纹皮蝇Hypodermin C基因原核表达及间接ELISA检测方法的建立  

Prokaryotic expression of Hypodermin C gene of Hypoderma lineatum and establishment of indirect ELISA

作　　者：王文龙[1] 刘春霞[2] 阮风雷[1] 呼和巴特尔[2] 

机构地区：[1]内蒙古农业大学兽医学院,呼和浩特010018 [2]内蒙古农业大学生命科学学院,呼和浩特010018

出　　处：《中国预防兽医学报》2011年第11期877-881,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：教育部"春晖计划"合作项目(Z2007-1-01011;Z2007-1-01018);国家自然科学基金(30360082)

摘　　要：为建立牛皮蝇蛆病的抗体检测方法,本研究从纹皮蝇Ⅰ期幼虫中提取总RNA,采用RT-PCR扩增了Hypodermin C(HC)基因。将该基因片段插入到原核表达载体pET-30a(+)中进行原核表达。获得了31 ku以包涵体形式表达的重组HC蛋白。Western blot结果表明表达产物能够与阳性牛血清IgG特异性结合,具有良好的抗原活性。以纯化的重组HC作为包被抗原建立了牛皮蝇蛆病间接ELISA诊断方法,实验确定抗原最佳包被浓度为15.63μg/mL,血清最佳稀释度为1∶80,阳性标准判定为待检血清OD值>0.256。所建立的诊断方法具有较好的特异性、敏感性和可重复性。应用该检测方法对230份临床血清进行检测,阳性率为20.6%。研究结果表明该间接ELISA方法是一种有效的牛皮蝇蛆病血清学检测方法。To establish the method for detecting Hypoderma lineatum,the Hypodermin C（HC） gene of H.lineatum was amplified by RT-PCR using specific primers designed according to the sequence of HC gene in GenBank,cloned into pET-30a（＋） and expressed in E.coli BL21（DE3）.Western blot analysis showed that the recombinant protein reacted specifically with H.lineatum larvae positive serum.An indirect ELISA was developed to detect H.lineatum larvae antibody using the purified recombinant HC protein as coating antigen.The assay was specific,sensitive and reproducible,which could be used as a rapid serology detection method for monitoring anti-H.lineatum larvae antibody and epidemiologic survey of Hypodermosis.

关 键 词：纹皮蝇 HYPODERMIN C 原核表达 间接ELISA 

分 类 号：S852.7[农业科学—基础兽医学]