基于HIV-1 pNL4-3构建含CRF07_BC V3的嵌合分子克隆

Construction and identification of pNL4-3-based chimeric molecular clone with CRF07_BC V3 region

目的在感染性克隆pNL4-3骨架上构建含CRF07_BC V3的嵌合分子克隆。方法通过融合PCR将CRF07_BC pXJDC13的V3环与pNL4-3的env骨架进行融合并克隆至pNL4-3上。经过鉴定后将阳性V3嵌合克隆pNL4-3/XJDC13V3转染到293T细胞进行病毒包装并检测病毒的感染性。V3嵌合病毒的细胞嗜性分别用Ghost细胞系和MT-2细胞进行测定。结果利用融合PCR克隆方法在pNL4-3骨架上成功构建了含CRF07_BC pXJDC13 V3的嵌合分子克隆pNL4-3/XJDC13V3。该嵌合病毒利用的辅助受体为CCR5,不能利用CXCR4,也不能在MT-2细胞上诱发合胞体。结论在pNL4-3骨架上成功构建了含CRF07_BC pXJDC13 V3嵌合分子克隆pNL4-3/XJDC13V3。该嵌合分子克隆为进一步研究CRF07_BC嗜性转变中V3关键位点提供了有利工具。

Objective To construct pNL4-3-based chimeric molecular clone with a V3 loop derived from CRF07_BC.Methods The V3 loop of CRF07_BC pXJDC13 was cloned into pNL4-3 backbone by fusion PCR and the chimera pNL4-3/XJDC13V3 was identified by sequencing. The chimeric virus was obtained by transfecting 293T cell with pNL4- 3/XJDC13V3and viral tropism was tested in Ghost and MT-2 cell lines. Results The pXJDC13 V3 loop was successfully cloned into pNL4-3 backbone. The chimeric virus NL4-3/XJDC13 was CCR5-tropic and could not induce syncitia formation in MT-2 cell. Conclusion Based on pNL4-3, a functional infectious clone with a V3 loop derived from CRF07_BC pXJDC13 was successfully constructed. This chimera will be useful to study the V3 role in coreceptor switching in CRF07_BC.