摘要
为建立快速检测鸭坦布苏病毒病抗体的血清学方法,本研究利用鹅坦布苏病毒JS804株非结构蛋白NS1基因序列将其克隆至原核表达载体pET32a上,建立重组表达pET32a-NS1,转化至BL21(DE3)中。经IPTG诱导得到NS1融合蛋白,融合蛋白均以包涵体形式存在,包涵体经过变性和复性,纯化的坦布苏病毒NS1蛋白作为包被抗原,在最佳检测条件基础上,建立了检测坦布苏病毒病(Tembusu virus disease,简称TVD)抗体的间接ELISA方法。结果在条件优化后抗原最适包被量为1.925μg·孔-1,抗原最佳包被条件为37℃放置1 h后4℃过夜,血清的最佳稀释度为1∶200,最佳封闭时间为1 h,酶标二抗最适稀释度为1∶1 000,显色时间10 min。阴阳性临界值判定标准为0.346。用建立的间接ELISA方法检测禽流感、新城疫、传染性支气管炎、TVD阳性血清样品均无交叉反应,表明该方法具有良好的特异性。板内和板间重复试验的最大变异系数分别为6.7%和8.2%,显示该方法具有很好的稳定性。用间接NS1-ELISA方法对81份疑似鸭坦布苏病毒病血清样品进行检测,有43份样品呈现阳性,E-ELISA有48份呈阳性结果,证明该方法具有较高的敏感性。NS1-ELISA方法的建立为TVB的诊断和流行病学调查提供了一种新的方法。
In order to establish a NS1-ELISA for rapid detection of antibodies against duck Tembusu virus (DTV), a series of studies were conducted .Firstly, the non-structural protein NS1 gene of goose Tembusu virus JS 804 strain was inserted into the prokaryotic vector pET 32a, then pET32a-NS1 were transformed into Escherichia coli BL12 (DE3)and the combination protein NS1 (His-NS1) was obtained with the induction of IPTG .The fusion protein was purified by extracting the inclusion bodies with urea , and the purified protein was used as coated antigen .Based on the best working condition , the indirect ELISA method ( NS1-ELISA) to detect the antibody of duck TVD was devel-oped.The optimized working condition included: the coating antigen of purified DTV-NS1 protein was 1.925μg·well^ -1 , the best package condition was 37℃for 1 h and then over night at 4℃, the best dilution of testing serum was 1∶200, the best closure time was 1 h, the dilution of HRP conjugated anti-duck lgG was 1∶1 000,the chromoge-nic time was 10 min, and the cut off-value judging negative or positive was 0.346(D450).The specific tests showed that there were no cross-reaction to the anti-sera against avian influenza virus , Newcastle disease virus , infectious bronchitis virus and DTV , which indicated that the NS 1-ELISA was specific to anti-sera against DTV .The intra-plate and inter-plate demonstrated that the coefficient of maximum variation was 6.7%and 8.2% respectively , which showed the method had good stability .A total of 81 samples from affected ducks were tested and 43 samples were positive detected by the NS1-ELISA, While 48 samples showed positive detected by the E-ELISA, which showed the method had a high sensitivity .The results revealed the NS1-ELISA could be used for laboratory diagnosis and sera-survey for duck Tembusu virus infection .
出处
《浙江农业学报》
CSCD
北大核心
2014年第1期135-140,共6页
Acta Agriculturae Zhejiangensis
基金
国家自然科学基金(31172345)
江苏省自然科学基金(BK2012376)
江苏省农业科技自主创新资金项目(CX(12)5052)