山羊痘病毒059蛋白表达及鉴定

Expression and Identification of Goatpox Virus 059 Protein

为研究山羊痘病毒的059蛋白,本研究用PCR技术扩增山羊痘病毒THX株059基因,并构建了融合表达His标签的原核表达载体,测序鉴定并分析了059基因的核苷酸序列特征和推测的氨基酸序列结构特征,将重组质粒转化大肠杆菌BL21,经IPTG诱导表达,用SDS-PAGE和Western-blot鉴定其蛋白表达情况。结果成功扩增并构建了山羊痘病毒059基因的原核表达载体PET-42b-059,THX株059基因全长为765 bp,与参考株的相似性高达99. 34%,与山羊痘病毒在亲缘关系上较近,而与疙瘩皮肤病病毒与绵羊痘病毒的亲缘关系较远。059蛋白会长255aa,由多个α螺旋和β片层交错构成,分子量大小为29 KDa,SDS-PAGE结果显示其以包涵体的形式表达,大小与理论相符,Western Blot结果证明059蛋白与His标签进行了融合表达。结果表明059蛋白可以较好原核表达,为进一步研究该蛋白的其他生物学特性奠定了基础。

To study protein 059 of goat pox virus, the 059 gene of goat pox virus THX strain was amplified by PCR, and its prokaryotic expression vector that fused expression His tag was constructed, and the target gene was identified by sequencing and the characteristics of the nucleotide sequence and amino acid structure were analyzed. Recombinant plasmids were transformed into E. coli BL21 and induced by IPTG, and the protein was identified using SDS-PAGE and Western blot. As results, the goat pox virus 059 gene and its prokaryotic expression vector (pET42b -059) were amplified and constructed successfully, and the 059 gene of THX strain was 765 bp and its homology was 99. 34% compared with reference strains. It is closely related to goat pox virus, but far related to pimple skin disease virus and sheep pox virus. 059 protein is composed of 255aa, which is composed of several alpha helix and beta lamellae. The molecular weight of 059 protein is 29 KDa. SDS-PAGE results show that it is expressed in the form of inclusion bodies. The size of 059 protein is consistent with the theory. Western Blot results show that 059 protein is fused with His tag. The results showed that 059 protein could be expressed in prokaryotic cells, which laid a foundation for further study of other biological characteristics of 059 protein.