古尔图病毒核蛋白的原核表达纯化及ELISA检测方法的建立

Prokaryotic expression and purification of nucleoprotein of Guertu virus and its establishment of ELISA detection method

目的获得纯化的GTV-rNP蛋白抗原,并建立快速准确检测GTV抗体的ELISA法。方法人工合成密码子优化的GTV-NP编码基因,克隆至pET32a(+)载体,构建重组表达质粒,转化至BL21(DE3)。将优化表达获得的蛋白经Ni柱纯化后用SDS-PAGE和Western blot鉴定。以纯化蛋白为抗原,建立并优化GTV IgG抗体间接ELISA检测方法,并对其进行评价和初步应用。结果成功构建原核表达质粒pET32a-NP,重组蛋白以包涵体形式在大肠杆菌中高效表达,大小约为44 kD,Western blot结果表明重组蛋白与GTV阳性血清具有良好的抗原性。建立的ELISA法特异性强、敏感性高、批内和批间的变异系数均小于10%具有较好的重复性;检测结果与IFA检测结果符合率达到92.77%。结论建立的GTV NP抗体检测ELISA方法的敏感性、重复性和特异性均较好。

Objective To obtain purified protein antigen of guertu virus(GTV)nucleoprotein(NP)and establish a rapid and accurate enzyme-linked immunosorbent assay(ELISA)method for detection of GTV antibody.Methods Codon optimized GTV NP encoding genes were synthesized,cloned into the pet32a(+)vector,and recombinant expression plasmids were constructed and transformed into BL21(DE3).Recombinant protein(rNP)obtained from the optimized expression were purified over a Ni column and identified by SDS-PAGE and Western blot.The purified protein was used as the antigen to optimize the reaction conditions,and an indirect ELISA assay for GTV IgG antibody was developed and optimized,which was evaluated and initially applied.Results The prokaryotic expression plasmid pet32a-NP was successfully constructed,the recombinant protein was highly expressed in E.coli in the form of inclusion bodies,the size was about 44 kD,and the results of Western blot indicated that the recombinant protein had good antigenicity with GTV positive serum.The optimized ELISA(GTV-rNP-iELISA)established in this study showed strong specificity,high sensitivity,and the coefficient of variation within and between batches is less than 10%,and has good repeatability;the detection results are consistent with the IFA detection results.Using the established ELISA method to detect 162 sheep sera from some regions of Xinjiang in 2017-2019,the total positive rate of antibodies was 39.8%.Conclusions The GTV NP antibody detection ELISA method has good sensitivity,reproducibility,and specificity and has the potential to be a powerful tool for the diagnosis and serological investigation of GTV infection.