禽呼肠病毒σC基因的表达及ELISA抗体检测方法的建立  

Development of ELISA for Antibodies Against Avian Reovirus Using σC Proteins Expressed in Escherichia coli

作　　者：董嘉文[1] 孙敏华[1] 李林林[1] 胡奇林[1] 

机构地区：[1]广东省农业科学院兽医研究所,广东省兽医公共卫生公共实验室,广东广州510640

出　　处：《动物医学进展》2012年第8期11-16,共6页Progress In Veterinary Medicine

基　　金：广东省重大科技项目(2009B020201008);广东省产学研结合项目(2010A09020029-10)

摘　　要：将RT-PCR扩增得到的禽呼肠病毒(ARV)S1133毒株的σC基因克隆至pMD-18T-Simple载体,经酶切及测序鉴定,证明该基因片段为ARVσC基因。将该基因亚克隆至pET32a(+)成功构建了表达载体pET32a-σC。该重组质粒在大肠埃希菌BL21中经1.0mmol/L IPTG诱导5h得到最佳表达。pET32a-σC蛋白的分子质量为55ku,Western blot分析表明,该重组蛋白能与ARV阳性血清发生特异性反应,表明其具有良好的反应原性。将纯化好的ARVσC蛋白作为包被抗原,确定了该方法的抗原最适包被浓度为2μg/mL,血清的最适稀释度为1∶400。用建立的ELISA方法检测接种过ARV疫苗的65份临床血清样品,测得44份为阳性,阳性率为67.7%;未接种ARV疫苗的30份血清样品检测结果均为阴性。TheσC gene of ARV S1133 was designed and amplified ,which was cloned into the vector pMD- 18T-Simple. The plasmid was identified by endonucleases digestion and sequenced. The result revealed that the insert gene fragment was the σC gene of ARV. Then the gene was inserted into the pET32a （＋） and indicated that fusion expression vector pET32a-σC was constructed. The recombinant fusion protein was highly expressed in E. coli BL21 induced by 1. 0 retool/ L IPTG for 5 hours in the form of inclusion bodies. The molecular weight of recombinant fusion protein is 55 ku. Western blot showed that the recom- binant protein has a favourable reactivity against ARV antibodies. The structural protein σC was used as coating antigen to establish a indirect enzyme-linked immunosorbnent assay （ELISA）. The results showed that the optimal coating concentration was 2.0 g/mL, the optimal dilution of primary antiserum was 1 ： 400. 65 clinical antiserum samples from vaccinated chickens and 30 samples from non- vaccinated chickens were detected by the developed ELISA. The results revealed that the correlation rates of positive and nega- tive were 67.7% and 100% respectively.

关 键 词：禽呼肠病毒 σC基因 克隆与表达 酶联免疫吸附试验 

分 类 号：S852.659.4[农业科学—基础兽医学] Q786[农业科学—兽医学]