山羊痘病毒疫苗株 ORF103基因的克隆、 序列分析及原核表达

根据GenBank已登录的GTPV基因组序列,设计并合成1对特异性引物,以GTPV疫苗株基因组DNA为模板,PCR扩增获得ORF103基因片段并进行序列分析,再将该基因片段亚克隆于原核表达载体pET-28a中,构建重组质粒pET-ORF103,经鉴定正确后转化BL21感受态细胞进行诱导表达,并进行SDSPAGE和Western blot检测。成功克隆GTPV疫苗株ORF103基因片段。重组菌经IPTG诱导后成功表达分子质量约为35ku的重组蛋白,该蛋白能与山羊痘阳性血清特异性结合,具有良好的反应原性。研究结果为GTPV的分子生物学特性研究提供资料,并为进一步研制GTPV抗体检测试剂盒奠定基础。

Cloning, Sequence Analysis and Prokaryotic Expression of ORF103 Gene of Goatpox Virus Vaccine Strain

According to the published sequence of ORF103 gene of GTPV, a pair of specific primers were designed and synthesized. The ORF103 gene was amplified by PCR from goatpox virus vaccine strain. ORF103 gene sequence was analyzed and cloned into pET-28a vector. The prokaryotic expression plasmid pET- ORF103 containing ORF103 gene was successfully constructed. The expression of recombinant plasmid pET- ORF103 in BL21 was induced and detected by SDS-PACE and Western blot analysis. ORF103 gene was successfully obtained. SDS-PAGE analysis showed that the fusion protein had molecular weight of about 35 ku by IPTG induction. Western blot analysis showed that the fusion protein had well reactiongenicity. These results provide basic information of ORF103 gene and protein in goatpox virus vaccine strain. Further more, the ORF103 protein can be used to prepare GTPV antibody detection kit.