牛单纯疱疹病毒Ⅰ型gB基因原核表达载体的构建及蛋白表达

2019-04-15 01:46:18 安徽农学通报2019年4期

张海威 翟璐 王丽姿 黄艳梅 涂伟 徐乐 宋佰芬

摘  要:目的:獲取牛单纯疱疹病毒Ⅰ型gB基因编码的蛋白。方法:根据GenBank中牛疱疹病毒Ⅰ型 VRL 27-FEB-2012株囊膜糖蛋白B(UL27)基因,设计并合成一对特异性引物,利用 PCR 方法扩增gB基因序列的第100~1197bp,将此目的片段其命名为1F8基因,随后将PCR扩增后的1F8基因克隆到pMD18-T载体上,重组质粒命名为pMD18-T-1F8;对构建的重组克隆质粒进行PCR、双酶切和测序鉴定正确后,将目的基因亚克隆到 pET-32a(+)原核表达载体上,并转化到BL21感受态细胞中,获得的重组表达质粒pET-32a(+)-1F8,再通过双酶切和测序进行鉴定;鉴定正确后,在37℃条件下用终浓度为0.1mM IPTG诱导重组菌4h,取诱导后产物进行 SDS-PAGE 分析和Western Blot 鉴定。结果:通过琼脂糖凝胶电泳实验结果表明,在1097bp处出现了目的条带;对pMD18-T-1F8重组质粒的PCR、酶切及测序结果表明,扩增的目的条带为牛疱疹病毒Ⅰ型gB基因序列;通过SDS-PAGE和Western Blot实验对表达蛋白的鉴定结果显示,在58.6kDa左右出现目的条带,与软件预测蛋白的大小相符,确定为牛疱疹病毒Ⅰ型gB蛋白。结论:研究成功构建了牛单纯疱疹病毒Ⅰ型gB基因部分片段1F8的重组表达质粒pET-32a(+)-1F8,并成功表达1F8蛋白,为后续牛单纯疱疹病毒Ⅰ型单克隆抗体的制备和检测方法的建立奠定了基础。

关键词:牛单纯疱疹病毒Ⅰ型;gB基因;原核表达

中图分类号 R373.11文献标识码 A文章编号 1007-7731(2019)04-0008-05

Abstract:Objective:To obtain the protein encoded by bovine herpes simplex virus type Ⅰ gB gene.Methods:According to the envelope protein glycoprotein B(UL27)gene of bovine herpesvirus type Ⅰ VRL 27-FEB-2012 in GenBank,a pair of specific primers was designed and synthesized,and the 100th bp to 1197th of gB gene sequence was amplified.The amplified partial gene fragment was named as the 1F8,and then the amplified 1F8 gene was cloned into the pMD18-T vector and recombinant plasmid was named as pMD18-T-1F8,pMD18-T-1F8 plasmids were identified by PCR and double enzyme digestion and sequencing.Positive cloning was inserted into pET-32a(+)expression vector and transferred into E.coli BL21 competent cells;The recombinant expression plasmid pET-32a(+)-1F8 was obtained,and then identified by double enzyme digestion and sequencing;the positive recombinant strain was induced by 0.1mM IPTG for 4 hours at 37℃.The induced product was subjected to SDS-PAGE and western blot analysis.Results:The result of agarose gel electrophoresis showed that the target band appeared around 1097bp,which was in accordance with the expectation.The results of PCR,enzyme digestion and sequencing of pMD18-T-1F8 recombinant plasmid showed amplified bovine herpes virus type ⅠgB gene sequences was correct.The results of SDS-PAGE and Western blot showed the size of target bands was about 58.6kD,which was in accordance with the expectation.These results confirmed expression protein was bovine herpes simplex virus type Ⅰ gB protein.Conclusion:This study successfully constructed the recombinant expression plasmid pET-32a(+)-1F8 of bovine herpes simplex virus type ⅠgB gene fragment 1F8,and successfully expressed and purified 1F8 protein,which is a follow-up method of herpes simplex virus Ⅰ.The establishment of the foundation laid the foundation.


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