CN102373226A - Methods for cloning, expressing and protein purifying of mutant HPV16 E7 (Human Papilloma Virus 16 Early Protein 7) - Google Patents
Methods for cloning, expressing and protein purifying of mutant HPV16 E7 (Human Papilloma Virus 16 Early Protein 7) Download PDFInfo
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Abstract
The invention relates to a method for expressing a mutant HPV16 E7 (Human Papilloma Virus 16 Early Protein 7) by using a prokaryotic cell expressing system. The method comprises the steps of: firstly, cloning an mE7 (mutant E7) gene, transforming T (Thymine) of a 70th basic group into G (Guanine), removing fifteen basic groups before a terminator codon, respectively constructing a cloning vector TA (Thymine Adenine)-mE7 and an expression vector pET-28a(+)-mE7 of the mE7, and prokaryotically expressing as well as isolating and purifying the mE7, wherein when the purified mE7 is compared with HPV16 E7 protein, Cys (Cysteine) of a 24th amino acid residue is transformed into Gly (Glycine), and five amino acid residues at C-terminal are removed, 94 positions of Cys are included, a Cys-X-X-Cys motif structure is destroyed, and thus, the transformation activity of the E7 is greatly reduced. The mE7 protein immunity is beneficial to 100% protection of a mouse from producing TC-1 (Tissue Culture No.1) tumor.
Description
Technical field
The invention belongs to the gene cloning and expression technical field, be specifically related to a kind of clone, expression and method for purifying proteins of HPV16 E7 gene of sudden change.
Background technology
Cervical cancer is the female reproductive system common malignancy; Sickness rate is positioned at second of female tumor, only comes after the mammary cancer, the annual whole world nearly 500,000 routine New Development cases of cervical cancer; About 200,000 people die from cervical cancer, in some developing countries even to have occupy women's cancer mortality the first.The research of molecular epidemiology confirmer's papilloma virus (humanpapillomavirus HPV) infects with cervical cancer very confidential relation is arranged.The infection of HPV high-risk-type (HPV16,18,31,45 types etc.) is the major reason that causes cervical cancer to take place and develop, and the recall rate of HPV DNA is up to 99% in the cervical cancer, and wherein HPV16 is about 60%.Therefore, the HPV vaccine is used for prevention and treats cervical cancer having become hot research in recent years.Although the HPV preventative vaccine comes out, in the face of numerous patients that infected HPV and pathology occurred, the preventative vaccine effect is little.
The expression product E6 of two early gene E6 of HPV16, E7, E7 albumen play an important role in tumour generation, cell cycle regulating and apoptosis are regulated, and are the major causes that cervical cancer takes place, and continuous expression in cancer cells also is called as E6, E7 cancer protein.Therefore E6 and E7 albumen can be used as the desirable target antigen of HPV16 related neoplasms therapeutic vaccine, and also having occurred with E6, E7 in succession is the multi-form vaccine of immunizing antigen.Because protein vaccine neither receives HLA restricted, has avoided virus vector or dna vaccination potential potential safety hazard again, be to generally acknowledge the most safe and effective vaccine at present.Report is arranged, and its activity of conversion can avoided or reduce to E6, the proteic two mutants of E7.Proteic the 2nd His of E7,24 Cys and two block Cys-X-X-Cys (being respectively 58-61 and 91-94) structure are the key positions of its activity of conversion; Change 24 Cys into Gly and can eliminate the proteic activity of conversion of E7 fully; For changing Gly into, Cys also can reduce proteic activity of conversion and change 2 His into Gly, 61 or 94 greatly, so clonal expression E6, the proteic two mutants of the E7 security that can further improve vaccine.
Summary of the invention
The problem that the present invention need solve is the HPV16 E7 method of protein for the security that has further improved the cervical cancer therapeutic vaccine is cloned, expressed and separation and purification suddenlys change.
The invention relates to a kind of HPV16 E7 gene (mutant E7, clone mE7), expression and method for purifying proteins of sudden change.Mainly form by following content:
1. the structure of recombinant cloning vector TA-mE7
(1) design of primers of HPV16mE7
With pET28a (+)-E7 is template, and through splicing pcr amplification mE7, (T → G), remove terminator codon 15bp before designs 4 primer mE7p1, mE7p2, mE7p1-G-3 ', mE7p2-G-5 ' to introduce the sudden change of E7 gene the 70th bit base.
Upstream primer mE7p1 contains restriction enzyme site Nde I, and downstream primer mE7p2 contains restriction enzyme site Hind III, and the primer of introducing the mutational site is respectively mE7p1-G-3 ', mE7p2-G-5 '.
mE7p1：5’-CG
CATATGGCTAGCATGACTGGTGGA-3’
mE7p1-G-3’：5’-TAATTGCTCATAACCGTAGAGATCAGTTG-3’
mE7p2-G-5’：5’-CAACTGATCTCTACGGTTATGAGCAATT-3’
mE7p2：5’-CG
AAGCTTTTAGATGGGGCACACAATTC-3’
(2) splicing pcr amplification HPV16mE7 gene fragment
With pET28a (+)-E7 is template, and through splicing pcr amplification mE7 gene fragment, (135bp is called Nde I-E7 for amplification Nde I and E7 gene the 84th base intermediary fragment earlier
84) and E7 gene 56-297 bit base sequence (be called E7
56-297).PCR reaction system (Pyrobest enzyme): 41.25 μ L sterilized waters, 5 μ L, 10 * buffer (contains MgCl
2), 1 μ L 10mM dNTPs, 1 μ L mE7p1/mE7p1-G-3 ', 1 μ L mE7p2-G-5 '/mE7p2,0.5 μ L pET28a (+)-E7,0.25 μ L Pyrobest enzyme increases behind the mixing immediately.Reaction conditions is: 94 ℃ of preparatory sex change 3min; Carry out following 35 circulations then: 94 ℃ of 45s, 56 ℃ of 1min, 72 ℃ of 45s; Extend 3min at 72 ℃ at last.
Above-mentioned PCR product is with 1% sepharose 90V constant voltage electrophoresis 20min, cuts to reclaim test kit with glue behind the glue and reclaim the purpose fragment, splices pcr amplification Nde I-mE7 sequence again.PCR reaction system (Pyrobest enzyme): 39.75 μ L sterilized waters, 5 μ L10 * buffer (contain MgCl
2), 1 μ L 10mM dNTPs, 1 μ L mE7p1,1 μ L mE7p2,1 μ L Nde I-E7
84, 1 μ L E7
56-297, 0.25 μ L Pyrobest enzyme increases behind the mixing immediately.Reaction conditions is: 94 ℃ of preparatory sex change 3min; Carry out following 35 circulations then: 94 ℃ of 45s, 56 ℃ of 1min, 72 ℃ of 45s; Extend 3min at 72 ℃ at last.
Agarose gel electrophoresis, glue reclaim dna fragmentation, add A for ease of extending with the Taq enzyme, and the elution buffer that glue reclaims uses and contains MgCl
2Taq enzyme buffer liquid.Reclaim the dna fragmentation of elution buffer wash-out pcr amplification with 25 μ L glue.The glue of getting 13.7 μ L reclaims elutriant, adds 1.2 μ L 10mM dNTP and 0.1 μ L Taq enzyme, and 72 ℃ are extended 6min, and the PCR product extends A.
(3) the TA clone connects
Get 1 μ L pMD18-T carrier, add the mE7 fragment of 2 μ L extend through A, add the connection damping fluid of 5 μ L again, 2 μ L sterilized waters complement to 10 μ L systems, connect 2hr in 16 ℃.
(4) the TA clone transforms
Above-mentioned TA is connected product 10 μ L to be transferred in the DH5 α competent cell; Leave standstill 30min on ice; 42 ℃ of heat shock 90s; Leave standstill 5min on ice again; Add 800 μ L LB, 37 ℃ of shaking table 120rpm shake 1hr; The centrifugal 3min of 4000rpm; In super clean bench, remove most of supernatant, light outstanding deposition is coated in the LB flat board that contains 50 μ g/mL penbritins (Amp); The LB flat board is inverted in 37 ℃ of incubator overnight cultures.
(5) TA clone's evaluation
4 TA positive colonies of picking insert 4mL LB (Amp at random
+) in; 37 ℃ of shaking table 220rpm shake and spend the night; Extracting plasmid TA-mE7 is stored in-80 ℃, and carries out double digestion and PCR evaluation.Choose all positive TA cloned plasmids order-checking of double digestion and PCR.
2. the structure of recombinant expression plasmid pET-28a (+)-mE7
(1) enzyme is cut and is reclaimed
EcoR I restriction enzyme site (introducing in the E7 upstream primer) is arranged in the mE7 gene order, with EcoR I/Hind III double digestion TA-mE7 and pET-28a (+) plasmid, use 1% agarose gel electrophoresis, glue reclaims target fragment.
(2) the T4DNA ligase enzyme connects and transforms
Linked system is: 1 μ L, 10 * ligase enzyme damping fluid, and 1 μ L double digestion pET-28a (+) plasmid, 7 μ L mE7 fragments, 1 μ L T4DNA ligase enzyme, 16 ℃ of connections are spent the night.
To connect product and change DH5 α competence over to, coat after the conversion on the LB flat board that contains 50 μ g/mL kantlex (Kan), be inverted overnight cultures for 37 ℃.
(3) positive colony is identified
4 positive colonies of picking at random insert the LB (Kan of 4mL
+) in; 37 ℃ of shaking table 220rpm shake and spend the night; The extracting plasmid carries out double digestion (EcoR I/Hind III) and PCR identifies.
3.mE7 induction expression of protein and evaluation
(1) recombinant plasmid transformed intestinal bacteria
Change above-mentioned male recombinant plasmid pET-28a (+)-mE7 that is accredited as over to competence Rosetta, coat LB flat board (Kan
+) on, be inverted overnight cultures for 37 ℃.
(2) the IPTG inducible protein is expressed
Choose mono-clonal in LB (Kan
+) in, 37 ℃ of shaking tables, the 220rpm overnight cultures to take over the night bacterium in 20mL LB (Kan at 1: 50
+) in, continue to cultivate about 3hr OD
600nmAdd IPTG to 1mM inducible protein when reaching 0.6-0.8 and express, respectively at collecting 1mL bacterium liquid behind 0hr, 1hr, 2hr, 3hr, 4hr, 5hr, the 6hr.
(3) SDS-PAGE identifies
Get the centrifugal 1min of 1mL bacterium liquid 12000rpm and collect thalline, add 1 * sample-loading buffer of 100 μ L, boil 5min, the centrifugal 1min of cooling back 12000rpm gets 10 μ L samples and carries out 12%SDS-PAGE, and deposition condition is 18mA, 1.5hr.Take off gel and in the Xylene Brilliant Cyanine G dye liquor, shake dyeing 1hr, shake wash-out in the immigration elutriant and spend the night.The variation of protein band before and after observation is induced, protein expression arrived the peak after 4hr was induced in result's demonstration.
4.mE7 proteic purifying and evaluation
(1) mE7 induction expression of protein
The Rosseta bacterium liquid that incubated overnight has been changed over to pET-28a (+)-mE7 to be connected to 1L LB (Kan at 1: 50
+) in, 37 ℃ of shaking tables, 220rpm are cultivated about 2hr, OD
600nmWhen reaching 0.6-0.8, add IPTG to 1mM, continue inducing culture 4hr, 4 ℃, the centrifugal 6min of 7000rpm collects thalline ,-80 ℃ of preservations.
(2) analysis of bacterioprotein expression-form
-80 ℃ of frozen thalline add bacterial lysate (0.5M NaCl, 20mM Tris, 10mM mercaptoethanol in the ratio of the wet bacterium 5-10mL of every gram; 1mM PMSF, pH7.9), carrying out ultrasonic bacteria breaking; 4 ℃, the centrifugal 20min of 8000rpm, inclusion body are deposited in the bottom; Get supernatant respectively, inclusion body is SDS-PAGE, the result shows that mE7 albumen mainly is present in the inclusion body.
(3) the proteic purifying of mE7
After the carrying out ultrasonic bacteria breaking, 4 ℃, the centrifugal 20min of 8000rpm abandons supernatant, the deposition with 50mL solution (0.5MNaCl, 20mMTris, 0.5%Triton, pH7.9) supersound washing once; 4 ℃, the centrifugal 20min of 8000rpm abandons supernatant.Deposition is that inclusion body adding 30mL solution (pH7.9), put more than 37 ℃ of dissolving 2hr for 0.5M NaCl, 20mM Tris by the 6M Guanidinium hydrochloride.4 ℃, the centrifugal 20min of 16000rpm gets on the supernatant appearance to using 0.5M NaCl, 20mM Tris in advance; The 6M Guanidinium hydrochloride, the nickel post that the solution equilibria of pH7.9 is good, respectively with solution 1., 2., 3. (1. 0.5M NaCl, 20mM Tris; The 5mM mercaptoethanol, 5mM imidazoles, 6M Guanidinium hydrochloride, pH7.9; 2. 0.5M NaCl, 20mM Tris, 5mM mercaptoethanol, 5mM imidazoles, 8M urea, pH7.9; 3. 0.5M NaCl, 20mM Tris, 5mM mercaptoethanol, 20mM imidazoles, 8M urea; PH7.9) 10 column volumes of washing are used elution buffer (0.5M NaCl, 20mM Tris, 5mM mercaptoethanol again; The 200mM imidazoles, 8M urea, pH7.9) wash-out is collected protein liquid; Put 4 ℃ 1 * PBS is fully dialysed, 4 ℃, the centrifugal 20min of 16000rpm gets supernatant.The albumen of results carries out purity check with SDS-PAGE, and the result shows that the purified proteins molecular weight is about 20kDa.
(4) the proteic evaluation of mE7
Western blot identifies: the SDS-PAGE electrophoresis takes off gel after finishing, electricity forward on the nitrocellulose filter (4 ℃, 100V, 1hr); Get film, with 5% skim-milk sealing 2hr, add mouse-anti people HPV16 E7 antibody, incubated at room 2hr in room temperature; Wash film 3 times with PBST, each 10min adds sheep anti-mouse antibody incubated at room 1.5hr, washes film 3 times with PBST again; Each 10min adds the substrate solution colour developing, development, photographic fixing in the darkroom.The result shows that the target stripe of mE7 has appearred in about 20kDa place.
Embodiment
Below through concrete embodiment the present invention is done further elaboration.
The nucleotide sequence of embodiment 1:mE7
The present invention has cloned the two mutants of E7; The 70th bit base is become G by T; And 15 bases before the removal terminator codon, successively having made up recombinant clone plasmid TA-mE7 and recombinant expression plasmid pET-28a (+)-mE7, the nucleotide sequence of mE7 is seen the next section of specification sheets.
The proteic aminoacid sequence of embodiment 2:mE7
Separation and purification of the present invention mE7 albumen, compare with HPV16 E7 albumen, the 24th amino acids residue changes Gly into by Cys, and has removed terminal 5 amino-acid residues of C-, comprises 94 Cys, has destroyed the block structure of Cys-X-X-Cys.The next section that the proteic aminoacid sequence of mE7 is seen specification sheets.
Embodiment 3:mE7 albumen epidemic prevention antitumor action
1.5nmol the subcutaneous immune C57BL/6 mouse of albumen (mE7 albumen and E7 albumen, the PBS group is contrast), two all pneumoretroperitoneum booster immunizations 1 time, week back subcutaneous vaccination 1 * 10
5TC-1 cell (through trysinization, PBS washing 2 times).Observe the tumor growth situation afterwards every day; Behind the 12d with vernier caliper measurement tumour major diameter and vertical diameter (every 3d measures 1 time); Continue observation till control group mice begins death, continue to observe, write down its lotus knurl situation and survival time up to all death of tumor-bearing mice; The whole PBS group of result mouse has grown tumour, and mE7 and E7 protein immunization group then 100% have protected mouse not generate tumour.It is long when planting knurl 40d that the knurl mouse is subcutaneous inoculates 2 * 10
5The TC-1 cell is observed the tumor growth situation with immune mouse not every day as control group afterwards; All dead up to control group mice; Write down its lotus knurl situation and survival time, the result all grows knurls after showing control group mice 6d, and does not still have the knurl bulk-growth behind mE7 and the E7 protein immunization group 50d.
Claims (4)
1. clone, expression and the method for purifying proteins of the HPV16 E7 gene of a sudden change; It is characterized in that " through the HPV16 E7 sequence of method clonal mutation of splicing PCR; make up prokaryotic expression carrier pET-28a (+)-mE7 of reorganization, the HPV16 E7 albumen of prokaryotic expression and purified mutant ".
2. clone, expression and the method for purifying proteins of the HPV16 E7 gene of sudden change according to claim 1; The cloning process of the HPV16 E7 gene that it is characterized in that suddenling change is: with pET28a (+)-E7 is template; Through splicing pcr amplification mE7 gene fragment; (135bp is called Nde I-E7 for amplification Nde I and E7 gene the 84th base intermediary fragment earlier
84) and E7 gene 56-297 bit base sequence (be called E7
56-297), reclaim test kit with glue and reclaim purpose fragment Nde I-E7
84And E7
56-297, splice pcr amplification Nde I-mE7 sequence again, make up recombinant clone plasmid TA-mE7 then.
3. clone, expression and the method for purifying proteins of the HPV16 E7 gene of sudden change according to claim 1; The construction process of the recombinant expression plasmid of the HPV16 E7 gene that it is characterized in that suddenling change is: EcoR I restriction enzyme site (introducing in the E7 upstream primer) is arranged in the mE7 gene order; With EcoR I/Hind III double digestion TA-mE7; With the directed expression vector pET-28a (+) that inserts of mE7 gene, make up recombinant expression plasmid pET-28a (+)-mE7.
4. clone, expression and the method for purifying proteins of the HPV16 E7 gene of sudden change according to claim 1; It is characterized in that the proteic expression and purification method of the HPV16 E7 that suddenlys change is: the Rosetta bacterium that will change pET-28a (+)-mE7 over to is 37 ℃ of cultivations, 1mM IPTG abduction delivering 4hr, ultrasonic split bacterium after; With ni-sepharose purification mE7 albumen; Compare with HPV16 E7 albumen, the 24th amino acids changes Gly into by original C ys in the E7 protein polypeptide chain of sudden change, and has removed terminal 5 amino-acid residues of C-; Comprise the 94th Cys; Destroyed the block structure of Cys-X-X-Cys, avoided or reduced the proteic activity of conversion of E7, safer reliable as the vaccine of cervical cancer treatment.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105177048A (en) * | 2014-08-12 | 2015-12-23 | 广东拓谱康生物科技有限公司 | Recombinant adeno-associated viral vector with human papillomavirus type 16 multi-point mutant E7<mm> antigen genes, method for constructing recombinant adeno-associated viral vector and application thereof |
| CN105177047A (en) * | 2014-08-12 | 2015-12-23 | 广东拓谱康生物科技有限公司 | Recombinant adeno-associated viral vector with human papillomavirus type 16 mutant E7<m94> antigen genes, method for constructing recombinant adeno-associated viral vector and application thereof |
| WO2016015684A1 (en) * | 2014-08-01 | 2016-02-04 | 广东拓谱康生物科技有限公司 | Nrecombinant adeno-associated virus vector carrying human papillomavirus type 16 mutation e7 antigen gene, construction method therefor, and application thereof |
| CN113637690A (en) * | 2021-08-18 | 2021-11-12 | 武汉华美生物工程有限公司 | Preparation method and application of human papilloma virus HPV16 type E7 active protein |
-
2010
- 2010-08-26 CN CN2010102629221A patent/CN102373226A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016015684A1 (en) * | 2014-08-01 | 2016-02-04 | 广东拓谱康生物科技有限公司 | Nrecombinant adeno-associated virus vector carrying human papillomavirus type 16 mutation e7 antigen gene, construction method therefor, and application thereof |
| CN105177048A (en) * | 2014-08-12 | 2015-12-23 | 广东拓谱康生物科技有限公司 | Recombinant adeno-associated viral vector with human papillomavirus type 16 multi-point mutant E7<mm> antigen genes, method for constructing recombinant adeno-associated viral vector and application thereof |
| CN105177047A (en) * | 2014-08-12 | 2015-12-23 | 广东拓谱康生物科技有限公司 | Recombinant adeno-associated viral vector with human papillomavirus type 16 mutant E7<m94> antigen genes, method for constructing recombinant adeno-associated viral vector and application thereof |
| CN113637690A (en) * | 2021-08-18 | 2021-11-12 | 武汉华美生物工程有限公司 | Preparation method and application of human papilloma virus HPV16 type E7 active protein |
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