克里米亚刚果出血热病毒抗体间接ELISA检测方法的建立

Establishment of an indirect ELSIA method of detecting antibodies to the Crimean Congo hemorrhagic fever virus

目的原核表达并纯化克里米亚-刚果出血热病毒(Crimean-Congo hemorrhagic fever virus, CCHFV)M基因编码的包膜糖蛋白片段Gn1和Gc1,以两段目的蛋白为包被抗原分别建立检测CCHFV抗体的间接ELISA方法。方法 PCR扩增CCHFV M基因的抗原保守区胞外域Gn1和Gc1基因片段,分别克隆至pET-30a(+)载体,转化大肠埃希菌BL21(DE3),并对不同诱导条件(温度、IPTG浓度、时间)进行优化,使用His-Ni柱纯化目的蛋白,进行SDS-PAGE和Western blot鉴定,分别以纯化的Gn1和Gc1蛋白作为包被抗原建立抗体ELISA检测方法。结果表达并纯化了CCHFV糖蛋白片段Gn1、Gc1,分子质量分别为35 ku和23 ku;Western blot检测两种重组蛋白均能被抗Gn1,Gc1抗体识别。以纯化蛋白为抗原建立的间接ELISA方法检测已知阳性血清CCHFV抗体滴度为1∶10 240,检测RVFV、WNV、JEV感染者血清均阴性,变异系数<8%。结论表达纯化的Gn1和Gc1蛋白纯度均在90%以上,两种蛋白均表现出良好的免疫原性,以两种蛋白为包被抗原建立的抗体间接ELISA检测方法,该方法具有良好的特异性和敏感性。重组蛋白的制备为克里米亚刚果出血热疫苗免疫效果评价、疫病监测和新型疫苗研发奠定了基础。

Objective To express the glycoproteins Gn1 and Gc1 of Crimean Congo hemorrhagic fever virus(CCHFV) in a prokaryotic expression system and to use those protein as a coating antigen to establish a method of indirect ELISA for detection of CCHFV antibodies. Methods The extracellular domains of the Gn1 and Gc1 fragments of the CCHFV M gene were amplified using PCR, cloned into a pET-30 a(+) vector, digested using EcoR I and Xho I, and transformed into BL21(DE3). Expression of Gn1 and Gc1 was induced using 0-1.0 mmol/L IPTG at different temperatures for 1-7 h. The target protein was purified on an His-Ni column and identified using SDS-PAGE and Western blotting. Purified Gn1 and Gc1 proteins were each used as coating antigens to establish an antibody indirect ELISA method of detection. The optimum concentration of the coated protein and the optimum dilution of serum were determined using chessboard titration. The specificity, sensitivity, and repeatability of the indirect ELISA method were tested. Results The recombinant plasmids were successfully constructed. The protein is mainly expressed in the form of inclusion bodies, and its expression was induced with 0.4 mmol/L IPTG at 37 ℃. The inclusion body proteins were denatured and renatured, and Gn1 and Gc1 were obtained after purification. SDS-PAGE and Western blotting indicated that the two recombinant proteins were expressed by the prokaryotic expression system, with respective molecular weights of 35 ku and 23 ku. The two recombinant proteins reacted with anti-Gn1 and anti-Gc1 antibodies. The optimum coating concentration of Gn1 in indirect ELISA was 10 g/ml, and the serum dilution was 1:640. The optimum coating concentration of Gc1 in indirect ELISA was 8 g/ml, and the serum dilution was 1:1,280. Conclusion The glycoprotein fragments Gn1 and Gc1 had good immunogenicity. An indirect ELISA method of antibody detection was established using two proteins as coating antigens. The method is specific and sensitive. This study has laid the foundation for evaluation of the immune effects of vaccination, disease surveillance, and research and development of new CCHFV vaccines.