坦布苏病毒囊膜蛋白(E蛋白)的截短表达及其抗体间接ELISA方法的建立与应用

Truncated expression of Tembusu virus envelope protein(E protein)and establishment and application of an indirect ELISA method of its antibody

为了大量表达坦布苏病毒囊膜蛋白(E蛋白)并建立一种可检测坦布苏病毒抗体的间接ELISA方法,试验首先将去除跨膜域的截短坦布苏病毒E基因克隆至原核表达载体pET-28a(+)上并转化至大肠杆菌BL21(DE3)感受态细胞中,用IPTG诱导表达重组蛋白,利用His标签对表达的重组蛋白进行纯化;然后以重组蛋白为包被抗原、鸭待测血清为一抗、HRP标记兔抗鸭IgG为二抗,通过反应条件[抗原包被质量浓度、抗原包被条件、封闭液、封闭时间、一抗(待测血清样品)稀释液、一抗(待测血清样品)稀释度、一抗(待测血清样品)孵育条件、二抗稀释度、二抗孵育条件和显色条件]的优化和临界值的确定建立间接ELISA方法;最后进行特异性、敏感性和重复性试验,并通过临床样品检测比较该方法与Western-blot方法的符合性。结果表明:PCR扩增得到大小为1227 bp的坦布苏病毒截短E基因,经双酶切与测序验证后,成功构建重组表达质粒pET28a-E-tr。诱导后的重组蛋白分子量大小为49 ku并以包涵体的形式表达,纯化后的重组蛋白条带单一,可与鸭源坦布苏病毒阳性血清发生特异性结合。建立的ELISA方法优化后的抗原包被质量浓度为5μg/mL,抗原包被条件为4℃过夜,封闭液为2%BSA,封闭时间为60 min,一抗(待测血清样品)稀释液为1%BSA,一抗(待测血清样品)稀释度为1∶800,一抗(待测血清样品)孵育条件为37℃、30 min,二抗稀释度为1∶7500,二抗孵育条件为37℃、90 min,显色条件为37℃、10 min,阴阳临界值为0.368;仅可检测出鸭源坦布苏病毒阳性血清,无法检测出鸭源鸭疫里默氏杆菌、新城疫病毒、禽腺病毒、禽流感病毒、细小病毒阳性血清;待测血清最高稀释度为1∶3200(Western-blot方法待测血清最高稀释度仅为1∶1600);批次内重复变异系数(CV)为1.7%~4.7%,批次间重复CV为1.4%~5.7%;对137份临床鸭血清样品进行检测,阳性率为81.7%,与Western-blot方法比较样品阳性率较高,两种方法的符合率为87.6%。说明成功表达了坦布苏病毒截短E蛋白,并建立了一种特异性良好、敏感性较高、重复性较好的可检测坦布苏病毒抗体的间接ELISA方法。

In order to express Tembusu virus envelope protein(E protein)in large quantities and to establish an indirect ELISA method that could detect duck Tembusu virus antibody,in the experiment,the truncated duck Tembusu virus E gene with the transmembrane domain removed was firstly cloned into the prokaryotic expression vector pET-28a(+)and transformed into E.coli BL21(DE3)competent cells.IPTG was used to induce the expression of recombinant protein,and the expressed recombinant protein was purified using His tag;then the recombinant protein was used as the encapsulated antigen,duck serum to be tested as the primary antibody,and HRP-labeled rabbit anti-duck IgG as the secondary antibody.The indirect ELISA method was established through the optimization of reaction conditions(antigen encapsulation mass concentration,antigen encapsulation conditions,closure solution,closure time,primary antibody[serum sample to be tested]dilution,primary antibody[serum sample to be tested]dilution,primary antibody[serum sample to be tested]incubation conditions,secondary antibody dilution,secondary antibody incubation conditions,and color development conditions)and the determination of critical values.Finally,the specificity,sensitivity and reproducibility tests were performed,and the conformity of the method with the Western-blot method was compared by clinical sample testing.The results showed that PCR amplification obtained 1227 bp Tembusu virus truncated E gene;the recombinant expression plasmid pET28a-E-tr was successfully constructed after double enzyme digestion and sequencing verification.The molecular weight of the induced recombinant protein was 49 ku and was expressed as inclusion body,and the purified recombinant protein had a single band,which could specifically bind to the positive serum of duck Tembusu virus.The established ELISA method was optimized with the antigen-coated mass concentration of 5μg/mL;the antigen-coated condition was overnight at 4℃;the sealing solution was phosphate buffer containing 2%BSA;and the sealing time was 60 min;the primary antibody(serum sample to be tested)dilution was phosphate buffer containing 1%BSA,the primary antibody(serum sample to be tested)dilution was 1∶800,and the primary antibody(serum sample to be tested)incubation condition was 37℃and 30 min;the secondary antibody dilution was 1∶7500;the secondary antibody incubation condition was 37℃and 90 min.The critical value of negative to positive was 0.368;only positive sera of duck Tembusu virus could be detected,but not positive sera of Riemerella anatipestifer,Newcastle disease virus,Fowl aviadenovirus,avian Influenza A virus,and Parvovirus.The highest dilution of serum to be tested was 1∶3200(the highest dilution of serum to be tested by Western-blot method was only 1∶1600);the coefficient of variation(CV)values of intra-batch duplicates ranged from 1.7%to 4.7%,and the CV values of inter-batch duplicates ranged from 1.4%to 5.7%.Testing of 137 clinical duck serum samples showed a high sample positivity rate(81.7%)compared with the Western-blot method,and the compliance rate between the two methods was 87.6%.The results indicated that the Tembusu virus truncated E protein was successfully expressed,and an indirect ELISA method with good specificity,high sensitivity and reproducibility was established to detect duck Tembusu virus antibody.