Ⅰ型人类免疫缺陷病毒包膜糖蛋白CD4结合位点修饰诱导小鼠体液免疫反应的实验研究  

Study of humoral immune responses of a mutant within CD4 binding site of human immunodeficiency virus type 1, envelop glycoprotein in mice.

作　　者：郭彩霞[1] 王甲业[1] 于昊彤[1] 田丹[1] 庄敏[1] 凌虹[2] 

机构地区：[1]哈尔滨医科大学微生物学教研室黑龙江省感染与免疫重点实验室黑龙江省教育厅病原生物学重点实验室,150081 [2]哈尔滨医科大学微生物学教研室黑龙江省感染与免疫重点实验室黑龙江省教育厅病原生物学重点实验室 150081 哈尔滨医科大学寄生虫学教研室,150081

出　　处：《国际免疫学杂志》2015年第3期214-218,共5页International Journal of Immunology

基　　金：黑龙江省卫生厅基金项目（2012-760）;高等学校博士学科点专项科研基金（20132307110023）;哈尔滨市科学技术局项目（2006RFLXS021）

摘　　要：目的 探讨Ⅰ型人类免疫缺陷病毒（HIV-1）包膜糖蛋白（Env） gp120的CD4结合位点（CD4BS）核心区第423、425和431位氨基酸三联突变ING/MKE对其诱导体液免疫反应的影响.方法 构建HIV-1原代毒株06044包膜gp120 ING/MKE三联突变表达载体pcT22-06044 gp120T-ING/MKE（gp120T-ING/MKE）,在体外转染的HEK293T细胞表达三聚化野生型gp120Twt蛋白和突变体gp120T-ING/MKE蛋白.免疫BALB/c小鼠后检测结合抗体、中和抗体和骨髓抗原特异性浆细胞.结果 获得真核表达载体gp120T-ING/MKE.转染后,在293T细胞培养上清中检测到了三聚化重组蛋白.末次免疫后14 d,gp120Twt和gp120T-ING/MKE免疫血清中结合抗体滴度都大于1∶1 000,但两组血清抗体滴度无显著差异.突变体组骨髓特异性浆细胞分泌水平和非特异浆细胞水平均低于野生型gp120免疫组.野生型和突变体免疫诱导血清抗体的中和活性均不强.结论 HIV-1包膜蛋白CD4结合区423、425和431位三联突变没有改善gp120蛋白的免疫原性.Objective To investigate the effect of the triple amino acid residues I423,N425 and G431 within CD4 binding site core region （CD4BScore） of human immunodeficiency virus type 1 （HIV-1） envelope glycoprotein （Env） on the Env immunogenicity,the humoral immune responses of an envelope gp120T-ING/MKE mutant was evaluated in BALB/c mice.Methods Thegp120 gene mutant ING/MKE at I423,N425 and G431 within gp120 CD4 binding site of HIV-1 06044 strain was PCR amplified.The recombinant plasmid pcT22-06044gp120T-ING/MKE （gp120T-ING/MKE） was constructed.HEK293T cells were transiently transfected with the recombinant plasmids to express the wild type （gp120Twt） and the mutant gp120 （gp120T-ING/MKE） trimeric proteins.BALB/c mice were immunized using DNA prime and protein boost regimen.Fourteen days after the last immunization,the binding and neutralizing antibody responses in mouse sera were measured by using an enzyme immunoassay and HIV-1 pseudovirus neutralization assay in TZM-bl cells.Antibody-secreting plasma cells in bone marrows were also detected by using B cell ELISPOT.Results The plasmid gp120T-ING/MKE was constructed and confirmed by DNA sequencing.The recombinant trimeric gp120 proteins were expressed in the culture supernatants.Day14 after the final immunization,sera from the immunized mice by gp120Twt and the mutant exhibited similar specific binding activities.The mutant-immunized mice had lower frequencies of specific and non-specific antibody-secreting cells than gp120Twt-immunized mice.Both gp120Twt and ING/MKE mutant induced weak neutralizing activities against SF162 pseudoviruses.Conclusion The triple mutation ING/MKE in CD4BS did not improve the immunogenicity of gp120.

关 键 词：人类免疫缺陷病毒 包膜糖蛋白 CD4结合位点 突变 免疫原性 

分 类 号：R392[医药卫生—免疫学]