Objective To study the function of anti-HIV activity of TRIM5α. Methods The full-length cDNA encoding TRIM5α in American green mnonkey(AGM) was obtained using the reverse transcription polymerase chain reaction (BT-PCB) method after extracting the total RNA fiom vero cells. The cDNA fragments corresponding to the B30.2 and coiled coil region of TRIM5α from human, rhesus monkey, and AGM were amplified with routine PCR method and ligated into the expression vector pEGFP-C3. After fusion expression with green fluorescence, the cellular location of the protein was observed under fluorescent microscope. Pseudotyped viruses were generated by co-transfection of HIV Aenv-expressing plasmid and VSV-G-expressing plasmid. The anti-HIV activity of TRIM5α and its fragments were quantitatively tested by the inhibition of pseudotyped virus infection. Results The plasmids expressing TRIM5α and its fraginents fused with GFP tag were constructed successfully. The TRIM5α and its fragments from human, rhesus monkey, and AGM were all located in the cytoplasn, among which the hTRIM5α, rhTRIM5α, AGMTRIM5α, rh-TRIM5α CC／B30.2, AGM-TRIM5α CC/B30.2 were distributed as oligomer. But h-TRIM5α CC／B30. 2 and TRIM5α B30. 2 from different species were distributed evenly in the cytoplasm. The inhibition rate of pseudotyped virus infection was about 70％ for rh-TRIM5α, AGM-TRIM5α, rh-CC／B30.2 and AGM-CC／B30.2, and that was less than 20％ for h-TRIM5α and different species B30.2. Conclusion TRIM5α CC/B30.2 domain of rhesus monkey and AGM could inhibit HIV-1. Reliable experimental data helping determine the TRIM5α functional domains were obtained.%目的 研究TRIM5α抗HIV-1功能.方法 从vero细胞中提取总RNA,采用反转录-聚合酶链式反应(RT-PCR)技术获得非洲绿猴TRIM5α全基因序列.用PCR技术从人、恒河猴和非洲绿猴TRIM5α全基因序列中扩增B30.2和coiled coil等功能片段.将测序鉴定过的目的基因克隆到真核表达载体pEGFP-C3上,与绿色荧光蛋白融合表达,荧光显微镜观察目标蛋白在细胞内的定位.利用VSV-G膜蛋白质粒和HIV-1骨架质粒包装假病毒,通过假病毒感染实验定量检测TRIM5α及其功能片段对HIV-1的抑制功能.结果 成功地构建了带有绿色荧光蛋白标签的TRIM5α及其功能片段真核表达质粒.人、恒河猴和非洲绿猴TRIM5α及其功能片段蛋白均定位于细胞质,其中hTRIM5α、rhTRIM5α、AGMTRIM5α、rh-TRIM5αCC/B30.2、AGM-TRIM5αCC/B30.2融合蛋白以高聚状态存在于胞质内,而h-TRIM5αCC/B30.2和不同种属TRIM5αB30.2融合蛋白则均匀分布于胞质内.恒河猴和非洲绿猴TRIM5α及CC/B30.2对HIV-1假病毒的抑制率均达70%,而人TRIM5α及各种属B30.2对假病毒的抑制率低于20%.结论 恒河猴和非洲绿猴TRIM5α CC/B30.2功能结构域对HIV-1有拮抗作用,为TRIM5α最小功能结构域的确定及结构的解析提供了可靠的实验依据.
展开▼
