携带中国株HIV-1 gp120基因的重组腺病毒的构建及其感染巨噬细胞的形态学研究

目的: 构建携带中国株HIV-1 gp120基因并可感染小鼠骨髓来源巨噬细胞(BMM)的重组腺病毒。方法: 利用AdMax腺病毒构建系统,以双质粒共转染人胚肾转化细胞293Ad5⁺细胞,完成重组腺病毒载体包装,并进一步扩增和纯化。通过ELISA测定gp120的蛋白产量,以确认目的基因重组与表达成功。以Karber法对病毒进 行TCID₅₀滴度测定,利用BMM进行感染复数(MOI)流式细胞术测定,并利用荧光显微镜观察细胞活化形态。结果: 成功构建AdMax-HIV-1 gp120(简称Ad-gp120)及其对照病毒(Ad-GFP),其滴度分别为10^8.3和10^8.1 TCID₅₀/mL。这些病毒可感染BMM,MOI测定结果表明2种重组腺病毒感染BMM和293Ad5⁺细胞的能力接近,验证了上述TCID₅₀滴度结果。Ad-gp120可在293Ad5⁺细胞中表达gp120蛋白,并可感染和诱导BMM相关形态改变。结论: 成功构建Ad-gp120,其感染可致BMM出现形态发生改变。

Morphological study of macrophages infected with constructed recombinant adenovirus carrying gp120 gene of Chinese HIV-1 strain

AIM: To construct a recombinant adenovirus carrying gp120 gene of Chinese HIV-1 strain,which can infect mouse bone marrow-derived macrophages(BMM).METHODS: Co-transfection of shuttle and backbone plasmids of AdMax system into 293Ad5+ cells was performed,followed by viral packaging,propagation and purification.These viruses were subject to Karber TCID50 titration.The expression of gp120 protein in 293Ad5+ cells was determined by ELISA.The viral titration was validated by a multiplicity of infection(MOI) test with BMM.RESULTS: The titers of the outcome viruses,including AdMax-HIV-1 gp120(Ad-gp120) and its vector control Ad-GFP,were 108.3 and 108.1 TCID50/mL,respectively.Both recombinant adenoviruses infected BMM with similar capacity of 293Ad5+ cell infection,which validated the TCID50 titration.The gp120 protein was positive in 293Ad5+ cell lysates.BMM activation was observed morphologically after Ad-gp120 infection as compared with Ad-GFP-infected cells.CONCLUSION: Functional adenovirus containing HIV-1 gp120 of prevalent strains in China was successfully constructed.Infection of Ad-gp120 causes BMM activation.