| Summary: | 碩士 === 國立屏東科技大學 === 生物科技研究所 === 98 === Glycoprotein G is translated from the US4 gene of human herpes simplex virus type 2 (HSV-2). The US4 gene has a secreted peptide (sgG) at the amino-terminal site and high-glycoslation protein at the C-terminal site. In order to get the modified Glycoprotein G proteins, we used Pichia pastoris and baculovirus system to express different G gene fragment initially, but the quantity of the expressed proteins was very low or insoluble. For the purpose of increasing Glycoprotein G expression, we chose a prokaryotic expression system to express sgG-His and sgG protein that is unique to HSV-2. The sgG protein about 45 kDa and sgG-His fusion protein about 56.4 kDa, were expressed by the prokaryotic expression system. BALB/c mice were immunized with the expressed sgG-His fusion protein to create hybridoma cells. Antibodies secreted from the hybridoma cells were screened by ELISA and further confirmed by immunoblot and Western blot. ELISA was performed to confirm that the produced monoclonal antibodies were truly against sgG or His protein. Nine monoclonal antibodies were prepared in this study, including 3 anti-His and 6 anti-sgG antibodies. Immuno blot and Western blot analysis suggested that these 9 monoclonal antibodies recognized a linear epitope. The outcome of the specificities of six sgG MAbs to detect HSV-2 virus, sgG-His fusion protein and pET32b-His confirmed that HSV-2 polyclonal antibodies and six sgG MAbs all could identify the G glycoprotein of HSV-2. The immunoglobulin (Ig) class of six anti-sgG MAbs was determined by the ELISA with a Thermo-MAb Kit. All six anti-sgG monoclonal antibodies belonged to IgG2b-subtypes. The anti-His MAbs included two IgG1 and one IgG2a antibodies. The result of the sensitivities of the six sgG MAbs showed that the antigen concentration was 0.008 ng/μl under the condition of P/N > 5. The dilution folds of the antibodies could reach as high as 80000-fold high and yield the same result. ELISA also confirmed that the six sgG MAbs have great specificities against sgG protein. The sensitivity of HSV-2 sgG protein could achieve 5 ng/μl by immunochromatography assay. In the future, the sgG MAbs will be applied to develop of immunochramatography strip for diagnosis of clinical cases of HSV-2 infections.
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