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.
出处
《动物医学进展》
CSCD
北大核心
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)