| Summary: | 碩士 === 國立臺灣大學 === 醫事技術學研究所 === 87 === AbstractAbstract
HSV is the most common and major virus in clinical infections. Serologic test is the only means to identify people with previous or present HSV infection. In addition, it's also useful in the sero-epidemiologic studies. HSV can be classified into to two serotypes: HSV-1 and HSV-2, and they are genetically 50% homologous to encode many similar proteins. This leads to extensive cross-reactivitive antibodies, so makes more difficult in sero-typing of HSV infection by serological assay. For this reason, developing tests to differentiate HSV-1 and HSV-2 antibodies of patient's serum is getting important and deliberate.
gG is an envelope glycoprotein of HSV and it can induce strong immune responses in host. Because the homology of gG between HSV-1 and HSV-2 is only 30%, it can induce type-specific antibodies and will be a good candidate for antigen. The cloning and expression of type-specific regions of HSV-1 and HSV-2 gG which can offer suitable recombinant proteins for developing serological diagnositic kits and other research purpose. 62~203 bp of HSV-1 gG-1 gene and 129~1300 bp, 1678~1783 bp of gG-2 gene were amplified by polymerase chain reaction (PCR). The PCR products are cloning and construct in plasmids. E.coli and Drosophila cell protein expression systems are used this study.
This study describes that the 62~203 bp of gG-1 gene and the 1678~ 1783 bp of gG-2 gene recombinant sequence can be translated in the E.coli system. The recombinant proteins, named gG-1 and gG-2S, are 19~20 KDa and 18~19KDa. But the 129-1300 bp of gG-2 gene fragment cannot be expressed in the E.coli system. The gG-1 and gG-2S recombinant proteins is confirmed by Western blot with monoclonal antibody for the tag of pThioHis vector, and then react with patient's serums. The gG-2S recombinant protein has specific reaction with patient serum having HSV-2 type-specific antibodies, and does not react with serums that have only HSV-1 antibodies. But the gG-1 recombinant protein cannot react with either.
In the Drosophila cell expression system, only few of gG-2 transfected cell is confirmed to be positive with monoclonal antibody, Anti-V5 for the tag of pMT/Bip/V5-His A vector by FA. It also has specific reaction with HSV-2 patient's serum and negative by HSV-1 serum only. So we can confirm the gG-2 recombinant protein is HSV-2 specific. The other 2 transfected cell: gG-1 and gG-2 do not see any expression.
To summarize our data, gG-1, gG-2, and gG-2S can express recombinant proteins, only the gG-2S expressed in E.coli system and gG-2 protein expressed in Drosophila system are HSV-2 type-specific antigen. Although both of gG-1 and gG-2 can produce HSV type-specific antigen, but the expression efficiency and quality of products is too low. It is need some efforts to improve this defect. For HSV-1 G gene clone (gG-1), it is need remodify our designation of gene sequencing of gG-1 to enhance the antigenicity of HSV-1 type specific antigen.
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