The Polyclonal Antiserum Preparation Of HCMV UL49

Object: To prepare specific polyclonal antiserum of Human cytomegalovirus UL49, This research has paved the way for further study on UL49 gene.Methods: Using bioinformatics methods to analyse the protein encoded by HCMV UL49 gene, a gene fragment (UL49j) be used for antigen was determined. The oligonucleotide primers were designed and used to amplify the gene fragment from AD169 cDNA.The amplified fragment was then cloned into E.coli expression vector pGEX-4T-3 to construct recombinant plasmid. The recombinant plasmid was transformed into E.coli expression strain BL21 (DE3).IPTG induces the expression protein of GST-pUL49j and the induced product was purified electroeluting.The PAGE mortar contained GST-pUL49j was used as antigen, injected intradermally to immunize male New Zealand white rabbit to get polyclonal antiserum.The titer and the specificity of the rabbit antiserum were detected by ELISA and Western blotting, respectively.Result: The pGEX-4T-3-UL49j E.coli expression plasmid was constructed successfully. The protein was overexpressed in E.coli expression strain BL21 (DE3). GST-pUL49j protein was also purified. The high titer of specific polyclonal antiserum was prepared successfully.Conclusion: The high titer of specific polyclonal antibody was prepared by E.coli expression protein GST-pUL49j, giving a good tool for further studies of HCMV UL49 gene function.