新城疫病毒F48E9株F1基因的克隆及其在大肠杆菌中的表达

Cloning of F1 Gene of Newcastle Disease Virus F48E9 Strain and Its Expression in Escherichia coli

根据GenBank中新城疫病毒(NDV)F48E9株F1基因序列和原核表达载体pET-28a的克隆位点,设计1对引物,通过PCR方法获得了F1基因;将F1双酶切产物插入原核表达载体pET-28a,得到的重组子命名为pET-F1。用IPTG进行诱导将其在大肠杆菌Rosetta(DE3)中进行了表达,收集菌液进行SDS-PAGE和Western-blot分析,结果表明,NDV F1在pET-F1中获得了高效融合表达,其表达蛋白的分子质量约为31 ku。表达的重组蛋白为进一步研究NDV的免疫特性和分子生物学功能奠定了基础。

According to the encoding sequence of the NDV F48E9 strain F1 protein and the multiple cloning sites characteristic of prokaryotic expression vector pET-28a in GenBank, appropriate primers specific to NDV F1 gene were designed. The target DNA fragments were obtained by PCR amplification. Then the F1 gene was subcloned into a prokaryotic expression vector pET-28a and the recombinant was designated as pET-F1. The target protein was expressed in E. coli Rosetta （DE3） induced with IPTG. The results of SDS-PAGE and Western-blotting indicated that the F1 gene was expressed from pET-F1 induced by IPTG at high level, and the expressed fusion protein was 31ku approximately in molecular mass. The result provided fundamental bases and materials for the further investigation on immunogenicity and molecular biological function of F1 of NDV.