牛乳头状瘤病毒13型L1基因的克隆及原核表达

本研究旨在克隆并表达牛乳头状瘤病毒13型(BPV13)L1基因。以BPV13基因组为模板,通过PCR技术扩增得到大小1 494 bp的目的片段,同时用BamHⅠ和Hind Ⅲ分别对目的片段和pET28a(+)载体进行双酶切,将双酶切后的L1基因片段克隆至原核表达载体pET28a(+),构建pET28a-L1重组质粒,双酶切和测序鉴定正确后转入大肠杆菌BL21(DE3)受体菌中,筛选出最佳IPTG浓度和最佳诱导时间后进行诱导表达,进行SDS-PAGE和Western blotting检测。结果表明,L1基因正确插入到原核表达载体pET28a(+)中;IPTG诱导后含重组质粒pET28a-L1的表达菌成功表达了带His标签的融合蛋白;SDS-PAGE电泳结果显示融合蛋白分子质量为60 ku,与预期大小一致,超声破菌后,SDS-PAGE电泳显示融合蛋白存在于沉淀中;Western blotting验证为带His标签的融合蛋白。本试验为进一步研究BPV13 L1基因的功能及为BPV13有效DNA疫苗的研制奠定基础。

Cloning and Prokaryotic Expression of L1 Gene of Bovine Papillomavirus Genotype 13

This experiment was conducted to clone and express L1 gene of bovine papillomavirus genotype 13 (BPV13).Specific primers were designed according to the published sequences of BPV13, and 1 494 bp L1 gene fragment was amplified by PCR.After digestion by BamHⅠand Hind Ⅲ, the fragment was inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant plasmid pET28a-L1.The recombinant plasmid pET28a-L1 was identified by double enzyme digestion and nucleotide sequencing, and was transformed into E.coli BL21(DE3).The expression of pET28a-L1 was induced by IPTG after selecting the best concentration and time.The results showed that L1 gene was exactly inserted into the prokaryotic expression vector pET28a(+), after induction, the target protein containing His-tag was successfully expressed by expression bacteria which included recombinant plasmid pET28a-L1;SDS-PAGE result showed that the molecular mass of the protein was 60 ku which was consistent with the expected size;After ultrasonic treatment, SDS-PAGE result showed that the protein existed in the precipitation;The target protein was a fusion protein with His-tag verified by Western blotting.This study laid a solid foundation for the research of function of BPV13 L1 gene and the development of effective DNA vaccine of BPV13.