小反刍兽疫病毒H蛋白原核表达的研究

Primary study on the eukaryotic expression of H protein of Peste des Petits Ruminants Virus

小反刍兽疫病毒属副粘病毒科麻疹病毒属,为一不分节段的负链RNA病毒。本研究以灭活的检测用抗原为材料,抽提基因组RNA作为模板,根据Gen Bank下载的序列及载体的需要设计其主要的抗原基因H基因的编码序列的RT-PCR扩增引物;然后将扩增基因片段与T载体连接克隆测序,再与原核表达载体pET100/D-TOPO连接、诱导表达。结果显示所设计引物对模板有预期扩增,序列测定表明为正确片段;片段经纯化与表达载体连接,已挑到阳性克隆。H基因的表达菌经IPTG诱导,SDS-PAGE电泳,结果显示:H基因有预期分子量大小的蛋白表达;用Anti-His抗体已检测到相应融合蛋白的表达,其原核表达的相应蛋白抗原性状况正在试验研究中,表达产物具有潜在的诊断抗原和免疫原价值。

Peste des Petits Ruminants Virus （PPRV） has been classified as a member of the Genus Morbillivirus in the Family Paramyxoviridae, containing a single-strand negative sense RNA genome. In this study, the genome RNA was extracted as template from the inactivated detection antigen in commercial c-ELISA kits. Based on both the sequences downloaded from GenBank and the expressing Vector requirements, the primers for RT-PCR amplification of the ORF of the major antigen epitope gene （ H gene）were designed. Then the amplified fragment was linked to pMD18-T vector and further confirmed by sequencing. The results showed the expected fragment had been amplified correctly. The purified fragment then was linked to the expressing vector pETIOO/D-TOPO and the positive clones were obtained. Through SDS-PAGE electrophoresis detection, the expressed protein, which was induced by IPTG, was showed containing the expected H protein and was also confirmed by Anti-His antibody. The other related biological characters of this expressed protein, e.g. the immunological antigen character, were still in studying.