禽网状内皮组织增生症病毒CA蛋白的表达纯化及多克隆抗体制备  

Expression and purification of reticuloendotheliosis virus CA protein and preparation of polyclonal antibody

作　　者：姜莉莉 王立成[1] 陈强 樊兆斌 JIANG Lili WANG Licheng CHEN Qiang FAN Zhaobin(Institute of Animal Husbandry and Veterinary, Jinzhou Medical University, Jinzhou 121001, China Drug Bioscience and Technology Department, Heze University, Heze 274015, China)

机构地区：[1]锦州医科大学畜牧兽医学院,辽宁锦州121001 [2]菏泽学院药物科学与技术系,山东菏泽274015

出　　处：《畜牧与兽医》2017年第9期67-70,共4页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(31402170);辽宁省自然科学基金项目(2014022045);菏泽学院博士基金(XY16BS09)

摘　　要：根据禽网状内皮组织增生症病毒(REV)DBYR1102株的前病毒基因组c DNA序列设计并合成1对引物,以p T-G质粒为模板,通过PCR扩增获得该病毒CA基因片段。将其克隆于pET-30a(+)中,构建原核表达质粒pET30a-CA。重组质粒经双酶切和测序鉴定,转化BL21(DE3),经IPTG诱导,SDS-PAGE鉴定蛋白表达情况。利用Ni-NTA His Bind Resin纯化重组蛋白,并经蔗糖浓缩,Western blot法检测重组蛋白的反应原性。纯化后的目的蛋白免疫新西兰白兔,制备兔抗CA多克隆抗体并对其进行鉴定。结果表明构建的重组质粒pET-30a-CA在大肠杆菌BL21中呈可溶性高效表达。经Ni柱纯化和蔗糖浓缩后得到纯度较高的融合蛋白,以纯化的融合蛋白免疫新西兰白兔制备多克隆抗体,抗体效价达1∶25 600。该研究为REV的检测及CA基因的深入研究奠定了基础。To express the CA protein of the reticuloendotheliosis virus（ REV）,the CA gene of REV DBYR1102 strain was cloned into prokaryotic expression vector pET-30a（ ＋）,and recombinant plasmid pET30a-CA was constructed. After double enzyme digestion and sequencing,the pET30a-CA was transformed into the Escherichia coli BL21（ DE3）. The transformants were induced by isopropylthio-β-Dgalactoside（ IPTG）,and SDS-PAGE was used to identify the expression of the CA protein. The His-tagged fusion protein REV-CA was ultrasonically treated and then purified through Ni-chelating affinity chromatography and sugar concentration. The purified CA was used to immune into New Zealand rabbit for preparing anti-CA polyclonal serum. The results showed that the recombinant plasmids pET30a-CA was successfully constructed,and the CA protein was expressed in soluble form. Polyclonal antibody was prepared and purified from immunized New Zealand white rabbit serum with the titer of 1 ∶ 25 600. This study laid the foundation for the development of the diagnostic methods and subunit vaccine for REV.

关 键 词：禽网状内皮组织增生症病毒 CA 原核表达 抗体 

分 类 号：S855.3[农业科学—临床兽医学]