Study On Quantitative Detection Of Targeted Hepatitis B Virus Core Protein

Hepatitis B is a worldwide infectious disease caused by hepatitis B virus,which mainly occurs in the liver.Clinically,through the detection of HBV serum markers to help judge whether HBV infection,evaluate the prognosis,treatment and drug response,but with more and more studies,hepatitis B virus core protein as a new method of detection of hepatitis B virus has been widely concerned.HBc monomers form homodimers,which further wrap pgRNA and HBV polymerase into regular icosahedral nucleocapsids.The nucleocapsid proteins composed of HBc not only act as viral structural proteins,but also detect the maturity of HBV core particles and HBV rcDNA,and finally interact with HBV surface proteins to form mature virus particles.In-depth study of the mechanism of HBV infection in the liver found that HBc is particularly important.At present,there is a consensus in the field of hepatitis B that the expression of HBc is positively related to the activity of HBV cccDNA in the liver nucleus.Quantitative detection of HBc can indicate whether cccDNA has been cleared or functionally inactivated in patients with chronic hepatitis B.In order to solve the above problems,we have carried out related research work on the new method of exogenous expression of HBc,targeted HBc specific antibody and quantitative detection of HBc.Part ? Secretory expression of hepatitis B virus core protein in Bacillus subtilis Objective Study on the feasibility of secretory expression of recombinant HBc in Bacillus subtilis Methods HBc gene was inserted into the MCS region of the pBES-DNA shuttle plasmid to obtain the pBES/HBc recombinant plasmid.The pBES/HBc recombinant plasmid was linearized and ligated with the signal peptide DNA library.The ligated products were transformed into stellar competent cells.All the positive colonies were harvested and the mixed plasmids were extracted.The mixed plasmids were transferred into Bacillus subtilis RIK1285 competent cells by chemical transformation,and the signal peptides were detected.The bacteria liquid containing different signal peptides were magnified and cultured,and the culture supernatant was collected to detect HBc secretion.Results Recombinant HBc exists in the culture supernatant of Bacillus subtilis and has different states;Westernblot found that Anti-HBc can bind to recombinant HBc in the supernatant with high specificity.Conclusion The recombinant HBc was secreted and expressed in Bacillus subtilis,and the recombinant HBc protein showed similar biological activity to the natural state.Part ? Construction single chain variable fragment(scFv)antibody against hepatitis B virus core protein Objective Establishment of HBc ScFv phage library by T7 phage screening.Method Recombinant HBc/183 and HBc/149 were used as antigens to immunize female Balb/c mice(6-8 weeks).After 8 weeks,mRNA,was extracted from mouse spleen cells and reverse transcribed into cDNA.Primers were designed and cDNA was used as template.After the first step of PCR reaction,the DNA fragments of light chain variable region(VL),heavy chain variable region(VH)gene were obtained respectively.After the second step of overlap extension PCR(SOE PCR)reaction,the light chain and heavy chain were spliced to obtain Single chain antibody(ScFv)gene library.ScFv gene fragment was digested with double endonuclease and ligated with T7Select10-3b to form a primary recombinant T7 phage library.Recombinant HBc/183 and HBc/149 were used as solid phase antigens for affinity screening with recombinant T7 phage.After three rounds of screening,the phage library binding to HBc/183 and HBc/149 was enriched.The recombinant T7 phage was selected for ELISA detection,and the recombinant T7 phage with high affinity was screened,and the scFv was sequenced.Results The amino acid sequences of(FR)and(CDR)in the frame region of ScFv gene were analyzed by igblast software.Highly enriched amino acid sequences are used to prepare ScFv antibody libraries.Conclusion The amino acid sequence of ScFv with high affinity binding to recombinant HBc/183 and HBc/149 was obtained,which was used as the basis for clinical detection of HBc.Part ? Screening of antibody pairs targeting hepatitis B virus core protein Objective The antibody pairs targeting HBc were screened for quantitative detection of HBc.Methods A total of 23 kinds of affinity antibodies targeting different gene fragments of HBc were prepared,including 1-14 targeting the C-terminal ARD region of HBc protein and 23-31 targeting the N-terminal region of HBc protein.Each ELISA plate formed an antibody pair with the ALP labeled antibody except itself,and the antibody-antigen-ALP labelled antibody mode fixed on the plate was used to detect HBc.PBS was used as control group and HBc sample group with different ng levels.The chemiluminescence of different combinations of antibody pairs was detected by chemiluminescence method,and the antibody pairs with high affinity were screened.Results The antibody pairs with high affinity were selected by statistical analysis.Conclusion Different antibody groups have different affinity for HBc protein,and different antibody groups have statistical significance for the detection of HBc protein with different detection limits.