Study On The Development And Application Of The Kit For Detecting Anti-P7 Protein Antibody In Serum Of HCV Infected Patients By The Chemiluminescence Method

Objective: To recombine and express hepatitis C virus(HCV)ion channel protein P7,develop a kit for detecting anti-P7 protein antibody in the serum of HCV infected patients using chemiluminescence assay(CIA),and its methodological evaluation and case application verification,which is to understand the immune response of P7 protein in the host,and provides technical support to further reveal the relationship between the changes of anti-P7 protein antibody content with the different genotypes and clinical types,progress in the course of clinical infection,before and after DAAs treatment of HCV infected patients.Methods:(1)Sequence comparison and selection of HCV 1b genotype P7 nucleic acid sequence(ACCESSION NO: AJ238800),construction of plasmid PUC-P7 containing the nucleic acid sequence,setting the plasmid PUC-P7 as the template,conducting two rounds of PCR amplification,obtaining the full length P7 gene of the PCR product,and conducting agarose gel electrophoresis to identify and recover the product.Restriction endonucleases Bam HI and Hind III digested PCR products and expression vector p GEX-KG,identified the size of the target fragment by electrophoresis again,cut agarose containing the target fragment for DNA fragment recovery.The recombinant plasmid p GEX-KG-P7 was constructed by linking the target fragment and expression vector p GEX-KG with T4 DNA ligase.(2)After the recombinant plasmid p GEX-KG-P7 was transformed into plasmid and expressed by prokaryotic induction,the target protein was extracted,purified and refolded.(3)Based on the indirect principle of enzyme-linked immunosorbent assay(ELISA),the anti-P7 protein antibody detection kit was established by the CIA,and the optimal coating concentration and enzyme marker working solution were selected.(4)The kit was evaluated with precision test,blank limit and detection limit,linear evaluation,accuracy evaluation,interference test,stability observation and other methodological indicators,as well as clinical example application.Results:(1)Successfully completed the connection between the expression vector p GEX-KG and the target fragment of P7,and constructed the recombinant plasmid p GEX-KG-P7.(2)The recombinant plasmid p GEX-KG-P7 was transformed into plasmid and expressed by prokaryotic induction to obtain P7 fusion protein with relative molecular weight of about 34 k Da.(3)Complete the development of the kit for the detection of anti-P7 protein antibody by the chemiluminescence method.The kit mainly includes 96-well anti-P7 protein antibody detection microplate,sample diluent,sample cleaning solution,anti-human Ig G enzyme solution,substrate solution A,substrate solution B,positive control,negative control,sealing membrane and self-sealing bag containing desiccant.The optimum coating concentration and enzyme marker working solution are 0.5 μg/ml and 0.05 μg/ml respectively,and the optimum sample adding amount is 100 μl.(4)Evaluate the methodological indicators of the prepared chemiluminescence assay kit for detecting anti-P7 protein antibody.In the precision test,the intra-assay variation coefficient is 2.97%-4.09%,and the inter-assay variation coefficient is 3.75%-5.52%,which all meet the requirements;The blank limit and detection limit are 1.56 RLIR and 5.99 RLIR respectively;The correlation coefficient r≥0.975,and the analytical measurement range(AMR)is 1.252-18.774RLIR;The average recovery rate was 97.8%;The positive serum samples of rheumatoid factor,hepatitis B virus surface antibody,hepatitis B virus e antibody,treponema pallidum antibody,human immunodeficiency virus type I and/or type II antibody have no cross reaction to this test;The kit is stable within 15 months.(5)The test kit was verified by clinical application.Serum samples from 45 HCV-infected persons and 50 healthy persons were randomly collected to detect serum anti-P7 protein antibody.The positive rate of serum anti-P7 protein antibody in 45 HCV-infected persons was 20%(9/45),and the positive rate of serum anti-P7 protein antibody in 50 healthy persons was 0.Conclusion: The indicators of the anti-P7 protein antibody kit in the serum of HCV-infected individuals detected by chemiluminescence method meet the requirements of clinical application.The detection of anti-P7 protein antibodies in the serum of HCV-infected individuals through clinical examples confirms the existence of a certain proportion of anti-P7 protein antibodies in HCV-infected individuals,indicating that there is an immune response phenomenon in the host body for P7 protein.The serum anti-P7 protein antibody detected by this kit can be used as an auxiliary screening indicator for HCV-infected individuals,but should not be the only basis for clinical diagnosis and treatment.It is suitable for monitoring the condition of HCV infection,disease mechanism,prognosis evaluation,and epidemiological research.