Influence Of S Gene Mutation On Antigencity Of HBsAg In Patients With HBsAg+/HBsAb+double Positive Result

Objective: To construct the expressing system of HBsAg with S gene mutationand to study the influence of S gene mutation on antigencity of HBsAg.Method:1. HBV DNA samples extracted from patients sera were assayed withPCR detection technique.then analyse the mutations of S gene region and to indentifygenotype. The cloning plasmid was constructed, and the positive clones weresequenced and analysed.2. To construct recombinant expressing plasmid and expressthe HBsAg with S gene mutation in prokaryotic host cell(E.coli.) and eukaryotic hostcell(CHO cell) to extract proteins.3. Detecting the protein content. The antigenicity ofrecombinant protein was confirmed by western blot, and change of antigencity wasestimated by the extent of combination of antigen and antibody.Result： The clone results show S gene of double positive patients mutated,amino acid mutation in the S gene region of genotype C were concentrated in loci194,68,3and126, while genotype B were concentrated in loci40,200,129and5. Themore amino acid mutation of a determinant, the lower content of HBsAg.Recombinant HBsAg was detected by chemiluminescence, and we find that content ofprotein with mutation at locus126were lower than that without mutation at locus126.Western blot results showed all the expression protein were responsed to monoclonalantibody and stripe were showed.Conclusion: Amino acid mutations occurred in the S gene region especially inthe first loop of the a determinant from patients with HBsAg+/HBsAb+doublepositive result. Mutated HBsAg which was expressed in prokaryotic and eukaryoticexpression system could react with antibody, and HBsAg had antigencity. Mutation atlocus126may affect the space conformation of protein, which decreasing thecombination with antibody, so the protein content was low in routine tests.