摘要
目的构建中国境内携带荧光素酶报告基因的B、B′C及AE亚型的HIV-1假病毒,研究该假病毒在检测病毒嗜性及中和抗体试验中的初步应用,为HIV-1的研究搭建一个方便、快捷且安全的平台。方法通过RT-PCR将3种亚型膜蛋白全长扩增,转化到表达质粒,构建膜蛋白质粒,与HIV骨架质粒共转染293T细胞,获得假病毒颗粒。假病毒感染GHOST细胞后,通过测定荧光值(RLU)来确定假病毒的感染效率、辅助受体使用情况及评价中和抗体作用。结果携带荧光素酶报告基因的HIV-1假病毒感染CD4+CCR5+CXCR4+GHOST细胞后,经检测被感染细胞的荧光值显著大于阴性对照组,证明假病毒能够在体外有效地感染GHOST细胞;利用辅助受体抑制剂方法确定了假病毒毒株的嗜性;假病毒感染GHOST细胞可被同亚型血清中和作用所抑制,抑制率随血清稀释度增加而减小。结论获得了携带荧光素酶报告基因的HIV-1假病毒,并初步证实了该假病毒可应用于病毒嗜性及中和抗体试验。
Objective The aim of this research is to construct luciferase report gene containing pseudovirus with Chinese HIV-1 epidemic subtypes of B,B′C and AE for viral tropism and neutralizing testing.Methods The full-length env-genes of HIV-1 subtypes were amplified by RT-PCR.The reconstructed HIV env-gene containing plasmids were co-transfected into 293T cells for HIV-1 pseudovirus harvesting.The infection efficiency and viral tropism of HIV-1 pseudovirus as well as the efficacy of neutralizing antibodies were measured in GHOST cells by relative light units（RLU） per milliliter.Results HIV-1 pseudovirus successfully infected GHOST cells in vitro.Viral tropism was defined by co-receptor inhibitors.The infection of pseudovirus could be inhibited by homologous neutralizing antibodies.The effect of inhibition was associated with antibody concentration.Conclusions Pseudotyped HIV-1 was successfully constructed and could serve as a platform for HIV research.
出处
《中国病毒病杂志》
CAS
2011年第4期269-272,共4页
Chinese Journal of Viral Diseases
基金
国家"十一五"艾滋病和病毒性肝炎等重大传染病防治科技重大专项(2008ZX10001-006)
