CAJ | 학술논문

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.
목적 탐토Ⅰ형인류면역결함병독(HIV-1)포막당단백(Env) gp120적CD4결합위점(CD4BS)핵심구제423、425화431위안기산삼련돌변ING/MKE대기유도체액면역반응적영향.방법 구건HIV-1원대독주06044포막gp120 ING/MKE삼련돌변표체재체pcT22-06044 gp120T-ING/MKE(gp120T-ING/MKE),재체외전염적HEK293T세포표체삼취화야생형gp120Twt단백화돌변체gp120T-ING/MKE단백.면역BALB/c소서후검측결합항체、중화항체화골수항원특이성장세포.결과 획득진핵표체재체gp120T-ING/MKE.전염후,재293T세포배양상청중검측도료삼취화중조단백.말차면역후14 d,gp120Twt화gp120T-ING/MKE면역혈청중결합항체적도도대우1∶1 000,단량조혈청항체적도무현저차이.돌변체조골수특이성장세포분비수평화비특이장세포수평균저우야생형gp120면역조.야생형화돌변체면역유도혈청항체적중화활성균불강.결론 HIV-1포막단백CD4결합구423、425화431위삼련돌변몰유개선gp120단백적면역원성.
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.