新城疫病毒F基因的克隆表达及重组蛋白的反应原性分析

Cloning, Expression and Western blotting Analysis of Newcastle Disease Virus F Gene

关键词:  新城疫病毒  F蛋白  原核表达  反应原性

Key words:  Newcastle disease virus（NDV）  fusion protein  prokaryotic expression  reactogenicity

摘要:

以携带有新城疫病毒（NDV）融合蛋白（F）及其突变体（Fm）基因的质粒为模板，设计引物，PCR扩增产物经双酶切后分别连入原核表达载体pET-SUMO、pET-28a，构建重组质粒pET-SUMO-F、pET-28a-Fm，将重组质粒转化入宿主菌Rosetta 2感受态细胞，在IPTG诱导下表达。通过SDS-PAGE检测，NDV-F和NDV-Fm可以在原核系统中表达，分别获得相对分子量(Mr)为64.7kD和48.7kD的重组蛋白。Western blotting分析结果显示，NDV-F和NDV-Fm蛋白能被抗NDV鸡阳性血清识别，具有良好的反应原性。

Abstract:

To design primer with plasmid carrying the gene of fusion protein and its mutants of Newcastle disease virus as template, DNA fragments were amplified from recombinant plasmids carrying the target gene. After treatment with double restriction enzyme,the product of PCR was inserted into pET-SUMO and pET-28a respectively, getting two recombinant plasmids named pET-SUMO-F and pET-28a-Fm. Recombinant plasmids were transformed into E. coli Rosetta 2 then induced by IPTG. The products of recombinant plasmids were verified by SDS-PAGE, which showed that both of the two recombinant plasmids can express in the prokaryotic expression system. The relative molecular weight of recombinantproteins was 64.7kD and 48.7kD respectively. And Western blotting results revealed that both of the recombinant proteins with good reactogenicity can be recognized by chicken positive serum.