| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 91 === Enterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease which most commonly affects children under the age of 5. In view of the time-consuming and laborious technique of virus isolation, a rapid and precise method for early diagnosis of EV 71 infection is required urgently. Using a specific primer pair designed for EV71 VP1 gene, a 342 nt DNA fragment was amplified by RT-PCR from EV 71 but not from the other 39 different serotyping enterovirus, including Coxsackievirus A、B and Echovirus. The DNA fragment was cloned into pQE30-UA vector containing 6×His tag. The recombinant plasmid was transformed into M15 E. coli host. A 14 Kda polypeptide was detected in lysates from M15 E. coli after induction with 1 mM IPTG. Western blot analysis using anti-6×His tag antibody confirmed the fusion of 6×His tag with the recombinant protein. The recombinant protein was purified by His-resin and injected into JCR strain mice to generate antibody. In addition to the recombinant protein, the raised antibody also recognized a 35 Kda polypeptide from a highly purified preparation of EV71. The result indicated that the recombinant protein was indeed a part of VP1 protein. The raised antibody was not able to display protection titer in neutralization test, indicating that the neutralization epitope was not in this region (aa 206-547) of VP1 protein. Immunofluorescence assay was utilized to check the specificity of the serum antibody. Due to high background in non-virus infected cells, the serum antibody has yet to be purified.
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