CN1696152A - LIE7 recombined protein of 16 type human papilomavirus expressed by bacillus coli - Google Patents

Abstract

A human papillomavirus' recombinant protein 16-L1E7 effectively expressed in colibacillus is prepared through combining the early-stage gene E7 of HPV16 with the chitoprotein gene L1 of HPV16 by gene engineering and purifying by chromatography. It is a pentamer with strong immune activity, so it can be used for preventing and treating the HPV16 infection and associated tumor.

Description

The HPV 16 L1E7 recombinant protein of escherichia coli expression

Technical field

1. medical biotechnology field 2. immunoprophylaxises and treatment

Background technology

Add up according to the World Health Organization, in the global woman cancer cause of the death, cervical cancer comes second, and in developing country, because the sanitary condition difference restriction of economic condition simultaneously makes the cervical exfoliated cell examination be difficult to popularize, cervical cancer rises in the woman cancer cause of the death and occupies first.The whole world has 200,000 women to die from cervical cancer every year approximately, and wherein 80% in developing country.China is one of cervical cancer state occurred frequently in the world, about 50,000 people of patient that die from cervical cancer every year.A large amount of research datas prove that the high-risk HPV of reproductive tract infects with cervical cancer very confidential relation.90% cervical cancer patient can find the existence of HPV DNA, and wherein the HPV16 type accounts for 50%, is the main virulence factor that causes cervical cancer.Although early cervical carcinoma patient radiotherapy at present and surgical result are better, the middle and advanced stage patient must give radiotherapy and certain chemotherapy on the basis of operative treatment.Because the huge side effect of radiotherapy and cervical cancer to the insensitivity of chemotherapy, still have 35% patient the present serious complication that still can't treat to occur, expense is big and recurrence rate is high.Everything shows and is necessary to develop infection and cervical cancer and the related neoplasms that more effective prevention and therapeutic vaccine are prevented and treated HPV.

The main capsid protein L 1 of HPV16 expresses and is self-assembled into virus-like particle (VLP) in multiple expression body system.Can bring out the neutralizing antibody that produces high titre, be comparatively ideal preventative vaccine, its caused cellular immunization a little less than, can not reach effective therapeutic action.Early protein E6, E7 continuous expression in cervical cancer cell is the desirable target antigen of tumor therapeutic vaccine.There is the people E7 early gene to be inserted the C-terminal of L1 in recent years abroad, in insect cell, develop L1 E7 recombinant protein, the Electronic Speculum structure is virus-like particle (cVLP), it not only produces the neutralizing antibody of high titre, but also generation is at the ctl response of E7, possessing prevention and treatment dual function, is more promising vaccine.Yet because the cVLP of insect cell expression yields poorly, poor stability, thereby the cost height, be difficult to accomplish widespread use, especially in developing country.Therefore whether explore can enough prokaryotic expression systems to develop simple, economical and to have immunocompetent L1E7 recombinant protein and become main purpose of the present invention.

Reference

1.Stoler?MH.Human?papillomavirus?and?cervieal?neoplasia：a?model?for?carcinogenesis.Int?J?Gynecol?Pathol.2000；19(1)：16-28.

2. grandson Asia. human papillomavirus (HPV), cancer and vaccine progress. the 5th national academic meeting paper compilation .2000 of gynecological tumor Professional Committee of Chinese Anti-Cancer Association, 15-17.

3.Robert?C，Wendy?I.White，Maolin?Li.Et?al.Human?Papillomavirus?Type?11?RecombinantL1?Capsomeres?Induce?Virus-Neutralizing?Antibodies.J?Virol.1998；72(7)：6151-6154.

4.Greenstone?HL，Nieland?JD，De?Visser?KE，et?al.Chimeric?papillomavirus?virus-likeparticles?elicit?antitumor?immunity?against?the?E7?oncoprotein?in?an?HPV16?tumor?model.Proc?Natl?Acad?Sci?USA.1998；95(4)：1800-1805.

5.Jochmus?I.，Schafer?K.，Faath?S.，et?al.Chimeric?papillomavirus?virus-likeparticles?of?the?human?papillomavirus?type?16(HPV16)as?a?prophylactic?andtherapeutic?vaccine.Arch?Med?Res.1999；30(4)：269-274.

6.Kaufman?RH，Adam?E，Vonka?V.Human?papillomavirus?infection?and?cervical?carcinoma.Clin?Obstet?Gynecol.2000；43(2)：363-80.

7.Ressing?ME，Sette?A，Brandt?RM，et?al.Human?CTL?epitopes?encoded?by?humanpapillomavirus?type?16?E6?and?E7?identified?through?in?vivo?and?in?vitroimmunogenicity?studies?of?HLA-A*0201-binding?peptides.J?Immunol.1995；154(11)：5934-5943.

Summary of the invention

The present invention is described in detail below in conjunction with Figure of description

Genetic engineering technique is adopted in this experiment, merge with the method for PCR and gene clone major antigen avtive spot HPV16 main glutelin L1 avtive spot and E7 early gene, again the fusion gene that makes up is inserted among the expression vector pQE30a, efficiently express the L1E7 recombinant protein by prokaryotic system, adopt the chromatography method protein isolate, and electron microscopic observation its be the penton structure.Immunocompetence with immune animal experimental evaluation L1E7 recombinant protein.

Description of drawings

Fig. 1 shows cloned plasmids " pMD18TL1E7 " structure diagram that has wherein inserted the HPV16L1E7 fusion gene

Fig. 2 shows expression plasmid " pQE30a L1E7 " structure diagram that has wherein inserted the HPV16L1E7 fusion gene

Fig. 3 show the HPV16L1E7 fusion gene in the intestinal bacteria system through the L1E7 of abduction delivering recombinant protein product,

Fig. 4 (A) shows that the result (B) that HPV16L1E7 fusion gene expression product detects with L1 antibody shows the result that HPV16L1E7 fusion gene expression product detects with E7 antibody

Fig. 5 shows that L1E7 recombinant protein sample is added on the copper mesh with 2% phospho-wolframic acid negative staining, knows observed penton spline structure under the electron microscope thoroughly at philips EM410

After Fig. 6 shows L1E7 recombinant protein immune mouse, the result of L1 antibody and E7 antibody titers in the detection mice serum

After Fig. 7 shows L1E7 recombinant protein immune mouse, the result of antitumor transplantation experiments

Embodiment

The object of the present invention is achieved like this:

The structure of example 1, L1E7 fusion gene cloning

Utilize round pcr with L1 coding region gene 3 ' terminal deletion, merge with 5 ' the end E7 coding region gene of removing activity of conversion then, be inserted into the pMD18T cloning vector again and obtain fusion gene cloning plasmid pMD18TL1E7 (see figure 1).

The structure of example 2, prokaryotic expression plasmid pQE30a L1E7

PMD18TL1E7 plasmid enzyme restriction digestion back obtains L1E7 fusion gene segment, is inserted into prokaryotic expression carrier pQE30a, obtains expression plasmid pQE30a L1E7 (see figure 2)

Example 3, the expression of L1E7 recombinant protein in intestinal bacteria

Expression plasmid pQE30a L1E7 is transformed JM109 calcification bacterium, through being coated with ware, choosing the spot abduction delivering and filter out high-expression clone bacterial strain 03B, the analysis of SDS-PAGE protein electrophoresis glue has a newly-increased protein band at the 61KD place, the results are shown in Figure 3

The Western-Blot of example 3, L1E7 recombinant protein identifies

Behind the high-expression clone bacterial strain 03B abduction delivering, walk SDS-PAGE protein electrophoresis glue, electricity changes nitrocellulose filter, detects with L1 antibody and E7 antibody respectively.The results are shown in Figure 4

The purifying of example 4, L1E7 recombinant protein

(1) preparation of bacterium

Bacterial classification inoculation in 37 ℃ of shaking culture of 300ML substratum 5 hours, is added IPTG to final concentration 0.84mM/ liter, and 37 ℃ of abduction deliverings 7 hours are collected bacterial sediment, and-20 ℃ frozen standby.

(2) proteic purifying

1. the processing of sample

A. get-20 ℃ of frozen thalline, be dissolved in the lysis buffer, add N,O-Diacetylmuramidase, after room temperature is placed 45 minutes, ultrasonic in ice bath (400 watts 12 times) 12 seconds, 60 seconds at interval.Centrifugal (12000rmp 20 minutes, 4 ℃) get precipitation.

B. precipitation is resuspended in the buffer A ultrasonic in ice bath (200 watts 10 times) 12 seconds, 30 seconds at interval.Centrifugal (12000rmp 20 minutes, 4 ℃) get precipitation.And step b repeated once again.

C. precipitation is resuspended in the buffer B, and vibration was dissolved one hour in room temperature.It is standby then suspension centrifugal (12000rmp 20 minutes, 20 ℃) to be got supernatant.

2. ion exchange chromatography

The supernatant adding has been used on the damping fluid C equilibrated ion exchange column, with having added the speed wash-out of 100mM sodium-chlor buffer A with 1 milliliter of per minute.

3. affinity chromatography

To transfer PH to 8.0 from the sample that ion-exchange obtains, add the Ni-NTA affinity column of crossing with the buffer B balance then,, collect target protein with containing the flow velocity wash-out of the buffer B of imidazoles with 1 milliliter of per minute.

(3) proteic dialysis renaturation and concentrated

1. proteic dialysis:

Place the dialysis tubing of handling well to seal in the albumen of last wash-out, put into the dialyzate D that contains urea of 100 times of volumes, changed dialyzate in per 6 hours, through 4,2,1, the urea gradient of 0M dialyses, carry out dialysis in the phosphate buffered saline(PBS) more at last.

2. proteic concentrating:

The albumen taking-up centrifugal (12000rmp 20 minutes, 4 ℃) that dialysis is good is got supernatant and is sealed with dialysis tubing again, is embedded in polyoxyethylene glycol (PEG 8000) particle, treat that it is concentrated into taking-up in 1/3 o'clock of volume just, albumen centrifugal (12000rmp 20 minutes, 4 ℃) is got supernatant, and packing stores.

Example 5, recombinant protein structure electronic microscope photos: L1E7 recombinant protein sample is added on the copper mesh,, knows thoroughly at philipsEM410 and to observe penton structure (see figure 5) under the electron microscope with 2% phospho-wolframic acid negative staining.

Example 6, the experiment of L1E7 recombinant protein animal immune

1. L1E7 recombinant protein immune serum antibody test: L1E7 recombinant protein immune mouse, two weeks back reinforcement, one pin, serum detects L1 antibody respectively with euzymelinked immunosorbent assay (ELISA) and the E7 antibody horizontal the results are shown in Figure 6 after getting in two weeks.The heavy albumen of L1E7 can bring out that to produce titre be 1: 450 L1 antibody and 1: 200 E7 antibody as can be seen from Figure.

2. the antitumor transplantation experiments of L1E7 recombinant protein: with L1E7 recombinant protein immunity C57 mouse, behind the booster immunization 10 days, with 1 * 10 ⁴The tumor growth situation was observed three months by TC-1 tumor cell injection mouse inguinal region place, the results are shown in Figure 7.The heavy albumen of L1E7 has immanoprotection action as can be seen from Figure, can not only will become the knurl time retardation, and protects 50% mouse opposing TC-1 tumour cell to attack, and through trimestral observation, proves that this protection is secular.

Claims (4)

1, the recombinant protein HPV16PL1E7 of the HPV 16 virus of expression in escherichia coli, it is characterized by: recombinant protein HPV16PL1E7 system utilizes genetic engineering technique that the immunocompetence part of the main capsid protein gene L1 of HPV 16 and the immunocompetence meromixis of early gene E7 are configured to fusion gene L1E7, and the L1E7 fusion gene is expressed the product that obtains in escherichia expression system.

2, according to claim 1, the electron microscope observation structure of HPV16PL1E7 recombinant protein is a penton.

3, according to claim 1, the HPV16L1E7 recombinant protein can be made pharmaceutical composition with pharmaceutically acceptable medicine, the infection that is used for prevention of human papillomavirus 16 types is infected relevant disease therewith with treatment, also can be used for developing the vaccine of prevention or treatment HPV 16 infection.

4, medicine, pharmaceutical composition and the vaccine of the feature generation of HPV16PL1E7 recombinant protein according to claim 1 and claim 2, the 3 HPV16P L1E7 recombinant proteins of describing.

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