科研动态

为建立一种检测非洲猪瘟病毒（ASFV）抗体的间接ELISA（iELISA）方法，对构建的ASFV P30基因表达工程菌诱导表达后，将获取的重组ASFV P30蛋白进行纯化和Western-blot检测，然后以纯化的重组蛋白为抗原，建立了ASFV抗体iELISA检测方法，并进行了特异性、灵敏性、重复性试验；同时与基于ASFV P30-2His6蛋白（N、C末端各融合1个His6标签）的iELISA方法进行兽医临床样本比较试验。结果显示：重组ASFV P30蛋白和重组ASFV P30-2His6蛋白在Western-blot检测中，均能与猪ASFV阳性血清产生特异性杂交带；基于重组ASFV P30蛋白iELISA的最佳反应条件为，抗原蛋白包被质量浓度20 μg/mL、血清样品稀释度1:1 000、酶标二抗稀释度1:40 000、血清样品检测OD450阳性结果临界值0.22。该方法仅对ASFV阳性血清呈特异性反应，1:3 200稀释的阳性血清仍可检出，批内试验和批间试验变异系数均小于10%，可以消除His标签所造成的假阳性反应。本研究建立的ASFV P30 iELISA检测方法为ASFV抗体检测提供了一种有效手段。

Establishment and Application of the iELISA for Detection of Antibodies against ASFV

In order to establish an indirect enzyme-linked immunosorbent assay（iELISA）for detecting antibodies against African swine fever virus（ASFV），the gene expression engineering strain of recombinant ASFV P30 was induced and expressed，followed by purification and Western-blot detection of the recombinant proteins，then the iELISA method was established by taking the purified recombinant proteins as antigens，its specificity，sensitivity and repeatability were subsequently evaluated. Meanwhile，it was compared with the iELISA based on ASFV P30-2His6 protein（one His6 tag was fused respectively at N and C terminals）for veterinary clinical samples. The results showed that，by Western-blot detection，both the recombinant proteins of ASFV P30 and ASFV P30-2His6 could produce specific hybrid band with ASFV positive serum；the optimal reaction conditions of iELISA based on the recombinant ASFV P30 proteins were 20 μg/mL mass concentration of antigen protein coating，1:1 000 dilution of serum samples，1:40 000 dilution of goat anti-swine IgG conjugate，and 0.22 critical value of positive serum samples OD450. The established method was specific only to ASFV positive serum，1:3 200 diluted positive serum could still be detected，and the coefficients of variation（CV）of intra assay and inter assay were both less than 10%，which could eliminate any false positive reaction caused by His tag. Therefore，an effective tool was provided for detecting antibodies against ASFV by the established ASFV P30 iELISA in the study.

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