CN1175637A

CN1175637A - Expression of human hepatitis C virus protein in engineering bacteria and use of expression protein

Info

Publication number: CN1175637A
Application number: CN 96119615
Authority: CN
Inventors: 叶林柏; 郑金荣; 孟小林; 徐进平
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 1996-09-01
Filing date: 1996-09-01
Publication date: 1998-03-11

Abstract

HCV envelope protein E1-CDNA and E2-CDNA and core protein C-CDNA are cloned to pREST His vector by means of gene engineering and E1, E2 and C proteins are respectively expressed in colibacillus BL21. After ultrasonic breaking, Ni2NTA agarose chromatographic purification and SDS-PEAG examination, E1 protein is a polypeptide with 26KDa of molecular weight, E2 protein is a polypeptide with 20 KDa of molecular weight and C is a polypeptide with 26 KDa of molecular weight. Said three high-purity natural proteins are the necessary antigen components in test box to diagnose the HCV antibody.

Description

The expression of human hepatitis C virus protein in engineering bacteria and the application of expressing protein

The present invention relates to viruses of human hepatitis C (HCV) E ₁, E ₂, the expression technology of C albumen in engineering bacteria, particularly relate to and can be used to detect HCV antibody or antigen.

Main menses of viruses of human hepatitis C (HCV) and blood source goods infect, be blood transfusion back third liver main diseases because of, after HCV infects, have patient more than half to develop into chronic hepatitis approximately, and closely related with liver cirrhosis and liver cancer.

Viruses of human hepatitis C HCV is a kind of single positive chain RNA virus, belongs to flaviviridae in classification, and its gene structure is 5 ' hold to be structural area, 3 ' hold to be non-structural area, genomic put in order be 5 '-C-E ₁-E ₂/ NS ₁-NS ₂-NS ₃-NS ₄-NS ₅-3 ', at present known each genome albumen can both induce body to produce antibody effectively, but because the singularity of HCV, and each antigen fragment is induced antibody time of occurrence that body produces and in time that body continues and strong and weak each is variant.Therefore, the antigen of used kit should be diversified, and HCV envelope protein antigen is in viral outermost layer, and directly the infection with virus is relevant, is one of viral topmost antibody induction thing.

In recent years,, the molecular biology of HCV virogene is furtherd investigate, adopted gene engineering method on this basis, in engineering bacteria, express hepatitis C virus (HCV) E along with modern molecular biology technique is introduced the virological investigation field ₁, E ₂, C albumen, and be that compatibility prepares the HCV detection kit with expressing protein antigen, detect blood and blood source goods, control HCV infects human body.

Document Virology (1994) No.205:141-150 has reported and has used gene engineering method, has expressed HCV envelope protein antigen E at eukaryotic cells ₁And E ₂The 10th the 4th phase of volume of domestic viral journal, also reported and used gene engineering method, at the prokaryotic organism expression in escherichia coli HCV structural area PROTEIN C ₂₂, non-structural area NS ₃, NS ₄, and NS ₅Albumen, and with C ₂₂, NS ₃, NS ₅Proteantigen assembling HCV antibody assay kit detects main contagium blood of HCV and blood source goods.But, because the HCV envelope protein is to the toxicity of prokaryotic cell prokaryocyte, the mentioned reagent box lacks HCV envelope protein antigen, particularly each antigen fragment of HCV is induced antibody time of occurrence that body produces and in time that body continues and strong and weak each is variant, the present HCV detection kit poor specificity of using, sensitivity is low.Though polymerase chain reaction,PCR (PCR) can detect HCV effectively, the PCR complicated operation costs an arm and a leg, and accuracy rate is low, is difficult in clinically to be used widely.

The objective of the invention is structure and can in prokaryotic organism, efficiently express HCV envelope protein and core protein intestinal bacteria system, and the HCV envelope protein of expressing is nontoxic to prokaryotic cell prokaryocyte, do not influence the normal growth breeding of prokaryotic cell prokaryocyte.

Another object of the present invention is that the HCV envelope protein and/or the core protein that propose to efficiently express in prokaryotic organism are antigen, makes to detect the test kit of HCV, and this test kit is highly sensitive to HCV, and high specificity is easy to use, cheap.

For realizing that the technical measures that purpose of the present invention is taked are:

Recipient bacterium of the present invention is prokaryotic organism, specifically is exactly e. coli strain bl21, and this bacterial strain contains the plasmid of energy expression of HCV envelope protein E1 or E2 or core protein C.HCV envelope protein E1 CD-NA897-1467 nucleotide sequence is cloned the restriction enzyme site in pRSET HisA Pst-ECOR I, HCV E2CDNA1379-1847 nucleotide sequence is cloned the restriction enzyme site in pRSET His A EcoR I-Hin d III, HCV core protein CCDNA342-915 nucleotide sequence is cloned the restriction enzyme site in pRSET HisA ECOR I-Hisd III, transform respectively in the e. coli bl21 recipient bacterium, obtain the engineering strain that transformed respectively through screening, put the glycerine cryopreservation, the mono-clonal bacterial strain is inoculated in test tube respectively, shaking culture, change the one-level triangle over to and shake bottle, 37 ℃ are cultured to logarithmic phase, by ten of inoculum size/change the big triangle of secondary again and again over to shake bottle, replace the conventional inducing culture of IPTG with about 5% lactose, when overcoming the HCV envelope protein and in prokaryotic cell prokaryocyte, efficiently expressing to the toxicity of prokaryote itself.Collect culture, add the normal intestinal bacteria lysate, ultra-sonic oscillation, the centrifugal again cell debris that goes, supernatant liquor is crossed Ni ²⁺NTA agarose affinity chromatography post obtains the engineered protein of purifying respectively.Through electrophoresis detection, HCV E1 albumen is a band, and molecular weight is 26K Da, and E2 albumen is an electrophoresis band, and molecular weight is 20KDa.Conventional grading extraction can reach the purifying purpose at the HCV of expression in escherichia coli C albumen xln, electrophoresis detection, and HCV C albumen is a band, molecular weight is 26kDa.

With core protein C and three kinds of proteantigens of HCV envelope protein E1, E2 of efficiently expressing in engineering bacteria is compatibility, and the HCV antibody assay kit of preparation can detect with core protein C Nonstructural Protein NS ₃, NS ₅The HCV positive serum that can not detect for the test kit of compatibility.

Compared with prior art, the beneficial effect that adopts technical measures of the present invention to reach: the present invention first by gene engineering method prokaryotic expression and adopt up-to-date expression vector and affinity chromatography system purifying HCV envelope protein E1 and E2, overcome HCV E1, E2 is to colibacillary toxicity, and purifying is after electrophoresis detection, HCV E1 albumen is a band, molecular weight is 26K Da, and E2 albumen is an electrophoresis band, and molecular weight is 20K Da, HCV C albumen is a band, and molecular weight is 26kDa.With core protein C and three kinds of proteantigens of HCV envelope protein E1, E2 of efficiently expressing in engineering bacteria is compatibility, and the HCV antibody assay kit of preparation can detect with core protein C Nonstructural Protein NS ₃, NS ₅Be the HCV positive serum that the test kit of compatibility can not detect, the absolute value of the sensitivity of detection improves 2%, and relative sensitivity improves 25%.

Following examples describe the present invention in detail:

Glycerine is preserved bacterium HCV BL21C, BL21 E1 and BL21E2 bacterial strain are inoculated in respectively in 25mL (IYT) substratum, every bottle graft kind 10uL, 200rpm/min, 37 ℃ of shake-flask culture spend the night, switching 12mL (included the AMP250uL/ bottle in 250ml IYT culturing bottle in the 2nd day, 5% lactose/bottle, 37 ℃ of 150rpm shaking culture 5～6 hours, the centrifugal 10min of 5000rpm, collecting precipitation bacterial classification, respectively with the 30mL IMAC-5 sedimentary thalline that suspends, one 20 ℃ freezing repeatedly 3～6 times, with S250 ultrasonication crusher machine thalline, with the microscopic examination cellular lysate to bacterium 90% cracking.Behind cell rupture, with the rinsing repeatedly of IMAC liquid, the centrifugal cell debris that goes, Ni on the supernatant liquor ²⁺NTA agarose chromatography post, per minute flow velocity 1.5mL after sample is crossed post, uses 200mL IMAC-40 successively, 50mL IMAC-60,50mL buffered soln PBS (PH7.2) wash-out obtains three kinds of HCV albumen elutriants.Add 2%NaN ₃4 ℃ of preservations.Collect the IMAC-60 elutriant, detect purity of protein with SDS-PAGE.

Claims

1. one kind can efficiently express the proteic bioengineered strain of viruses of human hepatitis C HCV system, and it is characterized in that: bioengineered strain is prokaryotic organism, and contains the plasmid of energy expression of HCV envelope protein E1 or HCV envelope protein E2 or HCV core protein C.

2. according to the described bioengineered strain of claim 1, it is characterized in that: described bioengineered strain is that prokaryotic organism are e. coli strain bl21.

3. according to the described bioengineered strain of claim 1, it is characterized in that: described HCV envelope protein E1 plasmid is for to clone the restriction enzyme site in pRSET HisA Pst-ECOR I with HCV envelope protein E1CDNA897-1467 nucleotide sequence.

4. according to the described bioengineered strain of claim 1, it is characterized in that: described HCV envelope protein E2 plasmid is for to clone the restriction enzyme site in pRSET His A EcoR I-Hin d III with HCV E2CDNA1379-1847 nucleotide sequence.

5. according to the described bioengineered strain of claim 1, it is characterized in that: described HCV core protein C plasmid is for to clone the restriction enzyme site in pRSET HisA ECOR I-Hisd III with HCV core protein CCDNA342-915 nucleotide sequence.

6. the method for any expressed HCV envelope protein described in the purifying claim 1 to 4, it is operating as: culture is added the intestinal bacteria lysate, through ultra-sonic oscillation, the centrifugal cell debris that goes, it is characterized in that: supernatant liquor is crossed Ni ²⁺NTA agarose chromatography post.

7. one kind with E1, the HCV diagnostic kit that three kinds of HCV albumen of E2 and C are formed as the antigen component compatibility.