摘要
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108.3和108.1TCID50/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。
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.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2012年第6期1034-1038,共5页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81000516)
教育部博士点基金资助项目(No.20104401120008)
中央高校基本科研业务费专项资金资助项目(No.21610602)
