人甲3型流感病毒基质蛋白及其诱导抗体在临床检测中的应用

Expression of recombinant M_1 protein of human influenza virus A3 in E. Coli and the application of anti-rM_1 antibody in clinical tests

目的探讨抗重组人甲3型流感病毒基质蛋白(M1)抗体在临床检测中应用的可行性。方法在生物信息学分析的基础上,经逆转录-聚合酶链反应(RT-PCR)从人甲3型流感病毒感染细胞中获得目的基因,构建pET-28c(+)/IFV.M1P2重组质粒并在BL21DE3中诱导表达;重组蛋白经亲和层析纯化后免疫家兔制备多克隆抗体。采用间接免疫荧光法检测重组蛋白抗体与病毒感染细胞和临床标本的反应性,并与流感病毒凝血抑制试验结果比较分析,确定重组蛋白抗体的灵敏度和特异性。结果间接免疫荧光法检测抗M1P2重组蛋白抗体与人甲3型﹑甲1型流感病毒反应的抗体滴度可达1∶1200和1∶1000,与乙型流感病毒及其他呼吸道常见病毒无特异性反应。同样方法检测262例上呼吸道感染患儿鼻咽分泌物标本,阳性18例(6.87%),凝血抑制试验阳性24例(9.16%)。与凝血抑制试验相比,间接免疫荧光法检测敏感性为62.5%,特异性为98.7%。结论重组M1P2蛋白具有较强的免疫原性,可模拟天然病毒蛋白诱导产生较强的抗人甲3型流感病毒抗体,该抗体具有较高的反应特异性和一定的灵敏度。

Objective To analyze the antigenicity of matrix protein and explore the application of anti-recombi- nant M1 protein antibody in clinical tests. Methods Based on the intratype conservative region of M, gene, the target gene was cloned into pET-28e （＋）to construct pET-28c （＋）/IFV.M1P2, and induced to express in BL21 DE3 with lmmol/L IPTG. The anti-rM1P2 antibody was prepared in rabbits with purified recombinant MIP2 （rM1P2）. The reactivity or crossreactivity of anti-rM1P2 antibody to IFV-A3 or other common respiratory viruses were assessed with indirect immunofluorescent assay （IFA）. The sensitivity and specificity of anti-rM1P2 antibody were analyzed in 262 specimens, with reference to hemaglutination-inhibition test （HI）. Results The anti-rMiP2 antibody （1：1200） could react with IFV-A3 by IFA and erossreact with IFV-A1 at 1：1000 without any positive reaction with other common respiratory viruses. 18 out of 262 （6.87%） clinical specimens were diagnosed as positive, with a sensitivity of 62.5% and specificity of 98.7% compared with HI test. Conclusions The rM1P2 proteins may mimic natural viral proteins to induce anti-IFV-A3 antibody with high specificity and sensitivity. And the antibody may be used for laboratory test of IFV infections.