CN107475298A - CdtB gene overexpressions slow virus carrier and its construction method and the slow virus comprising cdtB genes and its application - Google Patents
CdtB gene overexpressions slow virus carrier and its construction method and the slow virus comprising cdtB genes and its application Download PDFInfo
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Abstract
Slow virus the invention provides cdtB gene overexpressions slow virus carrier and its construction method and comprising cdtB genes and its application, belong to gene engineering technology field.A kind of cdtB gene overexpressions slow virus carrier pLVX AcGFP1 N1 cdtB, based on pLVX AcGFP1 N1 Lentivirals, cdtB genes are introduced at multiple cloning sites.Present invention also offers the slow virus containing cdtB genes, the pLVX AcGFP1 N1 cdtB carriers obtained using structure are mixed with slow virus packaging system VSVG, pCMV △ R8.2, realize slow virus packaging process, obtain being capable of the slow virus of direct infection eukaryotic, it is achieved thereby that the purpose of cdtB genes normal expression in eukaryotic, to further appreciate that the function of cdtB genes is laid a solid foundation.
Description
Technical field
The invention belongs to gene engineering technology field, and in particular to cdtB gene overexpressions slow virus carrier and its structure side
Method and the slow virus comprising cdtB genes and its application.
Background technology
CDT is that one kind has cytotoxic protein, including campylobacter jejuni, Mou Xie great as caused by some gram-negative bacterias
Enterobacteria, shigella dysenteriae, haemophilus and helicobacter hepaticus.Complete CDT albumen is by tri- subunits of cdtA, cdtB, cdtC
Composition, wherein cdtA combinations cell membrane, cdtC helps cdtB to enter cell, and then cdtB causes cytotoxicity.CdtB is Mg2+、
Ca2+Dependence neutrality nuclease, containing DNA hydrolysis and cation binding structural domain, triggered by hydrolytic cleavage phosphodiester bond
The DNA damage of host cell, and cause the extension of cytoplasm and nucleus, active cell cycle checkpoint blocks the cell cycle
The G2/M phases, ultimately result in cellular swelling and apoptosis.
The specific effect acted to further study cdtB to cell, it is former that existing method mainly builds cdtB
Nuclear expression carrier, by inducing Escherichia coli vivoexpression cdtB albumen, cdtB albumen, the cdtB eggs of gained are obtained by purifying
Pass through Microinjected cells in vain or be directly added into cell culture fluid, for observing effects of the cdtB to cell.But protokaryon table
The cdtB protein structures reached can occur necessarily to change, and may influence the function of cdtB albumen, and individually cdtB albumen is difficult
Enter into the cell, while microinjection condition requires higher and influences cell state, it is usually used in order to overcome above mentioned problem
Heavy dose of cdtB albumen and cell co-cultivation, but this method needs substantial amounts of cdtB albumen, this hinder significantly on
The progress that cdtB gene pairs cells act.
The content of the invention
In view of this, it is an object of the invention to provide cdtB gene overexpressions slow virus carrier and its construction method and bag
The slow virus of the gene containing cdtB and its application, make cdtB genes normal expression in eukaryotic.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The invention provides a kind of cdtB gene overexpressions slow virus carrier pLVX-AcGFP1-N1-cdtB, with pLVX-
Based on AcGFP1-N1 Lentivirals, cdtB genes are introduced at multiple cloning sites；The cdtB genes have such as sequence
Nucleotide sequence in list shown in SeqID No.1.
Preferably, the multiple cloning sites are XhoI and BamHI restriction enzyme site.
Preferably, the multiple cloning sites are MCS sequences.
The invention provides a kind of construction method of cdtB gene overexpressions slow virus carrier, comprise the following steps：
1) performing PCR amplification cdtB genes are entered using primer from helicobacter hepaticus genome, described primer has such as sequence
SEQ IDNo.1 and SEQ ID No.2 nucleotide sequence in table；
2) PCR primer obtained in the step 1) is connected in carrier T, obtains the carrier T with cdtB fragments；
3) with restriction enzyme XhoI and BamHI respectively to the carrier T with cdtB and slow virus carrier Plvx-
AcGFP1-N1 carries out double digestion, respectively obtains linearisation cdtB genetic fragments and linear slow virus carrier fragment；
4) linearisation cdtB genetic fragments that the step 3) obtains and linear slow virus carrier fragment are attached, obtained
To cdtB gene overexpression slow virus carriers pLVX-AcGFP1-N1-cdtB.
Preferably, the reaction system that PCR is expanded in the step 1) is as follows：
| Archaeal dna polymerase | 10μL |
| 10 μm of ol primers SEQIDNo.1 | 1μL |
| 10 μm of ol primers SEQIDNo.2 | 1μL |
| 100ng/ μ L helicobacter hepaticus genomes | 1μL |
| ddH2O | 7μL |
| Amount to | 20μL |
The response procedures that PCR is expanded in the step 1) are as follows：98 DEG C of 1min30s, 98 DEG C of 30s, 34 circulations, 50 DEG C
30s, 72 DEG C of 30s, 72 DEG C of 1min30s.
Preferably, the digestion system of double digestion is as follows in the step 3)：
| Carrier T or pLVX-AcGFP1-N1 | 10μL |
| BamHI | 1μL |
| XhoI | 1μL |
| 10x enzyme cutting buffering liquids | 2μL |
| ddH2O | 6μL |
| Amount to | 20μL |
The temperature of the double digestion is 37 DEG C, and the time of the double digestion is 12h.
Preferably, the system of connection is as follows in the step 4)：
| Linearize cdtB genetic fragments | 5μL |
| Linear slow virus carrier pLVX-AcGFP1-N1 fragments | 2μL |
| T4DNA ligases | 0.5μL |
| T4DNA ligase buffer solutions | 1μL |
| ddH2O | 1.5μL |
| Amount to | 10μL |
The temperature of connection is 16 DEG C in the step 4)；The time of the connection is 12h.
Preferably, also include after being connected in the step 4)：Recombinant plasmid is obtained to the connection and carries out positive bacterium colony
Structure and identification.
The invention provides the slow virus containing cdtB genes, it is prepared by following steps：
I, by the pLVX-AcGFP1-N1-cdtB that described pLVX-AcGFP1-N1-cdtB or methods described are built with
VSVG, pCMV Δ R8.2 are according to mass ratio 2：1：1 mixing, obtains plasmid mixed liquor；
The plasmid mixed liquor and liposome are 8 according to volume ratio by II,：15 ratio mixing, stands, after standing
Mixed liquor is added in the cell of DMEM cultures of serum-free, after cultivating 7h, replaces medium to complete medium；
III, cells are cultivated 48 hours under conditions of complete medium, are collected cell supernatant filtering, are obtained containing cdtB
The slow virus of gene.
Should in the cell line for establishing expression cdtB albumen present invention also offers the slow virus containing cdtB genes
With.
The invention provides a kind of cdtB gene overexpressions slow virus carrier pLVX-AcGFP1-N1-cdtB, with pLVX-
Based on AcGFP1-N1 Lentivirals, cdtB genes are introduced at multiple cloning sites.The present invention is by by foreign gene
CdtB replaces non-essential sequence in multiple cloning sites, and to build cdtB gene overexpression slow virus carriers, the described carrier is taken
Band CMV strong promoters, can be overexpressed the gene of its carrying in eukaryotic, at the same the carrier after polyclonal carrier with green
Color fluorescence sequence is used to screen positive cell with puromycin, does not change the original protein structure of cdtB albumen, makes cdtB albumen
Directly expressed in eukaryotic.
The invention provides the slow virus containing cdtB genes, the pLVX-AcGFP1-N1-cdtB obtained using structure is carried
Body mixes with slow virus packaging system VSVG, pCMV Δ R8.2, realizes slow virus packaging process, and obtaining being capable of direct infection eucaryon
The slow virus of cell, it is achieved thereby that the purpose of cdtB genes normal expression in eukaryotic, to further appreciate that cdtB bases
The function of cause is laid a solid foundation.
Brief description of the drawings
Fig. 1 is that PCR of the cdtB specific primers using helicobacter hepaticus as template expands electrophoretogram in embodiment 1；
Fig. 2 is the recombinant plasmid pLVX-AcGFP1-N1-cdtB figures that success is built in the embodiment of the present invention 4；
Fig. 3 is the luciferase expression figure of slow virus packaging 24h in the embodiment of the present invention 6；
Fig. 4 is the westernblot figures that Cdt albumen is identified in the embodiment of the present invention 7.
Embodiment
The invention provides a kind of cdtB gene overexpressions slow virus carrier pLVX-AcGFP1-N1-cdtB, with pLVX-
Based on AcGFP1-N1 Lentivirals, cdtB genes are introduced at multiple cloning sites；The cdtB genes have such as sequence
Nucleotide sequence in list shown in SeqID No.1.
In the present invention, the multiple cloning sites are preferably XhoI and BamHI restriction enzyme site.The multiple cloning sites are preferred
For MCS sequences.
The invention provides a kind of construction method of cdtB gene overexpressions slow virus carrier, comprise the following steps：
1) performing PCR amplification cdtB genes are entered using primer from helicobacter hepaticus genome, described primer has such as sequence
SEQ IDNo.1 and SEQ ID No.2 nucleotide sequence in table；
2) PCR primer obtained in the step 1) is connected in carrier T, obtains the carrier T with cdtB fragments；
3) with restriction enzyme XhoI and BamHI respectively to the carrier T with cdtB and slow virus carrier Plvx-
AcGFP1-N1 carries out double digestion, respectively obtains linearisation cdtB genetic fragments and linear slow virus carrier fragment；
4) linearisation cdtB genetic fragments that the step 3) obtains and linear slow virus carrier fragment are attached, obtained
To cdtB gene overexpression slow virus carriers pLVX-AcGFP1-N1-cdtB.
The present invention enters performing PCR amplification cdtB genes from helicobacter hepaticus genome using primer, and described primer has such as
SEQ ID No.1 and SEQ ID No.2 nucleotide sequence in sequence table.
The present invention is not limited the bacterial strain of the helicobacter hepaticus, using any one plant well-known to those skilled in the art
Helicobacter hepaticus.
In the present invention, the primer is as follows：
SEQ IDNo.1 CCGCTCGAGATGAGAATACTATTATGCTT
XhoI
SEQ IDNo.2 CGCGGATCCCTCCTAAAATCGTCCAAA
BamHI
Nucleotide sequence in SEQ ID No.1 includes XhoI restriction enzyme site；Nucleotide sequence in SEQ ID No.2
Restriction enzyme site comprising BamHI, it is follow-up digestion and connection so that pcr amplification product carries XhoI and BamHI restriction enzyme site
Operation site is provided.
In the present invention, the method for the PCR amplifications is not particularly limited, using PCR well-known to those skilled in the art
Amplification method, while there is no any restrictions to the PCR instruments for expanding use yet, expanded using PCR known in the art
Instrument.
In the present invention, the reaction system of the PCR amplifications is preferably as follows：
| Archaeal dna polymerase | 10μL |
| 10 μm of ol primers SEQIDNo.1 | 1μL |
| 10 μm of ol primers SEQIDNo.2 | 1μL |
| 100ng/ μ L helicobacter hepaticus genomes | 1μL |
| ddH2O | 7μL |
| Amount to | 20μL |
The response procedures of the PCR amplifications are preferably as follows：98 DEG C of 1min30s, 98 DEG C of 30s, 34 circulations, 50 DEG C of 30s, 72
DEG C 30s, 72 DEG C of 1min30s.
In the present invention, after PCR amplifications terminate, electrophoresis detection is preferably carried out.The electrophoresis detection preferably uses 1% agarose
Detected through gel electrophoresis PCR primer, electrophoretic band meet expection in 830bp or so.The PCR primer is preferably subjected to glue reclaim,
The PCR primer purified.The agarose DNA gel recovery is preferably carried out using agarose DNA gel QIAquick Gel Extraction Kit.This
Invention is not limited the source of the agarose DNA gel QIAquick Gel Extraction Kit, using fine jade well-known to those skilled in the art
Lipolysaccharide DNA gel QIAquick Gel Extraction Kit.In the embodiment of the present invention, the agarose DNA gel QIAquick Gel Extraction Kit is purchased from Beijing
Tiangeng company.
After the PCR primer purified, the present invention is by PCR primer connection with carrier T, obtaining and carrying cdtB fragments
Carrier T.
In the present invention, the method for the connection is preferably carried out using carrier T connection kit.The present invention is to the carrier T
The source of connection kit is not particularly limited, and kit is connected using carrier T known in the art.The present invention is implemented
In example, the carrier T connection kit is purchased from Transgene companies.
After obtaining the carrier T with cdtB fragments, the carrier T with cdtB fragments is preferably transformed into greatly by the present invention
In enterobacteria competent cell, select positive bacterium colony and expand culture, obtain a large amount of positive bacterium colonies.
The present invention is not particularly limited to the method for the conversion, using method for transformation known in the art.This
In inventive embodiments, converted using electrotransformation.
In the present invention, the method for selecting positive bacteria expansion culture is not particularly limited, using the sun described in this area
Property bacterium expand culture method.The authentication method of the positive bacteria is preferably identified using bacterium colony PCR methods.The bacterium colony
The identification of PCR methods is divided to two sections of sequencings.It is preferred that with such as SEQ ID No.3 in sequence table and the neucleic acid sequence shown in SEQ ID No.4
The primer pair detection cdtB genetic fragment leading portion sequences of row；It is preferred that with such as SEQ ID No.5 and SEQ ID No.6 in sequence table
The primer pair detection cdtB genetic fragments of shown nucleic acid acid sequence.It is complete to be sequenced after two sections of obtained sequence alignments splice
CdtB sequences.
Obtain amplification to a large amount of positive bacterias of purpose band to fall behind, the present invention is preferably by a large amount of sun of amplification to purpose band
Property bacterium colony using plasmid extraction kit extraction with cdtB fragments carrier T.
The present invention is not particularly limited to the source of the plasmid extraction kit, is carried using plasmid known in the art
Take kit.In the embodiment of the present invention, the plasmid extraction kit is bought from Beijing Tiangeng company.
After obtaining carrier T and the slow virus carrier Plvx-AcGFP1-N1 with cdtB, present invention restriction enzyme
XhoI and BamHI carries out double digestion to the carrier T with cdtB and slow virus carrier Plvx-AcGFP1-N1 respectively, respectively obtains
Linearize cdtB genetic fragments and linear slow virus carrier fragment.
The present invention is not particularly limited to the source of the slow virus carrier Plvx-AcGFP1-N1, and using this area, institute is ripe
The Plvx-AcGFP1-N1 known.In the embodiment of the present invention, the slow virus carrier Plvx-AcGFP1-N1 is purchased from Addgene
Company.
The present invention is not particularly limited to the restriction enzyme XhoI and BamHI sources, and use is known in the art
XhoI and BamHI.In the embodiment of the present invention, the restriction enzyme XhoI and BamHI is purchased from NEB companies.
In the present invention, the digestion system of the double digestion is preferably as follows：
| Carrier T or pLVX-AcGFP1-N1 | 10μL |
| BamHI | 1μL |
| XhoI | 1μL |
| 10x enzyme cutting buffering liquids | 2μL |
| ddH2O | 6μL |
| Amount to | 20μL |
The temperature of the digestion is preferably 37 DEG C, and the time of the digestion is preferably 12h.
Obtain after linearizing cdtB genetic fragments and linear slow virus carrier fragment, the present invention will linearisation cdtB genes and
Linear slow virus carrier fragment is attached, and obtains cdtB gene overexpression slow virus carriers pLVX-AcGFP1-N1-cdtB.
The present invention is not particularly limited to the method for the connection, using connection method known in the art.This
In invention, the system of the connection is preferably as follows：
| Linear cdtB fragments | 5μL |
| Linear slow virus pLVX-AcGFP1-N1 fragments | 2μL |
| T4DNA ligases | 0.5μL |
| T4DNA ligase buffer solutions | 1μL |
| ddH2O | 1.5μL |
| Amount to | 10μL |
The temperature of the connection is preferably 16 DEG C；The time of the connection is preferably 12h.
In the present invention, preferably also include after the connection：By the positive bacterium colony of recombinant plasmid progress for connecting and obtaining
Structure and identification.The structure and authentication method of the positive bacterium colony be preferably：Obtained connection mixed liquor is transformed into large intestine bar
Bacterium competence cell, choosing colony carry out positive bacterium colony sequencing identification.Method of the present invention to the positive bacterium colony sequencing identification
It is not particularly limited, using authentication method well-known to those skilled in the art.The PCR of positive bacterium colony of the present invention expands
The primer of increasing includes two pairs.It is preferred that with such as SEQ ID No.3 in sequence table and the nucleic acid acid sequence shown in SEQ ID No.4
Primer pair detects cdtB genetic fragment leading portion sequences；It is preferred that with as shown in SEQ ID No.5 in sequence table and SEQ ID No.6
Nucleic acid acid sequence primer pair detection cdtB genetic fragments.It is complete cdtB to be sequenced after two sections of obtained sequence alignments splice
Sequence.
In the present invention, the sequence obtained after sequencing is preferably subjected to sequence alignment, by the positive restructuring that matching rate is 100%
Plasmid is used for subsequent operation.
The invention provides the slow virus containing cdtB genes, it is prepared by following steps：
The pLVX-AcGFP1-N1-cdtB that I, builds above-mentioned technical proposal and VSVG, pCMV Δ R8.2 is according to mass ratio
2：1：1 mixing, obtains plasmid mixed liquor；
Plasmid mixed liquor and liposome are 8 according to volume ratio by II,：15 ratio mixing, stands, by the mixing after standing
Liquid is added in the cell of DMEM cultures of serum-free, after cultivating 7h, replaces medium to complete medium；
III, cells are cultivated 48 hours under conditions of complete medium, are collected cell supernatant filtering, are obtained containing cdtB
The slow virus of gene.
The present invention is by the pLVX-AcGFP1-N1-cdtB and VSVG, pCMV Δ R8.2 of above-mentioned technical proposal structure according to matter
Measure ratio 2：1：1 mixing, obtains plasmid mixed liquor.The VSVG is expression plasmid, has a CMV promoter, beta Globulin introne,
VSVG encoder blocks and polyA tails, packed for false type；The pCMV Δs R8.2 is packaging plasmid, the Gag of coding HIV-1 genes,
Pol, Rev, Tat albumen, most of coating correlated series is eliminated, for providing structure and duplication needed for production slow virus
Albumen.
After obtaining plasmid mixed liquor, plasmid mixed liquor and liposome are 8 according to volume ratio by the present invention：15 ratio is mixed
Close, stand, the mixed liquor after standing is added in the cell of plasma-free DMEM medium culture, after cultivating 7h, change culture
Base is complete medium.
The present invention is not particularly limited to the method for the mixing, not special using mixed method known in the art
Limitation.
In the present invention, the time of standing is preferably preferably 20~30min.
The present invention is not particularly limited to the method for the culture, using the scheme of culture known in the art without spy
Different limitation.
In the present invention, complete medium is replaced medium to, to provide the necessary condition of cell propagation.The culture completely
Base is containing the DMEM complete culture solutions that volumetric concentration is 5% serum.
After changing complete medium, the present invention cultivates cell 48 hours under conditions of complete medium, multigelation
Afterwards, centrifuge, abandon cell fragment, collect cell conditioned medium, using filter filtration cell supernatant, obtain the slow disease containing cdtB genes
Poison.
In the present invention, the method for the filtering preferably uses micro-filtration.The aperture of the micro-filtration is preferably 0.2 μm.
In the present invention, the method for the centrifugation is not particularly limited, and is using cell centrifugation protocol known in the art
Can.
Should in the cell line for establishing expression cdtB albumen present invention also offers the slow virus containing cdtB genes
With.
In the present invention, the species of the cell line is preferably AML12 cells.
In the present invention, the method for the cell line for establishing expression cdtB albumen preferably includes following steps：It is thin in AML12
Born of the same parents converge rate for 50%~70% when, the slow virus liquid of half cell volume is added into Tissue Culture Dish, and add poly-
The solidifying final concentration of 5 μ g/ml of amine, superinfection is once after cultivating 24h；Nutrient solution is replaced by complete medium after 24h, cultivated
72h d, obtain expressing the cell line of cdtB albumen.
In the present invention, the cell line of the expression cdtB albumen is identified.It is thin that the authentication method preferably includes observation
Born of the same parents' luciferase expression efficiency and resistance screening.The resistance screening is the cell to expression cdtB albumen using 2 μ g/ml puromycins
System carries out resistance screening.
With reference to embodiment to cdtB gene overexpressions slow virus carrier provided by the invention and its construction method and bag
The slow virus of the gene containing cdtB and its application are described in detail, but they can not be interpreted as to the scope of the present invention
Restriction.
Embodiment 1
In the following example, the experimental method of unreceipted actual conditions, generally routinely condition, such as《Fine works molecular biosciences
Learn experiment guide》(F.M. Ao Sibai, R.E. James Kingstons, J.G. Sai Deman etc. are edited, and Ma Xuejun, Su Yuelong's translates Beijing：Section
Learn publishing house, 2004) described in method carry out.
Specific primer is designed, expands cdtB fragments, primer sequence is as follows：
CDTB-F CCGCTCGAGATGAGAATACTATTATGCTT
XhoI
CDTB-R CGCGGATCCCTCCTAAAATCGTCCAAA
BamHI
PCR reaction systems such as table 1：
PCR response procedures such as table 2：
1% agarose gel electrophoresis detects PCR primer, and band meets expected (Fig. 1) in 800bp or so.Wherein, band is big
Small is 837bp；1 is positive, and 2 be blank control, and 3 be 200bp Marker.
Embodiment 2
Gel reclaims PCR primer, using carrier T connection kit connection cdtB fragments and carrier T, is transformed into Escherichia coli
Competent cell, select positive bacterium colony and expand culture, use carrier T of the plasmid extraction kit extraction with cdtB fragments.
Embodiment 3
Double digestion, digestion body are carried out to the carrier T with cdtB and pLVX-AcGFP1-N1 plasmids using XhoI and BamHI
System such as table 3：
| Carrier T or pLVX-AcGFP1-N1 | 10μL |
| BamHI | 1μL |
| XhoI | 1μL |
| CutsmartBuffer10x | 2μL |
| DdH2O | 6μL |
| Amount to | 20μL |
Table 3
37 DEG C of digestion 12h, gel reclaims kit recovery, the cdtB linearized and pLVX-AcGFP1-N1 fragments.
Embodiment 4
The fragment of recovery is recombinated using T4DNA ligases, system such as table 4：
16 DEG C of connection 12h, connection mixed liquor being transformed into competent escherichia coli cell, choosing colony enters performing PCR identification,
Reaction system and condition such as embodiment 1, positive bacterium colony are cultivated through expanding, and pLVX-AcGFP1- is obtained using plasmid extraction kit
N1-cdtB recombinant plasmids.
Embodiment 5
The sequencing identification of pLVX-AcGFP1-N1-cdtB recombinant plasmids, the primer sequence used are as follows：
Seq-F1 cggtgggaggtctatataag Seq-R1 agccaatttatttgtggcat
Seq-F2 cacctgctttagttacagctgt Seq-R2 agaagtcgtgctgcttcatgtg
Compared through sequencing, sequence is identical with expection, accuracy 100%.
Embodiment 6
Slow virus is packed
10cm flat board bed boards, 2 × 106Individual cell, overnight growth are paved with bottom 90%-95%, add with PBS twice
Enter the DMEM nutrient solutions of serum-free, it is stand-by.
8 μ g, 4 μ g, 4 μ g PLVX-CDTB, VSVG, pCMV Δ R8.2 are added into the DMEM nutrient solutions of 1ml serum-frees,
30 μ L lip2000 liposomes are added dropwise in the DMEM of 1ml serum-frees, plasmid solution is added in liposome, gently mixed
It is even, it is stored at room temperature 20min.
Mixed liquor is added in the DMEM cell 10cm culture plates of prepared serum-free, after 37 DEG C of culture 7h, by serum-free
DMEM nutrient solutions change the DMEM complete culture solutions containing 5% serum into.Expressed after 24h using fluorescence microscope green fluorescence
Situation.48h collects cell conditioned medium, is filtered using 0.2 μm of filter, obtains the slow virus of cdtB genes.
Embodiment 7
Slow-virus infection
AML12 plating cells：Using 25cm plastic culture bottles, add 5ml and contain 2 × 106The complete medium of individual cell,
After 12h, cell culture medium is discarded, 2.5ml slow virus liquid and 2.5ml complete mediums are added into blake bottle, is added
Superinfection is once after polybrene final concentration of 5 μ g/ml, 24h.Complete medium is changed to after infection 24h, observation cell is glimmering
Light expression efficiency (such as Fig. 3), and add 2 μ g/ml puromycins and carry out resistance screening, final obtain expresses cdtB albumen
AML12 cells.Cell lysis, cell pyrolysis liquid is collected, carry out westernblot detections, using the anti-CdtB of mouse as primary antibody, horseradish mistake
The sheep anti-mouse igg of oxide enzyme mark is secondary antibody, and ECL (thermo companies) nitrite ion develops the color, as a result as shown in figure 4, in 31kd
There is single band in place, and the cdtB albumen for illustrating cell expression is correct, and wherein M is molecular weight standard, and 1,2 is expression cdtB
AML12 cells, 3,4 be control cell.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Sequence table
<110>Yangzhou University
<120>CdtB gene overexpressions slow virus carrier and its construction method and the slow virus comprising cdtB genes and its application
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<213>Artificial sequence (Artificial Sequence)
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atgagaatac tattatgctt tttaatgagt tttacctttg ctttagccaa tcttgaagat 60
tatagagtaa gcacttggaa tctacaaggt tcttcagcaa atactgagag taaatggaat 120
ataagtgtga gacaactcat cacaggagat aatcctgcta atattcttat ggtgcaagag 180
gctggcgcta tacctgcttc agcgagaaga actggtagaa tggttcagcc cggtggtaca 240
cctgtagagg agtttacgtg ggaacttgga acttattcta gaccaaatac agtttatatc 300
tattatgctc ctctagatgt gggagcaaga agggtgaatc tcgccatagt ttctgataga 360
agagctgatg aagttcttgt agtgcatcaa aatgttgttg ctacagaggc atcgcgtcca 420
gctattggta ttcgtatcgg caatgatgtg ttttttgata ttcacgcttt agcaagtgga 480
ggtggtgatg cacctgcttt agttacagct gtgcacgata attttattaa tatgccacaa 540
ataaattggc ttatcgctgg agattttaat cgcgatccag cgctcttgca atcagggcta 600
gatacaagaa tcgctaatca tatccgtatt actgctccaa actctgctac acattttagc 660
tctagaggaa caaatagaac acttgattat gcggtagtag gtagatcaag cccttctcgt 720
tccactatag tattgccgca aattgcagca atacttatgg cagcaaatat acgcgcacac 780
ctctcatctg accattcacc tgtgcatttt ggacgatttt ag 822
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
cggtgggagg tctatataag 20
<210> 3
<211> 20
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<213>Artificial sequence (Artificial Sequence)
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agccaattta tttgtggcat 20
<210> 4
<211> 22
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<213>Artificial sequence (Artificial Sequence)
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cacctgcttt agttacagct gt 22
<210> 5
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
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agaagtcgtg ctgcttcatg tg 22
Claims (10)
- A kind of 1. cdtB gene overexpressions slow virus carrier pLVX-AcGFP1-N1-cdtB, with pLVX-AcGFP1-N1 slow virus Based on expression vector, cdtB genes are introduced at multiple cloning sites；The cdtB genes have such as Seq ID in sequence table Nucleotide sequence shown in No.1.
- 2. pLVX-AcGFP1-N1-cdtB according to claim 1, it is characterised in that the multiple cloning sites are XhoI With BamHI restriction enzyme site.
- 3. pLVX-AcGFP1-N1-cdtB according to claim 1 or 2, it is characterised in that the multiple cloning sites are MCS sequences.
- 4. a kind of construction method of cdtB gene overexpressions slow virus carrier, comprises the following steps：1) performing PCR amplification cdtB genes are entered using primer from helicobacter hepaticus genome, described primer has as in sequence table SEQ IDNo.1 and SEQ ID No.2 nucleotide sequence；2) PCR primer obtained in the step 1) is connected in carrier T, obtains the carrier T with cdtB fragments；3) with restriction enzyme XhoI and BamHI respectively to the carrier T with cdtB and slow virus carrier Plvx-AcGFP1- N1 carries out double digestion, respectively obtains linearisation cdtB genetic fragments and linear slow virus carrier fragment；4) linearisation cdtB genetic fragments that the step 3) obtains and linear slow virus carrier fragment are attached, obtained CdtB gene overexpression slow virus carriers pLVX-AcGFP1-N1-cdtB.
- 5. construction method according to claim 4, it is characterised in that the reaction system that PCR is expanded in the step 1) is as follows：
Archaeal dna polymerase 10μL 10 μm of ol primer SEQ ID No.1 1μL 10 μm of ol primer SEQ ID No.2 1μL 100ng/ μ L helicobacter hepaticus genomes 1μL ddH2O 7μL Amount to 20μL The response procedures that PCR is expanded in the step 1) are as follows：98 DEG C of 1min30s, 98 DEG C of 30s, 34 circulations, 50 DEG C of 30s, 72 DEG C 30s, 72 DEG C of 1min30s. - 6. construction method according to claim 4, it is characterised in that the digestion system of double digestion is as follows in the step 3)：
Carrier T or pLVX-AcGFP1-N1 10μL BamHI 1μL XhoI 1μL 10x enzyme cutting buffering liquids 2μL ddH2O 6μL Amount to 20μL The temperature of the double digestion is 37 DEG C, and the time of the double digestion is 12h. - 7. construction method according to claim 4, it is characterised in that the system of connection is as follows in the step 4)：
Linearize cdtB genetic fragments 5μL Linear slow virus carrier pLVX-AcGFP1-N1 fragments 2μL T4 DNA ligases 0.5μL T4 DNA ligase buffer solutions 1μL ddH2O 1.5μL Amount to 10μL The temperature of connection is 16 DEG C in the step 4)；The time of the connection is 12h. - 8. according to construction method described in claim 4~7 any one, it is characterised in that also wrapped after being connected in the step 4) Include：Structure and identification that recombinant plasmid carries out positive bacterium colony are obtained to the connection.
- 9. the slow virus containing cdtB genes, it is characterised in that be prepared by following steps：I, is by the pLVX-AcGFP1-N1-cdtB described in claims 1 to 3 any one or claim 4~8 any one institute The pLVX-AcGFP1-N1-cdtB and VSVG, pCMV Δ R8.2 of method structure are stated according to mass ratio 2：1：1 mixing, obtains plasmid Mixed liquor；The plasmid mixed liquor and liposome are 8 according to volume ratio by II,：15 ratio mixing, stands, by the mixing after standing Liquid is added in the cell of the DMEM medium cultures of serum-free, after cultivating 7h, is replaced medium to complete medium and is continued to cultivate 48 hours；The clasmatosis that III, will be cultivated in the step II, cell supernatant filtering is collected, obtains the slow disease containing cdtB genes Poison.
- 10. the slow virus containing cdtB genes described in claim 9 is establishing the cell line application of expression cdtB albumen.
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| CN112442514A (en) * | 2020-11-25 | 2021-03-05 | 云舟生物科技（广州）有限公司 | Lentiviral packaging vector system, lentivirus, construction method of lentivirus and kit |
| CN112442514B (en) * | 2020-11-25 | 2022-04-12 | 云舟生物科技(广州)股份有限公司 | Lentiviral packaging vector system, lentivirus, construction method of lentivirus and kit |
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