牛單純皰疹病毒Ⅰ型gB基因原核表達載體的構(gòu)建及蛋白表達

張海威 翟璐 王麗姿 黃艷梅 涂偉 徐樂 宋佰芬

摘? 要：目的：獲取牛單純皰疹病毒Ⅰ型gB基因編碼的蛋白。方法：根據(jù)GenBank中牛皰疹病毒Ⅰ型 VRL 27-FEB-2012株囊膜糖蛋白B（UL27）基因，設(shè)計并合成一對特異性引物，利用 PCR 方法擴增gB基因序列的第100～1197bp，將此目的片段其命名為1F8基因，隨后將PCR擴增后的1F8基因克隆到pMD18-T載體上，重組質(zhì)粒命名為pMD18-T-1F8;對構(gòu)建的重組克隆質(zhì)粒進行PCR、雙酶切和測序鑒定正確后，將目的基因亞克隆到 pET-32a（+）原核表達載體上，并轉(zhuǎn)化到BL21感受態(tài)細胞中，獲得的重組表達質(zhì)粒pET-32a（+）-1F8，再通過雙酶切和測序進行鑒定;鑒定正確后，在37℃條件下用終濃度為0.1mM IPTG誘導(dǎo)重組菌4h，取誘導(dǎo)后產(chǎn)物進行 SDS-PAGE 分析和Western Blot 鑒定。結(jié)果：通過瓊脂糖凝膠電泳實驗結(jié)果表明，在1097bp處出現(xiàn)了目的條帶;對pMD18-T-1F8重組質(zhì)粒的PCR、酶切及測序結(jié)果表明，擴增的目的條帶為牛皰疹病毒Ⅰ型gB基因序列;通過SDS-PAGE和Western Blot實驗對表達蛋白的鑒定結(jié)果顯示，在58.6kDa左右出現(xiàn)目的條帶，與軟件預(yù)測蛋白的大小相符，確定為牛皰疹病毒Ⅰ型gB蛋白。結(jié)論：研究成功構(gòu)建了牛單純皰疹病毒Ⅰ型gB基因部分片段1F8的重組表達質(zhì)粒pET-32a（+）-1F8，并成功表達1F8蛋白，為后續(xù)牛單純皰疹病毒Ⅰ型單克隆抗體的制備和檢測方法的建立奠定了基礎(chǔ)。

關(guān)鍵詞：牛單純皰疹病毒Ⅰ型;gB基因;原核表達

中圖分類號 R373.11文獻標識碼 A文章編號 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.