Cloning, Expression And Antibody Preparation Of HBV Core-related Antigens

Hepatitis B virus (HBV) belongs to the hepadnavirdae family with an enveloped DNAgenome, which is relaxed circular, partially double stranded. Hepatitis B is transmittedhorizontally by blood transfusion, injection, and vertically through birth and breast feed.As a major public health threat, HBV chronically infects40millions of people and most ofthem are in developing countries, the infection may eventually develop into liver diseaseslike cirrhosis and hepatocellular carcinoma.The template for HBV pre genome RNAsynthesis:HBV cccDNA (covalenfly closed circular DNA) is responsible for theestablishment of viral infection and persistence, moreover, its existence is highly relatedwith development into chronic infection, anti viral treatment, and viral rebound aftertreatment. Therefore, the detection of HBV cccDNA level is essential for the evaluation ofdisease progression and treatment outcome. At present, HBV cccDNA is detected from liveliver biopsy samples and thus cannot be established as a regular test. Therefore, there isurgent need for a more convenient and highly sensitive serum detection marker for HBVcccDNA. Among the array of HBV serum markers, HBV core related antigen (HBcrAg)gradually entered the scope for intensive research for its importance in HBV clinicaldetection and treatment evaluation.Coded by pre C/C region, HBcrAg is composed of HBcAg, HBeAg, and p22cr(22kDa precore protein) as shown in Fig.1, the1149amino acids encoded by C gene is aregion shared by the three components. Coded by pre core region, p22cr includes28to atleast150amino acids, a part of N end un spliced signal peptide, but misses the C endarginine rich segment. p22cr is usually found in vacant virus particles lacking virus core.A detection method of HBcrAg is indispensable for its further research and the longterm aim of our group is to establish a double antibody sandwich ELISA detection methodof HBcrAg. In achieving this, obtaining HBcrAg related antibodies is the first step. Thisthesis describes the expression of HBcAg, HBeAg, and p22cr protein, as well as the shared149amino acid peptide (nominated here as the1149protein). Afterwards, mousemulti clonal antibodies against each proteinwere produced with respective antigen: forHBcAg using149—183amino acid peptide; for HBeAg using10—1amino acid peptide,for p22cr using28—10amino acid peptide.1—149protein was used as an antigen toproduce multi clonal and mono clonal antibodies. The preparation of the antibodies prepared way for further research on HBcrAg, especially in the direction ofHBVserological test.In his thesis, recombinant HBcAg, HBeAg, p22cr protein were expressed and purifiedin prokaryotic expression system, as well as the protein specific peptides from the threeproteins. Then, multi clonal antibodies were induced and hybridoma cell lines stablyexpressing mono clonal antibodies were obtained. The major results were as follows:①PCR primers for HBcAg, HBeAg, p22cr, and114protein based on the sequencepublished on GeneBank were designed, and the segments were amplified with the primersusing HBV BJ plasmid containing HBV genome as template. Construction of the segmentsinto pGEX4T1prokaryotic expression vector, which is tested by NdeI and Xho Idigestion and sequencing.②The optimization of protein induction conditions: therecombinant proteins are best expressed at37℃, induced by0.8mM/L IPTG for3.5hours.The peptides were purified and tested with immuneblotting analysis.③The peptides wereexpressed and purified by Shanghai yingji, and tested by HPLC. We found that thepeptides can binding with target antibodies in humen blood.④Each of HBcAg, HBeAg,p22cr were coupled with KLH before immunizing two Japanese white rabbit for5times toproduced high titre antibodies. The antibodies binding to target protein were tested byWestern blotting and ELISA.⑤The1149protein was used to immunize four Balb/Cmice to induce antibody producing B cells which were used for hybridoma cell lineconstruction and selection. Four cell lines were selected and amplified in mouse ascites.The antibodies were purified from the ascites with affinity purification. The antibodies’specific binding with target protein were tested with western blotting and ELISA.Ths thesis describes the successful construction of HBcrAg prokaryotic expressionvector, the induction and expression of target protein, and the preparation of bothmonoclonal and muti clonal antibodies that is high in titre, highly specific, and highlyaffinitive to target antigens. This thesis contributes to the establishment of a platform onwhich a highly specific and highly sensitive detection method of HBcrAg can beresearched, which would eventually benefit the monitoring, prognosis, and evaluation oftreatment for HBV patients.