绵羊痘病毒A11R基因的优化表达及抗体间接ELISA方法的建立

Optimization expression of A11R gene of sheeppox virus and establishment of indirect ELISA for antibody

本研究旨在对免疫原性目的蛋白基因A11R优化表达的基础上建立羊痘血清学检测方法,为羊痘防控提供技术支撑。绵羊痘病毒A11R基因经密码子优化后合成目的基因,连入pET-28a(+)载体,并转化至BL21(DE3)大肠杆菌,pET28a-A11R经鉴定后进行诱导表达。结果显示,经密码子优化后的A11R蛋白的表达量显著增加,更易从转化的大肠杆菌裂解上清液和经过复性的包涵体中大量纯化获得。将该蛋白作为包被抗原,通过优化反应条件,建立检测羊痘抗体的间接ELISA方法,并进行特异性和敏感性试验,用该方法与商业化试剂盒对临床样品进行检测。结果显示,A11R蛋白的最佳包被剂量为0.5μg/孔,待检血清最适稀释度为1∶100,家兔抗羊Ig G最适稀释度为1∶20 000。用建立的间接ELISA方法检测小反刍兽疫、羊口疮、口蹄疫的阳性血清,结果均为阴性,无交叉反应,具有良好的特异性。当羊痘阳性血清稀释到1∶1 600时抗体检测仍为阳性,具有较高的敏感性。用该ELISA方法与商业化试剂盒检测临床样品,符合率为98.71%,具有良好的一致性。上述结果表明,本研究建立了基于A11R蛋白的羊痘血清学免疫诊断方法,同时表明了密码子偏好性的优化可显著提高蛋白表达量。

An indirect serological method was established to detect specific antibodies for capripoxvirus in goat and sheep sera that based on immunogenic target protein A11R expression,which provide technical support for the prevention and control of sheep pox.Codon optimized A11R gene of SPPV was synthesized and was expressed in E.coli BL21(DE3) strain using p ET-28a(+) vector and evaluated by SDSPAGE and Western blotting.The results showed that the codon optimization was required for the mass expression of A11R protein.Meawhile,A11R protein was used and optimized as the indirect ELISA coating antigen.The optimized conditions were found to be 0.5 μg/well of A11R protein,1 ∶ 100 dilution of antibody,1 ∶ 20 000 dilution of rabbit anti-sheep antibody conjugate with HRP.In addition,the recombinant A11R protein showed high specificity and diagnostic sensitivity with capripoxvirus-specific antibodies although the positive antibodies were diluted in 1 ∶ 1 600,whereas no cross-reactions with positive sera from RPPV,orfv and FMDV-infected animals.Furthermore,the coincidence rate of the established indirect ELISA with commercial ELISA kit reached to 98.71%.These findings indicate that the indirect ELISA system based on A11R protein could potentially be used as a serological detection method in sero-surveillance of all capripoxviruses.