非洲猪瘟病毒P30蛋白的表达及抗体液相芯片检测方法的建立

Expression of ASFV P30 Protein and Development of ASFV Antibody Detection Method Based on x-MAP Technology

本研究旨在为建立高效、快速、准确检测非洲猪瘟病毒(African swine fever virus,ASFV)的抗体液相芯片检测方法。首先构建pET-28a-p30表达载体,利用大肠杆菌(Escherichia coli)原核表达系统表达ASFV重组P30蛋白,然后将重组P30蛋白与羧基化的荧光微球偶联,建立ASFV P30抗体液相芯片检测方法。经鉴定,重组质粒成功转入大肠杆菌BL21感受态细胞,并以包涵体形式表达,Western blot结果显示重组P30蛋白可以与ASFV阳性血清产生特异性反应,证明其具有良好的反应原性。建立的ASFV抗体液相芯片检测方法检测中值荧光强度(median fluorescent intensity,MFI)阈值为1575.7,最佳结合蛋白浓度为每1.25×10^(6)个微球6μg,最佳血清稀释度为1∶600,最佳二抗浓度为1μg·mL^(-1),该方法具有良好的特异性、灵敏性和重复性。用建立的方法和商品化ELISA试剂盒同时对92份临床采集的猪血清样本进行检测,结果显示二者符合率为92.39%。本研究成功表达重组P30蛋白,并建立了ASFV P30抗体液相芯片检测方法,为ASFV感染早期的血清学检测提供了新的方法,也为多种病原体的抗体检测奠定了基础。

This study was conducted to develop a rapid and accurate method for detection of African swine fever virus(ASFV)antibody based on x-MAP technology.In this our study,pET-28a-p30 expression plasmid was constructed,and ASFV recombinant P30 protein was expressed in Escherichia coli.Then the recombinant P30 protein was coupled with carboxylated fluorescent microspheres and an x-MAP multiple bead-based technology was developed for detection of ASFV P30 antibody.The recombinant plasmid was successfully expressed in inclusion body form in Escherichia coli BL21.Western blot ting analysis showed that recombinant P30 protein could react specifically with ASFV positive serum,indicating that it had good reactivity.The detection threshold of the x-MAP based ASFV antibody detection method is MFI 1575.7,and the optimal binding protein concentration is 6μg per 1.25×10^(6) microspheres,the optimal serum dilution is 1:600,and the optimal concentration of secondary antibody is 1μg·mL^(-1),the method has good specificity,sensitivity and repeatability.Ninety-two clinical pig serum samples were tested by x-MAP analysis and compared with the result tested by a commercial ELISA kit.The results showed that the consistency was 92.39%.The recombinant P30 protein was successfully expressed,and the ASFV antibody detection method based on x-MAP technology was established,which provides a new method for the early serological detection of ASFV infection.