乙型脑炎病毒NS1′蛋白移码序列的鉴定

摘要

The sequence resulted from -1 ribosomal frameshifl in the NS1 coding region was amplified using three primers and cloned in triplex into a prokaryotic expression vector pET-28a. The recombinant plasmid pET3NS was transformed into E.coli BL-21 competent cells. The target proteins was expressed with IPTG induction and purified with His-Bind Purification Kit. Mice were immunized with the purified proteins and polyclonal antibodies were prepared. The polyclonal antibodies were reacted in IFA with Japanese encephalitis virus （JEV） proteins in Vero cells infected with JEV. Total proteins of Vero ceils infected with JEV were prepared. Western blot results showed that the monoclonal antibodies against NS 1 of JEV reacted with both NS 1 and NS 1′, while the polyclonal antibodies reacted only with NS 1′. These results indicated that the sequence of the expressed protein was partially identical to that of NSI＇ and NS 1′ was the product resulted from -1 ribosomal frameshift in the NS1 coding region.%本文通过对猪乙型脑炎病毒（Japanese encephalitis virus，JEV）基因组中移码序列分析，设计合成三条引物，通过PCR扩增出假定的NS1′移码片段，并将移码片段三倍重复克隆入pET-28a表达载体中。重组质粒转化E．coliBL-21后，经IPTG诱导在37℃下实现目的片段的可溶性表达。纯化表达产物，并免疫小鼠制备了多抗血清，以多抗血清为一抗，对JEv感染的Vero细胞进行免疫荧光分析，结果发现抗移码片段表达蛋白的血清可识别JEV蛋白。以JEV感染Vero细胞，提取感染细胞总蛋白，分别以NSl单抗和VAI-制备的多抗血清为一抗，进行Westernblot分析。结果显示，NS1单抗为一抗，可染出48kDa的NS1条带和56kDa的NS1′条带：而以多抗血清为一抗，仅染出56kDa的条带。上述结果显示，多抗血清识别的NS1′蛋白为NS1′移码后片段表达的蛋白。