For The Preparation Of HCV Antigen And Antibody Integrated Detection Kit Related Reagents

Viral Hepatitis C is one of the ten major infectious diseases in the world that can cause death,which is also common in our country.Viral Hepatitis C or Hepatitis C,which is caused by the hepatitis C virus,is mainly transmitted via body fluids and blood.About 70%to 85%of people who are infected with HCV are the patients with chronic hepatitis C,60%to 70%of the patients with chronic infection will develop into chronic liver disease,including cirrhosis and hepatocellular carcinoma.1%to 4%of patients with cirrhosis resulted in liver cancer every year.HCV infection is the leading cause of cirrhosis and liver cancer.Another main reason for the great threat of hepatitis C to the people is because of no effective vaccine,which can prevent hepatitis C virus infection.In developing countries,because of the lack of effective methods for both diagnosis and treatment of hepatitis C,HCV infection leads to a gradual increase in mortality of the patients with hepatitis C.Due to the difficulty in getting a breakthrough in the short term for the development of HCV vaccine,therefore,the only way for controlling the development of HCV infection is to cut off the source of infection and deter a route of transmission.Due to the strong infectivity of HCV at the early stage of HCV infection,the most effective means to control the spread of hepatitis C is to have early discovery of HCV infection and an accurate and timely diagnosis.So,to establish the screening method for HCV infection with high sensitivity,specificity and stability is of great significance.The common method for HCV detection mainly includes(1)the detection of HCV antibody in serum,(2)the detection HCV core antigen in serum,(3)the detection of HCV RNA in blood.However,the antigen used in anti-HCV detection kit is genetic engineering antigen and synthetic peptide antigen with lower immunogenicity,therefore false-positive results may occur.In addition,as a result of the existence of "window period",people infected with the HCV might have no the production of antibodies at the beginning of the HCV infection.Therefore,early diagnosis of HCV infection needs additional assistance for making up for the deficiency of the fourth generation of anti-HCV detection kit.Hepatitis C virus core protein contains about 190 amino acids,and has a conserved amino acid sequence.The occurrence of HCV core antigen in blood is an important symbol for HCV infection.Thererfore the detection of HCV core antigen could be applied to the screening for "window period" of HCV infection,which could be reduced by about 50 days.The common method for the detection of HCV core antigen is to use double antibody sandwich method.Double antibody sandwich assay can be used in the detection of total or free core antigen in serum samples,which significantly shortens the window period and is not affected by the interference of the anti-HCV antibody in the test sample.In addition,double antibody sandwich assay is characteric of reliability for sample testing,simplicity with low requirements for environment,low rate of false positives and short process.When environmental exposure to HCV reaches 7 to 21 days,the presence of HCV RNA could be detected in peripheral blood of patients.The quantity of HCV RNA in serum before seroconversion can reach a peak titer between 1x105 to 1x108 copies/mL.In order to achieve early detection of HCV infection,reduce the infection rates,many countries have adopted nucleic acid assay with a higher sensitivity,but this technology needs high requirements for equipment,reagents,operation methods,and at the same time,the detection process is easy to make a cross contamination,hence it is difficult to use the technology in developing countries.To sum up,the combination of the anti-HCV antibody detection and HCV core antigen detection will become one of the effective ways to detect HCV infection.The project that may simultaneously detect HCV antigen and anti-HCV antibody of samples,which not only could improve positive detection rate,effectively shorten the window period of HCV detection,will be helpful for early diagnosis of HCV infection by the combination of antigen detection and antibody detection.By comparing antigen components among the former three kits of anti-HCV detection,the core and NS3-5 recombinant antigens were chosen to detect antibodies in the blood of the patients,at the same time,the monoclonal antibodies against two small peptides on both ends of conservative region present in HCV core antigen were used to detect antigns in patient’s blood using the principle of double-antibody sandwich assay.The concrete research contents of this topic of are as follows.(1)Expression and purification of HCV antigen used in ELISA kit for detecting antibody.HCV core-NS3-5 gene fragments were obtained using PCR method,then were subcloned into prokaryotic expression vector pRSET-BL.Recombinant prokaryotic expression vector pRSET-BL/HCV core-NS3-5 was obtained by enzyme digestion and sequencing.Then the plasmid was electrotransformed to E.coli BL21(DE3).The fusion protein 6His-HCV core-NS3-5 fusion protein was induced by IPTG,then purified by Ni-NTA affinity chromatography.The fusion protein was used as antigen components in ELISA kit.(2)Construction of prokaryotic expression vectors carrying hepatitis C virus core antigen C8-160 fragment,small peptide C8-22 and C101-115 fusion proteins respectively and their expression and purification.the gene encoding HCV C8-160 fragments,HCV C8-22 small peptide and HCV C101-115 small peptide were synthesized,respectively.Then the HCV C8-160 gene was subcloned into prokaryotic expression vector PGEX-4T-3,the resultant plasmid was named PGEX-4T-3/HCV C8-160.HCV C8-22 small peptide and HCV C101-115 small peptide were cloned into pET-28a/HBc-LacZ BS vector and pRSET-B/Grn E BS vector,respectively.The obtained plasmid was named pET-28a/HBc-HCV C8-22,pET-28a/HBc-HCV C101-115,pRSET-B/Grn E-HCV C8-22 and pRSET-B/Grn E-HCV C101-115,respectively.The five prokaryotic expression vectors above were electrotransformed into E.coli BL21(DE3)strains,respectively.Then target proteins were expressed by IPTG induction,and purified by Ni-NTA affinity chromatography,which were used to prepare for monoclonal antibodies agains different HCV core antigen epitope.(3)Construction of eukaryotic expression vectors carrying hepatitis C virus small peptide C8-22 and C101-115 fusion proteins respectively.HCV C8-22 small peptide and HCV C101-115 small peptide were cloned into pAd5/E1-CMV-H1H2-Grn E-Flag vector digested by BamHI and SfuI,respectively.The supernatants and cell lysates of HEK 293 transfected by pAd5/El-CMV-H1H2-Gm E-HCV C8-22-Flag or pAd5/El-CMV-H1H2-Grn E-HCV C101-115-Flag for 72 h were collected for western blot using monoclonal antibodies.(4)Preparation of monoclonal antibodies against human IgG Fc segment.Using human IgG protein purchased from Sigma as immunogen,mice were immunized for the first time by subcutaneous injection of the total amount of the immunogen per mouse was 40 μg with multipoint which were mixed with an equal volume of Freund’s complete adjuvant.Three weeks later,the mice were immunized with the immunogen mixed with an equal volume of Freund’s incomplete adjuvant for the second time,and immunized dose was the same as the first time,then two weeks later the third immunization was done by intraperitoneal injection of immunogen using the same dose as before without adjuvant.Two weeks later,serum antibody titer of blood from tail vein was detected by ELISA,two mice whose antibody titer of serum reached 1:64,000 or above were selected for the intraperitoneal booster immunization with the immunogen.One week later,the mice were boostered.The second day after booster immunization,the feeder cells were seeded in 96-well plates,then spleen cells from immunized mice with SP2/0 myeloma cells were fused according to 10:1 or 5:1 proportion by PEG4000 on the third day of booster immunization,and HAT/HT selective medium were applied for screening hybridoma,7 days to 10 days after fusion,positive wells were screened by ELISA,through subclones for 3 to 4 times,positive rate is 100%,now we have obtained secreting mouse anti-human IgG monoclonal antibody-producing hybridomas cells,then hybridoma cells were transferred to 24-well plates were expanded and frozen.6-week-old healthy female BALB/c mice were prepared to produceascites.Finally,the desired anti-human IgG mouse monoclonal antibodies were collected.(5)Preparation of monoclonal antibodies against different epitopes of HCV core antigen.To generate monoclonal antibodies against HCV C8-22,the GST-6His-HCV C8-160 obtained above as an initial immunogen,HBc-HCV C8-22 fusion protein was used as an immunogen for boost immunization,Gm E-HCV C8-22 fusion protein was used to screen positive hybridoma.The positive hybridomas were expanded and used for the preparation of ascites followed by the evaluation using immunoblotting and immunofluorescence staining,then monoclonal antibodies to HCV C8-22 peptide were obtained and diagnosed.Using similar strategy,monoclonal antibodies against HCV C101-115 peptide were obtained.(6)Establishment and application of the system for the detection of both HCV antigen and antibody.Monoclonal antibodies targeting different epitopes of HCV core antigen,Anti-HCV C8-22 or Anti-HCV C101-115 and HCV core-NS3-5 recombinant antigen were co-coated on enzyme-linked immunosorbent assay plate.If it was used to detect antibodies in the sample,HRP-labeled mouse anti-human IgG monoclonal antibody would be applied in the system.If it was used to detect antigen in the sample,anti-HCV core monoclonal antibody against the antigenic epitope that is different from monoclonal antibody coated on the plate was used for being labeled with horseradish peroxidase in the system.A qualitative or quantitative detection for antigens or antibodies in the samples was performed after color development.The following research results were obtained by these studies.(1)Successfully constructed pRSET-BL/HCV core-NS3-5 prokaryotic expres-sion vector and obtained 6His-HCV core-NS3-5 protein with the concentration of 1.0 mg/mL.(2)Successfully constructed PGEX-4T-3/HCV C8-160 prokaryotic expression vector,and purified GST-6His-HCV C8-160 fusion protein with the concentration of 1.0 mg/mL.(3)Successfully constructed pET-28a/HBc-HCV C8-22 and pRSET-B/Grn E-HCV C8-22 prokaryotic expression vectors,and purified HBc-HCV C8-22 fusion protein with the concentration of 0.1 mg/mL,Grn E-HCV C8-22 with the concentration of 0.25 mg/mL.(4)Successfully constructed pET-28a/HBc-HCV C101-115 and pRSET-B/Gm E-HCV C101-115 prokaryotic expression vectors,and purified HBc-HCV C101-115 fusion protein with the concentration of 0.1 mg/mL,Grn E-HCV C101-115 with the concentration of 1.2 mg/mL.(5)Successfully constructed pAd5/El-CMV-H1H2-Grn E-HCV C8-22-Flag and pAd5/E1-CMV-H1H2-Gm E-HCV C101-115-Flag eukaryotic expression vectors.(6)Successfully generated 3 strains hybridomas against hIgG Fc segment,which were named 33E5,30E1 and 28D11,respectively.The monoclonal antibodies secreting from hybridomas were confirmed by western blot.All of them are shown to be specific for hIgG Fc fragment.They are all IgGl subtypes and purified antibodies with the concentration of 2.28 mg/mL,1.07 mg/mL and 1.61 mg/mL with Protein G Plus/Protein A-Agarose.(7)Generated 3 strains hybridomas against HCV C101-115 through protein-protein combined immunization,which were named 39A6,43C6 and 73B6,respect-tively.The monoclonal antibodies secreting from hybridomas were confirmed by western blot and immunofluorescence staining.All of them are specific for HCV C1O1-115.They are all IgGl subtypes and purified antibodies with the concentration of 3.41 mg/mL,5.15 mg/mL and 1.09 mg/mL with Protein G Plus/Protein A-Agarose.These results provide specific antibodies and antigens for HCV diagnosis,and laid lay the foundation for the investigation of ng the prevention and treatment of HCV.It also provides a new and effective immune scheme strategy for the preparation of monoclonal antibody against the small peptide.