牛乳头状瘤病毒13型L1基因的克隆及原核表达  

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

作　　者：赵天靖 贾晓晓[1] 史巧芸[1] 郭莳雨[1] 庞峰[1] 朱华培[1] 徐开莲 李亚颖 彭冬梅[1] 李国华[1] 王凤阳[1] 

机构地区：[1]海南大学农学院,海南省热带动物繁育与疫病研究重点实验室,海口市动物基因工程重点实验室,海口570228

出　　处：《中国畜牧兽医》2015年第9期2286-2291,共6页China Animal Husbandry & Veterinary Medicine

基　　金：海南大学中西部计划项目(ZXBJH-XK002)

摘　　要：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 494bp的目的片段,同时用BamHⅠ和HindⅢ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60ku,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。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 L1gene fragment was amplified by PCR.After digestion by BamHⅠand HindⅢ,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.

关 键 词：牛乳头状瘤病毒13型(BPV13) L1 克隆 原核表达 

分 类 号：Q78[生物学—分子生物学]