摘要

       目的：从KSHV的免疫调节基因中筛选能够激活宿主抗艾滋病基因表达的特异基因。方法：以KSHV基因组为模板,PCR扩增待筛选的18条基因的开放阅读框,通过T-A克隆的方法将其分别克隆至PIRES2-EGFP真核表达载体。重组质粒电穿孔转染Jurkat细胞,24h后观察细胞荧光估计转染效率。36h后收集细胞,抽提细胞总RNA,荧光定量PCR检测细胞内五条抗艾滋病基因(A3G、A3F、 CCL5、 MX1、MX2)的表达水平,并最终从KSHV的18条免疫调节基因中筛选出具有激活宿主抗艾滋病基因表达的特异基因。结果：测序结果显示正确构建了各基因的表达载体。荧光显微镜显示各载体的电转效率均在40%以上。通过对各重组载体转染Jurkat细胞后对细胞内抗艾滋病相关基因表达影响的定量PCR结果分析,从18条KSHV免疫调节基因中共筛选出4条(K4,K6,K13,ORF4)能较强地上调宿主抗艾滋病相关基因表达的病毒序列。其中K4基因使Jurkat细胞内A3G、A3F、CCL5、MX1、MX2分别上调2.407、3.086、2.549、2.023、2.671倍。K6基因使Jurkat细胞内A3G、A3F、CCL5、MX1分别上调1.580、2.420、7.367、2.064倍。K13基因使Jurkat细胞内A3G、A3F、CCL5、MX2分别上调2.199、1.518、6.713、21.782倍。ORF4基因使Jurkat细胞内A3G、A3F、CCL5、MX1、MX2分别上调1.843、1.236、4.382、2.772、1.415倍。 结论：从KSHV免疫调节基因中共筛选出K4,K6,K13,ORF4四条能较强地激活Jurkat细胞A3G、A3F、CCL5等抗艾滋病相关基因表达的特异基因。

       Objective: To screen specific genes that can activate the expression of host anti-AIDS genes from the immune regulatory genes of KSHV. Methods: Using the KSHV genome as a template, the open reading frames of the 18 genes to be screened were amplified by PCR, and they were cloned into the PIRES2-EGFP eukaryotic expression vector by T-A cloning. The recombinant plasmid was transfected into Jurkat cells by electroporation. After 24 hours, the cell fluorescence was observed to estimate the transfection efficiency. After 36 hours, the cells were collected, and the total RNA of the cells was extracted. The expression levels of five anti-AIDS genes (A3G, A3F, CCL5, MX1, MX2) in the cells were detected by fluorescence quantitative PCR, and the 18 immunomodulatory genes of KSHV were finally screened out. A specific gene that activates the host's anti-AIDS gene expression. Result: The sequencing result showed that the expression vector of each gene was constructed correctly. Fluorescence microscope showed that the electroporation efficiency of each carrier was above 40%. Through the quantitative PCR results analysis of the effect of each recombinant vector on the expression of anti-AIDS-related genes in the cell after transfection of Jurkat cells, a total of 4 (K4, K6, K13, ORF4) were screened out of 18 KSHV immunoregulatory genes, which can strongly A viral sequence that up-regulates the expression of host anti-AIDS-related genes. Among them, K4 gene up-regulated A3G, A3F, CCL5, MX1, MX2 in Jurkat cells by 2.407, 3.086, 2.549, 2.023, 2.671 times, respectively. K6 gene up-regulated A3G, A3F, CCL5, and MX1 in Jurkat cells by 1.580, 2.420, 7.367, 2.064 times, respectively. K13 gene up-regulated A3G, A3F, CCL5, and MX2 in Jurkat cells by 2.199, 1.518, 6.713, and 21.782 times, respectively. ORF4 gene up-regulated A3G, A3F, CCL5, MX1, MX2 in Jurkat cells by 1.843, 1.236, 4.382, 2.772, 1.415 times, respectively. Conclusion: A total of four K4, K6, K13, ORF4 genes were screened from KSHV immunoregulatory genes that can strongly activate the expression of anti-AIDS-related genes such as A3G, A3F, and CCL5 in Jurkat cells.

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