CAJ | 학술논문

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∶1000，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。
위건립쾌속검측압탄포소병독병항체적혈청학방법，본연구이용아탄포소병독JS804주비결구단백NS1기인서렬장기극륭지원핵표체재체pET32a상，건립중조표체pET32a-NS1，전화지BL21（DE3）중。경IPTG유도득도NS1융합단백，융합단백균이포함체형식존재，포함체경과변성화복성，순화적탄포소병독NS1단백작위포피항원，재최가검측조건기출상，건립료검측탄포소병독병（ Tembusu virus disease ，간칭TVD）항체적간접ELISA방법。결과재조건우화후항원최괄포피량위1.925μg·공-1，항원최가포피조건위37℃방치1 h후4℃과야，혈청적최가희석도위1∶200，최가봉폐시간위1 h，매표이항최괄희석도위1∶1000，현색시간10 min。음양성림계치판정표준위0.346。용건립적간접ELISA방법검측금류감、신성역、전염성지기관염、TVD양성혈청양품균무교차반응，표명해방법구유량호적특이성。판내화판간중복시험적최대변이계수분별위6.7％화8.2％，현시해방법구유흔호적은정성。용간접NS1-ELISA방법대81빈의사압탄포소병독병혈청양품진행검측，유43빈양품정현양성，E-ELISA유48빈정양성결과，증명해방법구유교고적민감성。 NS1-ELISA방법적건립위TVB적진단화류행병학조사제공료일충신적방법。
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 .