| PreS1 region of large HBV surface antigen (LHBsAg) plays an important role inassembly and infection of HBV, holds abundant T- and B-cell epitopes, and possessesHBV hepatocyte receptor binding site in the PreS1 (21-47) fragment. Theincorporation of PreS1 region into the present HBV vaccines containing only S regionmay improve the immune response of new-designed vaccines. It is important toidentify the epitopes in PreS1 region for the design of the future vaccines. In addition,anti-PreS1 (21-47) antibody in the sera may be a new marker for hepatitis B diagnosis.Therefore, it can acquire great social and economic benefit to develop a new methodwith high sensitivity and stability for the detection of this antibody. In this study, eight PreS1 gene segments overlapped in PreS1 (21-119) region, i.e.,PreS1 (21-47), PreS1 (34-59), PreS1 (48-70), PreS1 (60-85), PreS1 (71-94), PreS1(86-109), PreS1 (95-119) and PreS1 (21-119), were fused with GST gene, and thenthe eight fusion proteins were highly expressed in E.coli. Using Western blotting andELISA, they were applied to investigate immunological properties and B-cell epitopesof mouse and human in PreS1 region, and to compare different anti-PreS1 antibodiesin the sera from different HBV-infected patients. The results showed that humanimmune response to PreS1 region was more complex than that of mouse. In PreS1(21-119) region, B-cell epitopes of mouse located in PreS1 (21-59) and PreS1 (95-119)fragments, however, B-cell epitopes of human located in most part of PreS1 (21-119)region, and the protective epitopes might locate in PreS1 (21-59) and PreS1 (95-119)fragments, which were in agreement with the previous studies. More importantly, amajor B-cell epitope located in PreS1 (34-59) region was first identified, which could 4induce strong immune response and was speculated as a conformational andprotective epitope. The above results will provide some useful information for thefuture research on immunological properties of HBV and design of new vaccinesagainst HBV. In order to improve the method detecting anti-PreS1 (21-47) antibody, a GSTfusion protein (GST-SLS) containing two copies PreS1 (21-47) fragments connectedby a flexible peptide (Gly4Ser)3 was successfully expressed in E.coli. Itsimmunoreactivity with anti-PreS1 antibody was compared with PreS1 (21-47)synthesized peptide and other two GST fusion proteins containing one copy PreS1(21-47) (GST-SI) and two copies PreS1 (21-47) connected directly (GST-SII). Theresults indicated that the immunoreactivity and stability of GST-SLS weresignificantly better than those of other PreS1 (21-47)-related peptide and proteins,which might be explained by that the flexible linker could increase the distancebetween the two PreS1 epitopes and make the greater freedom of orientation of thetwo PreS1 domains. Additionally, the result of preliminary clinical trial also showedthat the sensitivity and stability of GST-SLS were improved in relation to GST-SII,which suggested that it has potential to be developed into a commercial kit. Using His-PreS1 (21-119) fusion protein as an immunogen, two stains ofhybridoma that can secrete monoclonal antibody against PreS1 were obtained bycell-fusion technique. The isotypes of the two monoclonal antibodies were identifiedas IgG1, and their directed epitope located in PreS1 (34-47) identified by Westernblotting analysis. It may be beneficial to the future research on properties andapplications of the monoclonal antibodies. |
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