HIV-1中和抗体检测方法的建立

目的以荧光素酶作为报告基因,表达假病毒,建立快速、有效的HIV-1中和抗体检测方法。方法将HIV-1中国流行株B/C重组亚型和C亚型Envelope基因的真核表达质粒分别与带有荧光素酶报告基因的pNL4-3.Luc.R-E质粒共转染293T细胞,培养48h后收获含有假病毒的上清液,感染表达CD4受体和一个辅助受体CCR5的HOS细胞,培养72h后,裂解细胞,检测荧光素酶含量。结果HIV-1的B/C重组亚型和C亚型Envelope基因真核表达质粒分别与pNL4-3.Luc.R-E质粒共转染的293T细胞,均有HIV-1结构蛋白Gagpol和Env的表达,相对分子质量分别为55000和160000,用产生的假病毒感染HOS-CD4-CCR5/CXCR4细胞后,产生的荧光素酶含量明显高于对照组。结论已建立了瞬时转染检测HIV-1中和抗体的方法,为今后抗体样本及病毒样本的检测提供了参考。

Development of A Method for Determination of HIV-1 Neutralizing Antibody

Objective To express pseudovirus using luciferase as report gene and develop a rapid and effective method for determination of HIV-1 neutralizing antibody. Methods Transfect 293T cells by pNL4-3.Luc.R-E together with eukaryotic expression plasmids pcDNA3. 1 EnvB / C and VR1012EnvC respectively and culture for 48 h. The supernatant containing pseudovirus was harvested to infect HOS cells expressing receptor CD4 and accessory receptor CCR5. The infected cells were cultured for 72 h, then lysed and determined for luciferase content. Results HIV-1 structural proteins Gagpol and Env, with relative molecular mass of 55 000 and 160 000 respectively, were expressed in 293T cells co-transfected with pcDNA3. 1 EnvB / C and pNL4-3. Luc. R-E as well as those with VR1012EnvC and pNL4-3. Luc. R-E. The luciferase content in HOS-CD4-CCR5 / CXCR4 cells infected with the harvested pseudovirus was significantly higher than that in control. Conclusion A method for determination of HIV-1 neutralizing antibody by transient transfection was developed, which provided a basis for the determination of antibody and virus specimens.