, the clone of mice PNAS-4 (mPNAS-4) gene

(1) according to the protein sequence of people's PNAS-4, cDNA library by Blastp search mice, obtain the PNAS-4 homologous genes sequence (GenBank No.NM 024282) of a mice, at this called after mPNAS-4, ORF Finder software analysis with NCBI, this gene has complete ORF, and start codon meets kozak rule (AXXATGG), and 3 ' end has a tailing signal AATAAA (the black underscore is represented).Therefore this sequence meets the full-length cDNA feature.The ORF start codon is ATG, and termination codon is TAA.The 585bp altogether from 500bp to 1084bp contains 194 amino acid residues (sequence see Table 3, table 4).

The nucleotide sequence (SEQ ID NO.3) of table 3.mPNAS-4

(sequence in the black surround:

Be start codon,

Be termination codon).

ggg?gct?aac?cag?tta?gtg?gtg?ctc?aac?gtc?tac?gac?atg?tac?tgg?atg?aat?gaa?tac?acc?tca

tct?att?gga?att?gga?gtt?ttt?cat?tct?gga?att?gaa?gta?tat?ggc?aga?gag?ttt?gct?tat?ggt?ggc?cat

cca?tat?cct?ttt?tct?gga?ata?ttt?gaa?att?tcc?cca?gga?aat?gct?tct?gag?cta?gga?gaa?aca?ttt?aaa

ttt?aaa?gaa?gct?gtt?gtt?cta?gga?agt?acg?gac?ttt?cta?gaa?gat?gat?ata?gag?aaa?att?gta?gaa?gaa

ctg?ggg?aaa?gag?tat?aag?ggc?aac?gcc?tac?cat?ctg?atg?cac?aaa?aac?tgc?aat?cac?ttt?tct?tca

gct?tta?tca?gag?att?ctc?tgt?ggg?aaa?gag?att?cct?cgc?tgg?atc?aac?cgg?ctg?gcc?tac?ttc?agc

tcc?tgt?ata?ccc?ttt?cta?cag?agt?tgc?ctg?cca?aag?gag?tgg?ctc?act?cct?gct?gca?ctt?cag?tct?agt

gtc?agc?caa?gaa?ctc?cag?gat?gaa?cta?gaa?gaa?gca?gag?gat?gca?gcc?gca?tcc?agt?gcc?atg

gca?agt?gct?gca?gct?ggt?gcc?aga?act?ggg?cgt?cac?act?aaa?cta

Table 4.mPNAS-4 encoded protein matter sequence (SEQ ID NO.4)

Met?Gly?Ala?Asn?Gln?Leu?Val?Val?Leu?Asn?Val?Tyr?Asp?Met?Tyr?Trp

Met?Asn?Glu?Tyr?Thr?Ser?Ser?Ile?Gly?Ile?Gly?Val?Phe?His?Ser?Gly

Ile?Glu?Val?Tyr?Gly?Arg?Glu?Phe?Ala?Tyr?Gly?Gly?His?Pro?Tyr?Pro

Phe?Ser?Gly?Ile?Phe?Glu?Ile?Ser?Pro?Gly?Asn?Ala?Ser?Glu?Leu?Gly

Glu?Thr?Phe?Lys?Phe?Lys?Glu?Ala?Val?Val?Leu?Gly?Ser?Thr?Asp?Phe

Leu?Glu?Asp?Asp?Ile?Glu?Lys?Ile?Val?Glu?Glu?Leu?Gly?Lys?Glu?Tyr

Lys?Gly?Asn?Ala?Tyr?His?Leu?Met?His?Lys?Asn?Cys?Asn?His?Phe?Ser

Ser?Ala?Leu?Ser?Glu?Ile?Leu?Cys?Gly?Lys?Glu?Ile?Pro?Arg?Trp?Ile

Asn?Arg?Leu?Ala?Tyr?Phe?Ser?Ser?Cys?Ile?Pro?Phe?Leu?Gln?Ser?Cys

Leu?Pro?Lys?Glu?Trp?Leu?Thr?Pro?Ala?Ala?Leu?Gln?Ser?Ser?Val?Ser

Gln?Glu?Leu?Gln?Asp?Glu?Leu?Glu?Glu?Ala?Glu?Asp?Ala?Ala?Ala?Ser

Ser?Ala?Met?Ala?Ser?Ala?Ala?Ala?Gly?Ala?Arg?Thr?Gly?Arg?His?Thr

Lys?Leu

(2) RT-PCR amplification

For the further existence of the opening code-reading frame of checking mPNAS-4 gene, comprised that by design the specific PCR primer of start codon and termination codon carries out RT-PCR.Forward primer wherein is (SEQ IDNO.10):

5 '-GCGGATCCGCCACCATGGCCAACCAGCCCATCATC-3 '; Downstream primer is (SEQ IDNO.11): 5 '-CCGCTCGAGCTATAGTTTTGTGTGGCGCCCAGG-3 '.Total RNA with mouse liver is a template, adopts one-step method RT-PCR amplification, obtains mice PNAS-4 gene open reading frame (ORF), and the product that obtains is the 608bp (see figure 1), and the sequence that obtains after the order-checking and the sequence of expection are in full accord.

(3) structure of pcDNA3.1-mPNAS-4 expression plasmid and evaluation

The amplified production of above-mentioned gained is connected on the BamH I/Xho linearizing pcDNA3.1 of I (+) carrier for expression of eukaryon (Figure 2) with 16 ℃ of water-bath 16-20h of T4DNA ligase after with BamH I/Xho I double digestion, transformed into escherichia coli XL1-blue, several white colonies of random choose extract plasmid DNA on the amicillin resistance flat board, go out possible recon by the gel electrophoresis Preliminary screening, further limiting property restriction endonuclease BamHI/Xho I enzyme action is identified, the enzyme action result has obtained a fragment and the treaty 5400bp left and right sides fragment of a treaty 608bp, consistent with expected results, name pcDNA (+)-mPNAS-4, further serve Hai Boya biotech firm and check order, qualification result is consistent with expection.

3, the acquisition of Xenopus laevis PNAS-4 (xPNAS-4) gene

According to the protein sequence (GenBank No:NM_016076) that people's PNAS-4 gene is submitted at NCBI, adopt the cDNA library of Blastp retrieval Xenopus laevis, thereby obtain the PNAS-4 gene cDNA sequence of Xenopus laevis; The cDNA sequential design special primer of PNAS-4 by above acquisition,

Forward primer (SEQ ID NO.12): 5 '-GGATCCATGGCCAACCAGCCCATCATC-3 '

Downstream primer (SEQ ID NO13): 5 '-CTCGAGCTATAGTTTTGTGTGGCGCCCAGG-3 '

Total RNA with the Xenopus laevis testis is a template, adopts one-step method RT-PCR amplification to obtain Xenopus laevis PNAS-4 gene open reading frame (ORF), and the product that obtains is 591bp, and then be cloned on the pGEM-T easy carrier, at last by sequence verification, with expection consistent (seeing Table 5, table 6).

The xPNAS-4cDNA of table 5 Xenopus laevis: sequence (SEQ ID NO.5)

atggccaacc?agcccatcat?cctcaatgtg?tatgatatgt actggattaa?tgaatataca

tcatcccttg?gaataggagt?tttccactct?ggaattcagg tatatggaag?agagtttgct

tatggaggtc?acccctatcc?attctctggt?gtgtttgaaa tctctcctgg?agattccacc

gaactgggag?acacttttaa?atttaaagaa?gcgatagccc tggggagcac?agatttcaca

gaaaatgata?tcgaaaaaat?aatcgaggaa?ctaggaaaag aatacaaagg?aaacgcatat

catctgatgc?acaaaaactg?caaccacttc?tcctcagctt tatcggagat?cctgtgcggg

aaagagattc?ctcgttgggt?taaccgacta?gcctacttca gtacctgtgt?accgtttttg

caaagctgcc?tacccaagga?atggctgaca?ccggctgctt tgcagtctag?cataagtcag

gaactccaag?acgaactgga?ggaagctgaa?gatgccgctg catcagcctc?cacctctaca

actgcaatgc?ccagacctgg?gcgccacaca?aaactatag

The protein sequence (SEQ ID NO.6) that the xPNAS-4 of table 6 Xenopus laevis is coded

Met?Ala?Asn?Gln?Pro?Ile?Ile?Leu?Asn?Val?Tyr?Asp?Met?Tyr?Trp?Ile

Asn?Glu?Tyr?Thr?Ser?Ser?Leu?Gly?Ile?Gly?Val?Phe?His?Ser?Gly?Ile

Gln?Val?Tyr?Gly?Arg?Glu?Phe?Ala?Tyr?Gly?Gly?His?Pro?Tyr?Pro?Phe

Ser?Gly?Val?Phe?Glu?Ile?Ser?Pro?Gly?Asp?Ser?Thr?Glu?Leu?Gly?Asp

Thr?Phe?Lys?Phe?Lys?Glu?Ala?Ile?Ala?Leu?Gly?Ser?Thr?Asp?Phe?Thr

Glu?Asn?Asp?Ile?Glu?Lys?Ile?Ile?Glu?Glu?Leu?Gly?Lys?Glu?Tyr?Lys

Gly?Asn?Ala?Tyr?His?Leu?Met?His?Lys?Asn?Cys?Asn?His?Phe?Ser?Ser

Ala?Leu?Ser?Glu?Ile?Leu?Cys?Gly?Lys?Glu?Ile?Pro?Arg?Trp?Val?Asn

Arg?Leu?Ala?Tyr?Phe?Ser?Thr?Cys?Val?Pro?Phe?Leu?Gln?Ser?Cys?Leu

Pro?Lys?Glu?Trp?Leu?Thr?Pro?Ala?Ala?Leu?Gln?Ser?Ser?Ile?Ser?Gln

Glu?Leu?Gln?Asp?Glu?Leu?Glu?Glu?Ala?Glu?Asp?Ala?Ala?Ala?Ser?Ala

Ser?Thr?Ser?Thr?Thr?Ala?Met?Pro?Arg?Pro?Gly?Arg?His?Thr?Lys?Leu

Embodiment two can be used for the structure of the PNAS-4 recombinant vector of gene therapy

1. the structure of recombiant plasmid PNAS-4-pVAX1:

By this area routine techniques the PNAS-4 that is cloned among the embodiment one on the pcDNA3.1-hPNAS-4 is downcut with BamH I, Xho I enzyme double digestion, promptly, sepharose electrophoresis and sequence verification are all correct on the BamH I of pVAX1 plasmid that insertion market is bought, the XhoI site.

2, the preparation of the liposome complex of recombiant plasmid PNAS-4-pVAX1

Liposome (lipofectamine 2000) is purchased the company in Invitrogen.

Prepare the liposome complex of recombiant plasmid PNAS-4-pVAX1 according to the operation instruction of liposome lipofectamine 2000, carried out the screening of liposome consumption, determine liposome at last: plasmid=1: 3 (weight ratio).

3, structure, production and the purification of the structure pAd-DEST-hPNAS-4 of PNAS-4 recombinant adenovirus and pAd-DEST-mPNAS-4.

Use this area routine techniques, with Gateway  Vectors sell pAd/PL-DEST be the basis, use this area routine techniques, pAd-DEST-mPNAS-4 and pAd-DEST-hPNAS-4 adenovirus expression carrier have successfully been made up, and with the adenovirus expression carrier transfection production of increasing in the 293A cell, extract according to a conventional method then and the purification of Recombinant adenovirus, test in, the body further external to carry out.

A, hPNAS-4, mPNAS-4 are building up among the medial expression vector pENTR 11

PNAS-4 homologous genes sequence (GenBank No.NM_024282) design primer (referring to embodiment one) according to disclosed PNAS-4 full-length gene order (GenBank No.NM-016076) and mice among the NCBI, for the gene clone of being increased being gone in pENTR 11 carriers, on coded sequence, introduce EcoRI and Xho I restriction enzyme site in the downstream primer respectively, do not introduce the V5 label on the recombinant adenoviral expressing vector in order to make, so in primer, introduced a termination codon, utilize increase the respectively coded sequence of hPNAS-4 and mPNAS-4 gene of these two pairs of primers then, and the fragment (about 600bp) that is increased is connected in pENTR 11 carriers, through transforming, screening, enzyme action is identified, dna sequencing is identified, is obtained medial expression vector plasmid pENTR11-hPNAS-4 and pENTR11-mPNAS-4.

B, pAd-DEST-hPNAS-4 and pAd-DEST-mPNAS-4 construction of recombinant adenovirus containing

According to including attL recombinase site and pAd/CMV/V5-DEST carrier on the pENTR11 carrier this characteristic of attR recombinase site is arranged, with supercoiled pENTR11-hPNAS-4, pENTR11-mPNAS-4 and pAd/CMV/V5-DEST carrier, according to the product operation instruction, respectively at recombinase LR Clonase ^TMUnder the effect of II enzymemix (Invitrogen), the LR recombining reaction takes place, can be with the hPNAS-4 on medial expression vector plasmid pENTR11-hPNAS-4 and the pENTR11-mPNAS-4, the mPNAS-4 gene is connected respectively in the pAd/CMV/V5-DEST carrier, owing to introduced a termination codon, so the V5 label does not have function in the constructed recombinant adenoviral expressing vector, be named as pAd-DEST-hPNAS-4 and pAd-DEST-mPNAS-4 respectively, among LR recombining reaction liquid transformed into escherichia coli one TOP10 with 2～3 μ l, utilize ampicillin (ampicillin) and chloramphenicol (chloromycetin) screening to insert the recombinant adenovirus clone (detailed process is with reference to the products instruction of Invitrogen) of exogenous gene, it is entirely true to extract our constructed recombinant adenoviral expressing vector of also order-checking proof of recombinant adenovirus expression plasmid.

C, amplification recombinant adenovirus

Extract constructed recombinant adenovirus-pAd-DEST-hPNAS-4 and pAd-DEST-mPNAS-4 expression plasmid, it is linear to utilize Pac I to carry out enzyme action, with the ITRs district on the left and right arm that exposes virus, help recombinant adenovirus duplicating and packing in the 293A cell, utilize Lipofectamine 2000 then, by its description operation, with linearizing recombinant adenovirus plasmid transfection in the 293A cell, virus massive duplication, packing in the 293A cell, acquisition can produce 293 seed cells of recombinant adenovirus.

D, collection, purification of Recombinant adenovirus

Collect the 293A seed cell among the step C and obtain rough recombinant adenovirus lysate supernatant, resulting rough recombinant virus is split to clean infect 293A, can obtain a large amount of recombinant adenovirus lysate supernatants through progressively amplifying to infect.Adopt conventional two step CsCl density gradient ultracentrifugation purification Ad-E then, the first step adopts discontinuous CsCl gradient to remove the virion of most cell debris and defective (virion that does not promptly possess infection activity), second step adopted continuous CsCl density gradient thoroughly will possess the virion of infection activity and the virion of defective distinguishes, pass through dialysis desalting at last again, remove CsCl, obtain pAd-DEST-hPNAS-4, pAd-DEST-mPNAS-4 and pAd-DEST-Null.

E, recombinant adenovirus titer determination

(detailed method is with reference to list of references: LaBarre DD to measure the titre of recombinant adenovirus according to tissue culture's 50 3nfective dose (TCID50) method, Lowy RJ.Improvements in methods for calculating virustiter estimates from TCID50and plaque assays.J Virol Methods.2001,96 (2): 107-26.).In brief, promptly use viral dilution liquid to infect the HEK293 cell respectively by 10 times of dilution preparations, be put in 37 ℃ of cultivation 10d in the CO2 incubator, observation of cell pathological changes effect (CPE) situation under the fluorescence inverted microscope then, and utilizing the formula calculating recombinant adenovirus of this method and the titre of empty virus, the result shows that pAd-DEST-hPNAS-4, the pAd-DEST-mPNAS-4 of present embodiment preparation, the titre of pAd-DEST-Null are respectively 4.5 * 10 ¹⁰PFU/ml, 3.35 * 10 ¹⁰PFU/ml, 4.0 * 10 ¹⁰PFU/ml reaches requirement.

The antitumor test of test example one product m-PNAS-4-PcDNA3.1 plasmid liposome complex of the present invention

Body outer cell proliferation activity experiment (MTT) is found, the propagation that can suppress mice lung cancer LL2 and colon cancer C26 cell is expressed in crossing of mPNAS-4 gene, PI dyeing and dna fragmentation fractional analysis show that the apoptosis (seeing figure .5) that can promote LL2 and C26 cell is expressed in crossing of mPNAS-4 gene.

Whether also has Graft Versus Tumor in vivo in order to detect the mPNAS-4 gene, set up BALB/c mouse colon cancer (C26) and two kinds of animal tumor models of C57BL/6 Mice Bearing Lewis Lung Cancer (LL2), carried out the test of the antitumor action of pcDNA3.1-mPNAS-4 and liposome complex.In C26 and LL2 tumor model, from inoculated tumour (i.e. back 7 days of treatment beginning) beginning in the 17th day, treatment group mice (M group) growth of tumor is slow than other each group obviously, has all produced tangible Graft Versus Tumor, and the result has statistical significance (P＜0.01); The mean survival time (MST) of treatment group mice, also obviously be longer than matched group, and the result has statistical significance (P＜0.05) (Fig.6); Behind inoculated tumour the 30th day the time, the gross tumor volume significant difference of treatment group and matched group, tumour inhibiting rate is 64.3% in the C26 tumor model, tumour inhibiting rate is 45.7% (seeing figure .6) in the LL2 tumor model.

Simultaneously, after treatment 4 times, get each group tumor tissues carry out the HE staining analysis: the tumor cell of visible a large amount of necrosis or apoptosis in treatment group mouse tumor group, the fine and close pyknosis of nucleus, endochylema is red dyes, and the phenomenon of lymphocytic infiltration is arranged.And (seeing figure .7A-figure .7D) appears in other each no tangible necrosis of group or apoptosis.Utilize the analysis of original position TUNEL detection kit respectively to organize apoptosis of tumor cells level in the tumor tissues shown in figure (see figure .7E-figure .7H) fluorescence photo, in the remaining tumor of Lewis lung cancer mice, the apoptotic cell showed increased.

But intravital tumor experiment shows that tumor pcDNA3.1-mPNAS-4 eukaryon expression plasmid interior or the tail vein injection liposome can be expressed mPNAS-4 albumen, with remarkable inhibition growth of tumor, prolongs the life cycle of tumor-bearing mice.

The antitumor test of test example two product liposome hPNAS-4-pcDNA3.1 plasmids of the present invention

Experiment in vitro and result

Each experiment is made as 4 groups respectively, is followed successively by: A, negative control (normal saline) group; B, liposome group; C, pcDNA3.1 zero load+liposome group; D, hPNAS-4-pcDNA3.1+ liposome group.

Cell inoculation carries out transfection after adherent growth about 70% covers with in 6 holes or 96 orifice plates, wherein the A group is handled with serum-free medium; B, C, D group are carried out transfection with liposome, pcDNA3.1 zero load+liposome, hPNAS-4-pcDNA3.1+ liposome respectively.

The transfection step is as follows: get 8 aseptic EP pipes of 1.5ml, be divided into two groups of a, b, every group of 4 pipes are labeled as 1,2,3,4 respectively.Add the corresponding amount serum-free medium respectively according to the transfection needs, add liposome (lipofectamine 2000) in a group 2,3,4 pipes; B organizes in 3 pipes and adds pcDNA3.1, adds hPNAS-4-pcDNA3.1 in 4 pipes.Corresponding mixing a, b 4 managed for two groups separately after room temperature was placed 5min, were labeled as 1,2,3, No. 4 pipe of c group.Room temperature is placed 30min, behind the liquid-transfering gun mixing according to aforementioned groupings respectively transfection respectively organize cell.The transfection concentration of wherein pcDNA3.1 zero load, hPNAS-4-pcDNA3.1 is 4 μ g/ml, liposome: plasmid is 3:1.Cell continues to cultivate in 37 ℃, 5%CO2, incubator after the transfection, adds after 6-8 hour to contain the serum culture fluid.37 ℃ of backs, 5%CO2, incubator continue to cultivate.

The experiment in vitro result: after SKOV348 hour, apoptosis rate is 82.6% to flow cytometry hPNAS-4-pcDNA3.1 group through the liposome transfection ovarian cancer cell, and cell blocks the phase in G0/G1, and proliferative cell reduces relatively in the distribution of S phase.And all the other are respectively organized apoptosis rate and are respectively: A, negative control group: 17.7%; B, liposome group: 33.1%; C, pcDNA3.1 zero load+liposome group: 40.0%; As seen hPNAS-4-pcDNA3.1 organizes scalariform band clearly behind liposome transfection; Hoechst33528 dyeing shows behind liposome transfection, sees the dense fine and close graininess fluorescence piece that dyes in the nucleus of hPNAS-4-pcDNA3.1 group and the Cytoplasm, prompting apoptosis (the results are shown in Figure 8).

In vivo test and result

In vivo test is for being provided with negative control group, liposome group, pcDNA3.1 zero load+liposome group, hPNAS-4-pcDNA3.1 group behind liposome transfection through tail vein injection on the ovarian cancer nude mice model, according to the experiment in vitro result, consumption is by every each tail vein injection 100 μ g plasmid equivalents.Treatment is taked per 3 days once, treats 11 times, to investigate hPNAS anti-tumor in vivo effect and toxic and side effects thereof.PcDNA3.1 zero load+liposome suspension and hPNAS-4-pcDNA3.1+ liposome suspension are according to plasmid: liposome=configuration in 1: 3.

The interior therapeutic result of the test: the lotus tumor group nude mice behind the liposome hPNAS-4-pcDNA3.1 plasmid preparation tail vein injection of the present invention can be expressed hPNAS-4 albumen in tumor tissues, make gross tumor volume obviously dwindle (Fig. 9) than matched group; HE dyeing shows that treatment group tumor center is obviously downright bad, and cell quantity reduces, and cell is rounded, the karyon engrain, and kytoplasm concentrates, and chromatin becomes lumps, prompting apoptosis of tumor cells (the results are shown in Figure 10).Vitals do not have obvious damage; Tunel detects its apoptosis to be increased; SABC shows that CD31 expresses reduction, and MVD is lower than matched group.Each is organized nude mice oophoroma tumor volume-time graph and sees Figure 10.

Embodiment three reorganization adenovirus pAd-DEST-hPNAS-4 and pAd-DEST-mPNAS-4 experimental results

Experiment in vitro

Select for use pAd-DEST-hPNAS-4 to carry out experiment in vitro.

DNA ladder (dna ladder shape band) test experience result

DNA ladder result shows, utilize the pAd-DEST-hPNAS-4 of liposome parcel to handle the LL/2 cell, handle and find after 24 hours that tangible DNA ladder phenomenon appears in this group cell, and the matched groups such as pAd-DEST-Null of liposome parcel fail to observe DNA ladder phenomenon, and this result shows that the pAd-DEST-hPNAS-4 of liposome parcel can obviously induce the LL/2 apoptosis.

The apoptosis PI experiment of dyeing

Apoptosis PI dyeing experimental result sees that (Figure 11) shows, utilize the pAd-DEST-hPNAS-4 processing LL/2 cell of liposome parcel to find after 24 hours that this group cell has obvious apoptosis to take place, and the pAd-DEST-Null processing LL/2 cell of liposome parcel has only produced a spot of apoptotic cell, other two groups of matched groups are not seen phenomena of apoptosis, this experiment shows that the pAd-DEST-hPNAS-4 of liposome parcel can express hPNAS-4 albumen, obviously induces the LL/2 apoptosis.

Experiment-recombinant adenovirus pAd-DEST-hPNAS-4 treatment Lewis lung cancer experiment in the body

The animal tumor model method for building up:

Collect the Mice Bearing Lewis Lung Cancer LL/2 cell of exponential phase, the centrifugal 3min of 1500rpm, cell precipitation washs 1 time with the culture fluid of serum-free, antibiotic-free, and adjusting cell concentration after the counting cells quantity is 1 * 10 ⁷Deliver to animal housing under the/ml, aseptic condition.

Adopt the C57BL/6 mice female mice about 6～8 ages in week, 18g, with 0.1ml (1 * 10 ⁶) the LL/2 cell inoculation to the right side of mice armpit subcutaneous.

Experiment grouping and processing:

Behind the inoculated tumour cell the 7th day, when laying one's hand on and during the about 4～5mm of diameter of tumor, tumor-bearing mice being divided into 4 groups at random, 10 every group, the beginning packet transaction was specially:

1) recombinant adenovirus pAd-DEST-hPNAS-4 treatment group: by every mice 5 * 10 ⁸The consumption that the pAd-DEST-hPNAS-4 recombinant adenovirus of PFU is joined the liposome of 200 μ g wraps up, and then this parcel complex is expelled in each mouse tumor, and injection in per 3 days is once injected 6 times continuously;

2) empty viral pAd-DEST-Null matched group: by every mice 5 * 10 ⁸The consumption of the pAd-DEST-Null recombinant adenovirus of PFU and the liposome of 200 μ g wraps up, and then this parcel complex is expelled in the mouse tumor, and injection in per 3 days is once injected 6 times continuously;

3) Liposome matched group: every mice intratumor injection Liposome 200 μ g/ time, injection in per 3 days is once injected 6 times continuously;

4) PBS matched group: every mice intratumor injection PBS 100 μ l/ time, injection in per 3 days is once injected 6 times continuously.

Result of the test shows: the pAd-DEST-hPNAS-4 of experimental group-liposome parcel can be in the mice body continuous expression hPNAS-4 albumen, find behind the treatment mice lung cancer that the tumor-bearing mice gross tumor volume obviously dwindles (seeing Figure 12) than three matched groups, survival time of mice obviously prolongs (seeing Figure 13), has demonstrated good antitumor action.Test example four products of the present invention and the test of cisplatin (cisplatin) coupling antitumor

Experiment in vitro:

Associating mPNAS-4 and cisplatin are handled mice CT26 colon cancer and IL/2 lung carcinoma cell, main growing multiplication by tumor cell suppresses experiment (MTT), DNA ladder and apoptosis morphological analysis, and whether test mPNAS-4 gene and cisplatin have the synergistic antitumor effect;

1MTT?assay

In 96 orifice plates, pair cell behind IL/2 and CT26 cell transfection pcDNA3.1 of elder generation or the pcDNA3.1-mPNAS gene 48h is MTT analyzes.The transfection amount of wherein pcDNA3.1 zero load, hPNAS-4-pcDNA3.1 plasmid is 0.2 μ g/ hole, and the ratio of liposome and plasmid is 2.5: 1.Uniting group for mPNAS-4 and cisplatin then is to add 5ug/ml cisplatin effect 24h behind the first transfection 0.2 μ g pcDNA3.1-mPNAS plasmid 24h.

Experimental result shows (seeing Figure 14), compares with matched group, and independent cisplatin and mPNAS-4 all have the effect that suppresses tumor cell proliferation; The effect that has stronger inhibition tumor cell proliferation after mPNAS-4 and the cisplatin associating.

2DNA ladder (dna ladder shape band) test experience result

Method is a conventional method.The result is as follows: compare with matched group and pcDNA3.1 empty carrier processed group, tangible dna ladder shape band all appears in mPNAS-4 and cisplatin individual processing, more obvious dna ladder shape band then appears in mPNAS-4 and cisplatin Combined Treatment, and apoptosis gets more when showing mPNAS-4 and cisplatin Combined Treatment than individual processing.

Experiment in the body

In vivo, set up BALB/c mouse colon cancer (CT26) and two kinds of tumor models of C57BL/6 mice lung cancer (LL/2) with conventional method, observe the mPNAS-4 gene and detect by immunohistochemistry techniques such as CD31, TUNEL and HE then by the influence to tumor growth behind the tail vein injection tumor-bearing mice.

Experiment grouping and processing:

Behind the inoculated tumour cell the 7th day, when laying one's hand on and during the about 4～5mm of diameter of tumor, tumor-bearing mice being divided into 5 groups at random, 10 every group, the beginning packet transaction was specially:

1) contrast (normal saline) group: every mouse tail vein injection normal saline 100 μ of normal saline group l/ time, weekly twice, injected for 4 weeks;

2) pcDNA3.1 liposome group: plasmid is injected by the equivalent of every each tail vein injection 100 μ g plasmids of mice through Liposome parcel back (plasmid and liposome mass ratio are 1: 3), and weekly twice, injected for 4 weeks;

3) cisplatin group: use Cisplatin separately: pass through lumbar injection: weekly according to the consumption lumbar injection cisplatin of 5mg cisplatin/kg body weight, injected for 4 weeks.

4) mPNAS-4 group: mPNAS-4Plasmid is through Liposome parcel back (mass ratio of plasmid and liposome is 1: 3), and plasmid and liposome complex pass through tail vein injection, 100ug mPNAS-4Plasmid/mouse, and secondary injected for 4 weeks weekly.

5) mPNAS-4+ cisplatin group: mPNAS-4Plasmid wraps up back (mass ratio of plasmid and liposome is 1: 3) through Liposome, tail vein injection, and 100ug mPNAS-4Plasmid/mouse, secondary injected for 4 weeks weekly; In second day behind the plasmid for the second time of injection weekly, weekly according to the consumption lumbar injection cisplatin of 5mg cisplatin/kg body weight, injected for 4 weeks.

Experimental result

(1). tumor growth and mice survival curve

Result of the test as shown in figure 15, in BALB/c mouse colon cancer (CT26) and two kinds of tumor models of C57BL/6 mice lung cancer (LL/2), all draw following result: compare with matched group and pcDNA3.1 empty carrier treatment group, mPNAS-4 and cisplatin treatment group separately tumor growth obviously are suppressed, and survival time of mice obviously prolongs.

And mPNAS-4 and cisplatin therapeutic alliance group tumor growth further are suppressed even have the tumor regression phenomenon, and survival time of mice further prolongs.

(2) CD31 dyeing

After the CD31 of tumor tissue section dyeed, numeration CD31 positive staining band (vessel density) came the growing amount of quantitative new vessels.The result shows, compares with matched group and pcDNA3.1 empty carrier treatment group, and the tumor neogenetic blood vessels quantity of mPNAS-4 and cisplatin treatment group separately obviously reduces, and mPNAS-4 and cisplatin therapeutic alliance group tumor neogenetic blood vessels quantity further reduce.

(3) TUNEL analyzes

Use the Tunel test kit, by specification requires to handle, and the back that disposes is detected under inverted fluorescence microscope and taken a picture, and the excitation wavelength of inverted fluorescence microscope is: 450-500nm, video picture wavelength are 515-565nm.Wherein the nucleus video picture is that dirty-green is the Tunel positive (being light color on black and white picture).As shown in figure 16, compare with matched group and pcDNA3.1 empty carrier treatment group, mPNAS-4 and cisplatin treatment group separately all can be to the tumor cell induction apoptosis in the tumor tissues, the then a large amount of apoptosis of the tumor cell in the tumor tissues of mPNAS-4 and cisplatin therapeutic alliance group.The result shows that the therapeutic alliance group can the interior apoptosis of tumor cells of co-induction tumor tissues.

This result of the test shows product of the present invention and cisplatin (cisplatin) coupling, the effect with collaborative resistive connection intestinal cancer, pulmonary carcinoma.Show that product of the present invention both can be used as antitumor drug, also can be used as antitumour auxiliary drug and and cisplatin (cisplatin) connection and use.Certainly, also can be designed to comprise the dosage form sale use of cisplatin (cisplatin).Test example five products of the present invention and the test of honokiol (honokiol) coupling antitumor

Honokiol (honokiol) has cancer cell specific induction of apoptosis and anti-new vessels nucleus formation as a kind of natural micromolecular compound, all can inducing apoptosis of tumour cell in external, body and PNAS-4 is verified.This test example is used the honokiol associating of mPNAS-4 gene therapy and low dosage, estimates its antitumor action.

In vitro tests and result

External, associating mPNAS-4 and honokiol are handled mice CT26 colon cancer and IL/2 lung carcinoma cell, growing multiplication by tumor cell suppresses experiment (MTT), DNA ladder and apoptosis morphological analysis, sees whether mPNAS-4 gene and honokiol have the synergistic antitumor effect;

1MTT?assay

In 96 orifice plates, pair cell behind IL/2 and CT26 cell transfection pcDNA3.1 of elder generation or the pcDNA3.1-mPNAS gene 48h is MTT analyzes.The transfection amount of wherein pcDNA3.1 zero load, hPNAS-4-pcDNA3.1 is 0.2 μ g/ hole, and the ratio of liposome and plasmid is 2.5: 1.Unite after group then is first transfection 0.2 μ g/ hole pcDNA3.1-mPNAS plasmid 24h for mPNAS-4 and honokiol, add 10ug/mL honokiol effect 24h.Experimental result shows (seeing Figure 17), compares with matched group, and independent honokiol (10ug/mL) and mPNAS-4 all have the effect that suppresses tumor IL/2 and CT26 cell proliferation; The effect that has stronger inhibition tumor cell proliferation after mPNAS-4 and the honokiol associating.

2DNA ladder (dna ladder shape band) test

Result of the test shows, compare with matched group and pcDNA3.1 empty carrier processed group, tangible dna ladder shape band all appears in mPNAS-4 and honokiol individual processing, more obvious dna ladder shape band then appears in mPNAS-4 and honokiol Combined Treatment, more can promote the apoptosis of cell when showing mPNAS-4 and honokiol Combined Treatment than individual processing.

In vivo test

Modeling and packet transaction

In the body, set up BALB/c mouse colon cancer (CT26) and two kinds of animal tumor models of C57BL/6 mice lung cancer (LL/2), observe the mPNAS-4 gene and the influence of tumor growth is carried out detection as a result by immunohistochemistry techniques such as alginate experiment, CD31 dyeing, TUNEL and HE then after by the tail vein injection tumor-bearing mice.

Modeling and grouping are with reference to test example four

1) contrast (normal saline) group; Behind the tumor inoculation 10 days, every mouse tail vein injection normal saline 100 μ l/ time, secondary injected for 4 weeks weekly;

2) pcDNA3.1 empty carrier group: tumor inoculation is after 10 days, the each tail vein injection 100 μ g pcDNA3.1 plasmids of every mice of pcDNA3.1 empty plasmid group, and secondary injected for 4 weeks weekly;

3) Honokiol group: tumor inoculation was used the Honokiol begin treatment separately after 10 days, and lumbar injection (i.p.) 0.5mg/mouse/ time, once a day, treated for 4 weeks continuously.

4) pcDNA3.1-mPNAS-4 group: tumor inoculation is after 10 days, mPNAS-4Plasmid is through Liposome parcel back (mass ratio of plasmid and liposome is 1: 3), pcDNA3.1-mPNAS-4 plasmid tail vein injection, 100ug pcDNA3.1-m PNAS-4Plasmid/mouse/ time, secondary injected for 4 weeks weekly;

5) pcDNA3.1-mPNAS-4 adds the Honokiol group: tumor inoculation is after 10 days, mPNAS-4Plasmid is through Liposome parcel back (mass ratio of plasmid and liposome is 1: 3), tail vein injection 100ug mPNAS-4Plasmid/mouse, secondary injected for 4 weeks weekly; The honokiol of lumbar injection 0.3mg/mouse dosage once a day, treated for 4 weeks continuously.

Result of the test

1, tumor growth and mice survival curve

As shown in figure 18, in BALB/c mouse colon cancer (CT26) and two kinds of tumor models of C57BL/6 mice lung cancer (LL/2), all draw following result: compare with matched group and pcDNA3.1 empty carrier treatment group, mPNAS-4 and honokiol treatment group separately tumor growth obviously are suppressed, and survival time of mice obviously prolongs.And mPNAS-4 and honokiol therapeutic alliance group tumor growth further are suppressed even have the tumor regression phenomenon, and survival time of mice further prolongs.

2, alginate experimental result

Each is organized mice and implants alginate, and takes out the alginate granule after 15 days in treatment and observe.Use the alginate bag and tested the situation that to observe mouse interior tumor cell peripheral new vessels.The result shows that list obviously is less than PBS and pcDNA3.1 treatment group with mPNAS-4 with single CT26 cell peripheral and inner new vessels with hydrochlorate bag quilt in the honokiol treatment group, and mPNAS-4 and honokiol therapeutic alliance group blood vessel further are suppressed.

3、CD31stain

After CD31 by tumor tissue section dyeed, numeration CD31 positive staining band (vessel density) came the growing amount of quantitative new vessels.Compare with matched group and pcDNA3.1 empty carrier treatment group, the tumor neogenetic blood vessels quantity of mPNAS-4 and honokiol treatment group separately obviously reduces, and mPNAS-4 and honokiol therapeutic alliance group tumor neogenetic blood vessels quantity further obviously reduce.

4, detect the natural death of cerebral cells situation with TUNEL assay (breach end-labelling))

Use the Tunel test kit, by specification requires to handle, and the back that disposes is detected under inverted fluorescence microscope and taken a picture, and the excitation wavelength of inverted fluorescence microscope is: 450～500nm, video picture wavelength are 515～565nm.Wherein the nucleus video picture is that dirty-green is the Tunel positive (being light color on black and white picture).Result of the test is seen as Figure 19, compare with matched group and pcDNA3.1 empty carrier treatment group, mPNAS-4 and honokiol treatment group separately all can be to the tumor cell induction apoptosis in the tumor tissues, the then a large amount of apoptosis of the tumor cell in the tumor tissues of mPNAS-4 and honokiol therapeutic alliance group.The result shows that the therapeutic alliance group can the interior apoptosis of tumor cells of co-induction tumor tissues.

This result of the test shows product of the present invention and honokiol (honokiol) coupling, the effect with collaborative resistive connection intestinal cancer, pulmonary carcinoma, and can significantly reduce the consumption of honokiol.Show that product of the present invention both can be used as antitumor drug, also can be used as antitumour auxiliary drug and honokiol (honokiol) coupling.Certainly, also can be designed to comprise the dosage form use of honokiol.

Test example six security of products preliminary assessments of the present invention

Also used HE dyeing whether impaired in the efficacy test process of product of the present invention to reach other vital tissues of mice around the detection tumor.

(1) hPNAS treatment mouse ovarian cancer test group

In the process of the test of embodiment, in order to observe the tumor tissues form, understand each important organ toxicity simultaneously, tumor tissues and vitals have been carried out HE dyeing.Wherein the tumor tissues paraffin section HE of hPNAS-4-pcDNA3.1+ liposome therapeutic group dyeing shows, the middle section cell quantity appears in tumor tissues to be reduced, and cell is rounded, the karyon engrain, and kytoplasm concentrates, and chromatin becomes lumps, the prompting apoptosis of tumor cells.Its excess-three group tumor growth is normal, and cell number is many than the treatment group, does not see textural anomaly, does not see necrotic zone, does not have a large amount of apoptosis signs.

Get the hPNAS-4-pcDNA3.1+ liposome therapeutic group heart, liver, spleen, lung, nephridial tissue, carry out conventional H E dyeing, detect its treatment back side effect.Organize each vitals paraffin section HE dyeing as seen from treatment, the heart, spleen, lung, nephridial tissue structural integrity are not seen obvious damage or tissue necrosis.It is big that liver organization small part cell volume becomes, and endochylema is loose to be netted or transparent, for the balloon sample becomes.This is the performance of liver organization reversibility damage common behind the drug administration.But do not see obvious necrosis or fibrosis degeneration in the liver organization.This results suggest h-PNAS-4-PcDNA3.1+ Liposomal formulation has certain liver side effect, but this damages and be the reversibility damage, and no significant threat is a kind of safer antitumor drug, can guarantee patient's quality of life well.

(2) observe about the toxic and side effects of mPNAS treatment mice lung cancer and colon cancer

Observe the ordinary circumstance of tumor-bearing mice in the therapeutic process, comprise fur, body weight, activity and feed situation etc., relatively do not find significantly difference between each group.Put to death mice after treatment, get internal organs such as the brain of respectively organizing mice, lung, the heart, liver,spleen,kidney, pancreas, small intestinal, bone marrow, carry out perusal, and make pathological section, HE dyeing back is observed under optical microscope, do not find significantly unusual.

The above more excellent specific embodiment is that the present invention is further illustrated, but be not limitation of the scope of the invention, though provide the specific embodiment of using expression vector in the embodiment of the invention, but can learn in conjunction with this area general knowledge again according to content disclosed by the invention, directly use the expressed albumen of PNAS-4 gene also can reach proximate effect.Those skilled in the art can make various modification or improvement according to basic thought of the present invention.Such as by this area general knowledge as can be known, if different requirements is arranged, the consumption of antineoplastic pharmaceutical compositions of the present invention and antitumour auxiliary drug compositions and occupation mode can change in a big way at one.Those skilled in the art can be according to some known factors, such as the kind of disease, and the degree that is in a bad way, patient body weight, dosage form, selected routes of administration etc. are determined at an easy rate.Only otherwise break away from basic thought of the present invention, these changes are all within the scope that spirit of the present invention and claims of being enclosed define.

Purposes _ ST25SEQUENCE the LISTING of PNAS-4 gene in preparation antitumor and antitumour auxiliary drug

＜110〉Sichuan University

＜120〉purposes of PNAS-4 gene in preparation antitumor and antitumour auxiliary drug

<130>A070149

<160>13

<170>PatentIn?version?3.4

<210>1

<211>585

<212>DNA

<213>Homo?sapiens

<220>

<221>CDS

<222>(1)..(585)

<400>1

atg?ggg?gct?aac?cag?tta?gtg?gtg?ctc?aac?gtg?tac?gac?atg?tat?tgg 48

Met?Gly?Ala?Asn?Gln?Leu?Val?Val?Leu?Asn?Val?Tyr?Asp?Met?Tyr?Trp

1 5 10 15

atg?aac?gaa?tat?acc?tca?tcc?att?gga?att?gga?gtt?ttt?cat?tca?gga 96

Met?Asn?Glu?Tyr?Thr?Ser?Ser?Ile?Gly?Ile?Gly?Val?Phe?His?Ser?Gly

20 25 30

att?gaa?gtc?tat?ggc?aga?gaa?ttt?gct?tat?ggt?ggc?cat?cct?tac?ccc 144

Ile?Glu?Val?Tyr?Gly?Arg?Glu?Phe?Ala?Tyr?Gly?Gly?His?Pro?Tyr?Pro

35 40 45

ttt?tct?gga?ata?ttt?gaa?att?tcc?cca?gga?aat?gct?tct?gaa?cta?gga 192

Phe?Ser?Gly?Ile?Phe?Glu?Ile?Ser?Pro?Gly?Asn?Ala?Ser?Glu?Leu?Gly

50 55 60

gaa?aca?ttt?aaa?ttt?aaa?gaa?gct?gtt?gtt?tta?ggg?agc?acg?gac?ttc 240

Glu?Thr?Phe?Lys?Phe?Lys?Glu?Ala?Val?Val?Leu?Gly?Ser?Thr?Asp?Phe

65 70 75 80

cta?gaa?gat?gat?ata?gaa?aaa?att?gta?gaa?gaa?ctg?gga?aaa?gaa?tac 288

Leu?Glu?Asp?Asp?Ile?Glu?Lys?Ile?Val?Glu?Glu?Leu?Gly?Lys?Glu?Tyr

85 90 95

aaa?ggc?aat?gct?tat?cat?tta?atg?cat?aaa?aac?tgc?aat?cat?ttt?tct 336

Lys?Gly?Asn?Ala?Tyr?His?Leu?Met?His?Lys?Asn?Cys?Asn?His?Phe?Ser

100 105 110

tca?gct?tta?tca?gag?att?ctt?tgt?ggg?aaa?gag?att?cct?cgc?tgg?atc 384

Ser?Ala?Leu?Ser?Glu?Ile?Leu?Cys?Gly?Lys?Glu?Ile?Pro?Arg?Trp?Ile

115 120 125

aat?cga?ctt?gcc?tac?ttc?agc?tcc?tgt?ata?ccc?ttt?cta?cag?agt?tgc 432

Asn?Arg?Leu?Ala?Tyr?Phe?Ser?Ser?Cys?Ile?Pro?Phe?Leu?Gln?Ser?Cys

130 135 140

ctc?ccg?aag?gag?tgg?ctc?acg?ccc?gca?gcc?ctg?cag?tct?agt?gtc?agc 480

Leu?Pro?Lys?Glu?Trp?Leu?Thr?Pro?Ala?Ala?Leu?Gln?Ser?Ser?Val?Ser

145 150 155 160

caa?gaa?ctc?cag?gat?gaa?ctg?gag?gaa?gca?gag?gat?gct?gcc?gca?tcc 528

Gln?Glu?Leu?Gln?Asp?Glu?Leu?Glu?Glu?Ala?Glu?Asp?Ala?Ala?Ala?Ser

165 170 175

gct?tcc?gtg?gca?agc?act?gca?gca?ggc?tcc?aga?ccc?ggg?cgc?cac?act 576

Ala?Ser?Val?Ala?Ser?Thr?Ala?Ala?Gly?Ser?Arg?Pro?Gly?Arg?His?Thr

180 185 190

aaa?cta?taa 585

Lys?Leu

Purposes _ the ST25 of PNAS-4 gene in preparation antitumor and antitumour auxiliary drug

<210>2

<211>194

<212>PRT

<213>Homo?sapiens

<400>2

Met?Gly?Ala?Asn?Gln?Leu?Val?Val?Leu?Asn?Val?Tyr?Asp?Met?Tyr?Trp

1 5 10 15

Met?Asn?Glu?Tyr?Thr?Ser?Ser?Ile?Gly?Ile?Gly?Val?Phe?His?Ser?Gly

20 25 30

Ile?Glu?Val?Tyr?Gly?Arg?Glu?Phe?Ala?Tyr?Gly?Gly?His?Pro?Tyr?Pro

35 40 45

Phe?Ser?Gly?Ile?Phe?Glu?Ile?Ser?Pro?Gly?Asn?Ala?Ser?Glu?Leu?Gly

50 55 60

Glu?Thr?Phe?Lys?Phe?Lys?Glu?Ala?Val?Val?Leu?Gly?Ser?Thr?Asp?Phe

65 70 75 80

Leu?Glu?Asp?Asp?Ile?Glu?Lys?Ile?Val?Glu?Glu?Leu?Gly?Lys?Glu?Tyr

85 90 95

Lys?Gly?Asn?Ala?Tyr?His?Leu?Met?His?Lys?Asn?Cys?Asn?His?Phe?Ser

100 105 110

Ser?Ala?Leu?Ser?Glu?Ile?Leu?Cys?Gly?Lys?Glu?Ile?Pro?Arg?Trp?Ile

115 120 125

Asn?Arg?Leu?Ala?Tyr?Phe?Ser?Ser?Cys?Ile?Pro?Phe?Leu?Gln?Ser?Cys

130 135 140

Leu?Pro?Lys?Glu?Trp?Leu?Thr?Pro?Ala?Ala?Leu?Gln?Ser?Ser?Val?Ser

145 150 155 160

Gln?Glu?Leu?Gln?Asp?Glu?Leu?Glu?Glu?Ala?Glu?Asp?Ala?Ala?Ala?Ser

165 170 175

Ala?Ser?Val?Ala?Ser?Thr?Ala?Ala?Gly?Ser?Arg?Pro?Gly?Arg?His?Thr

180 185 190

Lys?Leu

<210>3

<211>585

<212>DNA

<213>Mus?musculus

<220>

<221>CDS

<222>(1)..(585)

<400>3

atg?ggg?gct?aac?cag?tta?gtg?gtg?ctc?aac?gtc?tac?gac?atg?tac?tgg 48

Met?Gly?Ala?Ash?Gln?Leu?Val?Val?Leu?Ash?Val?Tyr?Asp?Met?Tyr?Trp

1 5 10 15

atg?aat?gaa?tac?acc?tca?tct?att?gga?att?gga?gtt?ttt?cat?tct?gga 96

Met?Asn?Glu?Tyr?Thr?Ser?Ser?Ile?Gly?Ile?Gly?Val?Phe?His?Ser?Gly

Purposes _ the ST25 of PNAS-4 gene in preparation antitumor and antitumour auxiliary drug

20 25 30

att?gaa?gta?tat?ggc?aga?gag?ttt?gct?tat?ggt?ggc?cat?cca?tat?cct 144

Ile?Glu?Val?Tyr?Gly?Arg?Glu?Phe?Ala?Tyr?Gly?Gly?His?Pro?Tyr?Pro

35 40 45

ttt?tct?gga?ata?ttt?gaa?att?tcc?cca?gga?aat?gct?tct?gag?cta?gga 192

Phe?Ser?Gly?Ile?Phe?Glu?Ile?Ser?Pro?Gly?Asn?Ala?Ser?Glu?Leu?Gly

50 55 60

gaa?aca?ttt?aaa?ttt?aaa?gaa?gct?gtt?gtt?cta?gga?agt?acg?gac?ttt 240

Glu?Thr?Phe?Lys?Phe?Lys?Glu?Ala?Val?Val?Leu?Gly?Ser?Thr?Asp?Phe

65 70 75 80

cta?gaa?gat?gat?ata?gag?aaa?att?gta?gaa?gaa?ctg?ggg?aaa?gag?tat 288

Leu?Glu?Asp?Asp?Ile?Glu?Lys?Ile?Val?Glu?Glu?Leu?Gly?Lys?Glu?Tyr

85 90 95

aag?ggc?aac?gcc?tac?cat?ctg?atg?cac?aaa?aac?tgc?aat?cac?ttt?tct 336

Lys?Gly?Asn?Ala?Tyr?His?Leu?Met?His?Lys?Asn?Cys?Asn?His?Phe?Ser

100 105 110

tca?gct?tta?tca?gag?att?ctc?tgt?ggg?aaa?gag?att?cct?cgc?tgg?atc 384

Ser?Ala?Leu?Ser?Glu?Ile?Leu?Cys?Gly?Lys?Glu?Ile?Pro?Arg?Trp?Ile

115 120 125

aac?cgg?ctg?gcc?tac?ttc?agc?tcc?tgt?ata?ccc?ttt?cta?cag?agt?tgc 432

Asn?Arg?Leu?Ala?Tyr?Phe?Ser?Ser?Cys?Ile?Pro?Phe?Leu?Gln?Ser?Cys

130 135 140

ctg?cca?aag?gag?tgg?ctc?act?cct?gct?gca?ctt?cag?tct?agt?gtc?agc 480

Leu?Pro?Lys?Glu?Trp?Leu?Thr?Pro?Ala?Ala?Leu?Gln?Ser?Ser?Val?Ser

145 150 155 160

caa?gaa?ctc?cag?gat?gaa?cta?gaa?gaa?gca?gag?gat?gca?gcc?gca?tcc 528

Gln?Glu?Leu?Gln?Asp?Glu?Leu?Glu?Glu?Ala?Glu?Asp?Ala?Ala?Ala?Ser

165 170 175

agt?gcc?atg?gca?agt?gct?gca?gct?ggt?gcc?aga?act?ggg?cgt?cac?act 576

Ser?Ala?Met?Ala?Ser?Ala?Ala?Ala?Gly?Ala?Arg?Thr?Gly?Arg?His?Thr

180 185 190

aaa?cta?taa 585

Lys?Leu

<210>4

<211>194

<212>PRT

<213>Mus?musculus

<400>4

Met?Gly?Ala?Asn?Gln?Leu?Val?Val?Leu?Asn?Val?Tyr?Asp?Met?Tyr?Trp

1 5 10 15

Met?Asn?Glu?Tyr?Thr?Ser?Ser?Ile?Gly?Ile?Gly?Val?Phe?His?Ser?Gly

20 25 30

Ile?Glu?Val?Tyr?Gly?Arg?Glu?Phe?Ala?Tyr?Gly?Gly?His?Pro?Tyr?Pro

35 40 45

Phe?Ser?Gly?Ile?Phe?Glu?Ile?Ser?Pro?Gly?Asn?Ala?Ser?Glu?Leu?Gly

50 55 60

Glu?Thr?Phe?Lys?Phe?Lys?Glu?Ala?Val?Val?Leu?Gly?Ser?Thr?Asp?Phe

65 70 75 80

Leu?Glu?Asp?Asp?Ile?Glu?Lys?Ile?Val?Glu?Glu?Leu?Gly?Lys?Glu?Tyr

85 90 95

The purposes ST25 of PNAS-4 gene in preparation antitumor and antitumour auxiliary drug

Lys?Gly?Asn?Ala?Tyr?His?Leu?Met?His?Lys?Asn?Cys?Asn?His?Phe?Ser

100 105 110

Ser?Ala?Leu?Ser?Glu?Ile?Leu?Cys?Gly?Lys?Glu?Ile?Pro?Arg?Trp?Ile

115 120 125

Asn?Arg?Leu?Ala?Tyr?Phe?Ser?Ser?Cys?Ile?Pro?Phe?Leu?Gln?Ser?Cys

130 135 140

Leu?Pro?Lys?Glu?Trp?Leu?Thr?Pro?Ala?Ala?Leu?Gln?Ser?Ser?Val?Ser

145 150 155 160

Gln?Glu?Leu?Gln?Asp?Glu?Leu?Glu?Glu?Ala?Glu?Asp?Ala?Ala?Ala?Ser

165 170 175

Ser?Ala?Met?Ala?Ser?Ala?Ala?Ala?Gly?Ala?Arg?Thr?Gly?Arg?His?Thr

180 185 190

Lys?Leu

<210>5

<211>579

<212>DNA

<213>Xenopus?laevis

<220>

<221>CDS

<222>(1)..(579)

<400>5

atg?gcc?aac?cag?ccc?atc?atc?ctc?aat?gtg?tat?gat?atg?tac?tgg?att 48

Met?Ala?Asn?Gln?Pro?Ile?Ile?Leu?Asn?Val?Tyr?Asp?Met?Tyr?Trp?Ile

1 5 10 15

aat?gaa?tat?aca?tca?tcc?ctt?gga?ata?gga?gtt?ttc?cac?tct?gga?att 96

Asn?Glu?Tyr?Thr?Ser?Ser?Leu?Gly?Ile?Gly?Val?Phe?His?Ser?Gly?Ile

20 25 30

cag?gta?tat?gga?aga?gag?ttt?gct?tat?gga?ggt?cac?ccc?tat?cca?ttc 144

Gln?Val?Tyr?Gly?Arg?Glu?Phe?Ala?Tyr?Gly?Gly?His?Pro?Tyr?Pro?Phe

35 40 45

tct?ggt?gtg?ttt?gaa?atc?tct?cct?gga?gat?tcc?acc?gaa?ctg?gga?gac 192

Ser?Gly?Val?Phe?Glu?Ile?Ser?Pro?Gly?Asp?Ser?Thr?Glu?Leu?Gly?Asp

50 55 60

act?ttt?aaa?ttt?aaa?gaa?gcg?ata?gcc?ctg?ggg?agc?aca?gat?ttc?aca 240

Thr?Phe?Lys?Phe?Lys?Glu?Ala?Ile?Ala?Leu?Gly?Ser?Thr?Asp?Phe?Thr

65 70 75 80

gaa?aat?gat?atc?gaa?aaa?ata?atc?gag?gaa?cta?gga?aaa?gaa?tac?aaa 288

Glu?Asn?Asp?Ile?Glu?Lys?Ile?Ile?Glu?Glu?Leu?Gly?Lys?Glu?Tyr?Lys

85 90 95

gga?aac?gca?tat?cat?ctg?atg?cac?aaa?aac?tgc?aac?cac?ttc?tcc?tca 336

Gly?Asn?Ala?Tyr?His?Leu?Met?His?Lys?Asn?Cys?Asn?His?Phe?Ser?Ser

100 105 110

gct?tta?tcg?gag?atc?ctg?tgc?ggg?aaa?gag?att?cct?cgt?tgg?gtt?aac 384

Ala?Leu?Ser?Glu?Ile?Leu?Cys?Gly?Lys?Glu?Ile?Pro?Arg?Trp?Val?Asn

115 120 125

cga?cta?gcc?tac?ttc?agt?acc?tgt?gta?ccg?ttt?ttg?caa?agc?tgc?cta 432

Arg?Leu?Ala?Tyr?Phe?Ser?Thr?Cys?Val?Pro?Phe?Leu?Gln?Ser?Cys?Leu

130 135 140

Purposes _ the ST25 of PNAS-4 gene in preparation antitumor and antitumour auxiliary drug

ccc?aag?gaa?tgg?ctg?aca?ccg?gct?gct?ttg?cag?tct?agc?ata?agt?cag 480

Pro?Lys?Glu?Trp?Leu?Thr?Pro?Ala?Ala?Leu?Gln?Ser?Ser?Ile?Ser?Gln

145 150 155 160

gaa?ctc?caa?gac?gaa?ctg?gag?gaa?gct?gaa?gat?gcc?gct?gca?tca?gcc 528

Glu?Leu?Gln?Asp?Glu?Leu?Glu?Glu?Ala?Glu?Asp?Ala?Ala?Ala?Ser?Ala

165 170 175

tcc?acc?tct?aca?act?gca?atg?ccc?aga?cct?ggg?cgc?cac?aca?aaa?cta 576

Ser?Thr?Ser?Thr?Thr?Ala?Met?Pro?Arg?Pro?Gly?Arg?His?Thr?Lys?Leu

180 185 190

tag 579

<210>6

<211>192

<212>PRT

<213>Xenopus?laevis

<400>6

Met?Ala?Asn?Gln?Pro?Ile?Ile?Leu?Asn?Val?Tyr?Asp?Met?Tyr?Trp?Ile

1 5 10 15

Asn?Glu?Tyr?Thr?Ser?Ser?Leu?Gly?Ile?Gly?Val?Phe?His?Ser?Gly?Ile

20 25 30

Gln?Val?Tyr?Gly?Arg?Glu?Phe?Ala?Tyr?Gly?Gly?His?Pro?Tyr?Pro?Phe

35 40 45

Ser?Gly?Val?Phe?Glu?Ile?Ser?Pro?Gly?Asp?Ser?Thr?Glu?Leu?Gly?Asp

50 55 60

Thr?Phe?Lys?Phe?Lys?Glu?Ala?Ile?Ala?Leu?Gly?Ser?Thr?Asp?Phe?Thr

65 70 75 80

Glu?Asn?Asp?Ile?Glu?Lys?Ile?Ile?Glu?Glu?Leu?Gly?Lys?Glu?Tyr?Lys

85 90 95

Gly?Asn?Ala?Tyr?His?Leu?Met?His?Lys?Asn?Cys?Asn?His?Phe?Ser?Ser

100 105 110

Ala?Leu?Ser?Glu?Ile?Leu?Cys?Gly?Lys?Glu?Ile?Pro?Arg?Trp?Val?Asn

115 120 125

Arg?Leu?Ala?Tyr?Phe?Ser?Thr?Cys?Val?Pro?Phe?Leu?Gln?Ser?Cys?Leu

130 135 140

Pro?Lys?Glu?Trp?Leu?Thr?Pro?Ala?Ala?Leu?Gln?Ser?Ser?Ile?Ser?Gln

145 150 155 160

Glu?Leu?Gln?Asp?Glu?Leu?Glu?Glu?Ala?Glu?Asp?Ala?Ala?Ala?Ser?Ala

165 170 175

Ser?Thr?Ser?Thr?Thr?Ala?Met?Pro?Arg?Pro?Gly?Arg?His?Thr?Lys?Leu

180 185 190

<210>7

<211>31

<212>DNA

<213>Artificial

Purposes _ the ST25 of PNAS-4 gene in preparation antitumor and antitumour auxiliary drug

<220>

<223>Artificial

<400>7

gcggatccaa?gatggcagat?tttttgaaag?g 31

<210>8

<211>32

<212>DNA

<213>Artificial

<220>

<223>Artificial

<400>8

cgcgatatct?taagtgtcct?cgtgcatgtc?tg 32

<210>9

<211>37

<212>DNA

<213>Artificial

<220>

<223>Artificial

<400>9

gcgccaccgg?atccaagatg?gcagattttt?tgaaagg 37

<210>10

<211>35

<212>DNA

<213>Artificial

<220>

<223>Artificial

<400>10

gcggatccgc?caccatggcc?aaccagccca?tcatc 35

<210>11

<211>33

<212>DNA

<213>Artificial

<220>

<223>Artificial

<400>11

ccgctcgagc?tatagttttg?tgtggcgccc?agg 33

<210>12

<211>27

<212>DNA

<213>Artificial

<220>

<223>Artificial

<400>12

ggatccatgg?ccaaccagcc?catcatc 27

<210>13

<211>30

<212>DNA

<213>Artificial

<220>

<223>Artificial

<400>13

Purposes _ the ST25 of PNAS-4 gene in preparation antitumor and antitumour auxiliary drug

ctcgagctat?agttttgtgt?ggcgcccagg 30

Claims (14)

1.PNAS-4 the purposes of gene in preparation antitumor drug thing and antitumour auxiliary drug.

2. purposes according to claim 1 is characterized in that: the sequence of described PNAS-4 gene is shown in SEQ ID NO.1, SEQ ID NO.3 or the SEQ ID NO.5.

3.PNAS-4 the purposes of albumen in preparation antitumor drug and antitumour auxiliary drug.

4. purposes according to claim 3 is characterized in that: the proteic sequence of described PNAS-4 is shown in SEQ ID NO.2, SEQ ID NO.4 or the SEQ ID NO.6.

5. a recombinant vector is characterized in that containing the PNAS-4 gene that sequence is SEQ ID NO.1, SEQ ID NO.3 or SEQID NO.5.

6. recombinant vector according to claim 5 is characterized in that: described carrier is a carrier for expression of eukaryon.

7. host cell that contains claim 5 or 6 described recombinant vectors.

8. claim 5 or the 6 described recombinant vectors purposes in preparation antineoplastic pharmaceutical compositions or antitumour auxiliary drug compositions.

9. antineoplastic pharmaceutical compositions or antitumour auxiliary drug compositions are that albumen by PNAS-4 gene or PNAS-4 gene code adds pharmaceutically as main active that the acceptable adjuvant is prepared from.

10. antineoplastic pharmaceutical compositions or antitumour auxiliary drug compositions are to add pharmaceutically by claim 5 or 6 described recombinant vectors as main active that the acceptable adjuvant is prepared from.

11. according to claim 9 or 10 described antineoplastic pharmaceutical compositions or antitumour auxiliary drug compositions, it is characterized in that: described active ingredient in pharmaceutical is by quilt that liposome wraps.

12., it is characterized in that also containing tumor chemotherapeutic drug as active component according to each described antineoplastic pharmaceutical compositions of claim 9～11.

13. a method for preparing the described recombinant vector of claim 5 is characterized in that may further comprise the steps:

A, go into the recombinant vector that carrier obtains containing PNAS-4 with PNAS-4 is gene constructed;

B, the recombinant vector that a step gained is contained PNAS-4 change host cell over to, amplification preparation recombinant vector.

14. one kind prepares described antineoplastic pharmaceutical compositions of claim 10 or antitumour auxiliary drug method for compositions, it is characterized in that may further comprise the steps:

A, under aseptic condition, get water for injection, add raw material by following proportioning again: reorganization PNAS-4 adenovirus 1 * 10 ⁹～9 * 10 ¹¹IU/ml, 5% glucose solution;

B, under aseptic condition with step a product mix homogeneously, regulate pH value to 7.5～8.5 with buffer;

C, will be distributed into injection after the aseptic filtration of step b product, or make lyophilized formulations.

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