筛选上调宿主ARGs表达的KSHV免疫调节基因
发布时间：2018-09-18 10:21
【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。
[Abstract]:Aim: to screen specific genes that can activate the expression of host anti-AIDS genes from the immune regulatory genes of KSHV. Methods: the open reading frame of 18 genes was amplified from KSHV genome and cloned into eukaryotic expression vector of PIRES2-EGFP by T-A cloning. The estimated transfection efficiency of Jurkat cells was observed 24 hours after transfection with recombinant plasmid electroporation. The total RNA, fluorescence quantitative PCR was used to detect the expression level of five anti-AIDS genes (A3GnA3F, CCL5, MX1,MX2) in the cells. Finally, 18 immunomodulatory genes of KSHV were screened out to activate the expression of host anti-AIDS genes. Results: the results of sequencing showed that the expression vectors of each gene were constructed correctly. Fluorescence microscope showed that the electrotransposable efficiency of each carrier was above 40%. The effect of recombinant vectors on the expression of anti-AIDS related genes in Jurkat cells was analyzed by quantitative PCR. Four of the 18 KSHV immunomodulatory genes (K4 / K6 / K13ORF4) were screened out to up-regulate the expression of host anti-AIDS-related genes. 鍏朵腑K4鍩哄洜浣縅urkat缁嗚優鍐匒3G,A3F,CCL5,MX1,MX2鍒嗗埆涓婅皟2.407,3.086,2.549,2.023,2.671鍊，
本文编号：2247605
[Abstract]:Aim: to screen specific genes that can activate the expression of host anti-AIDS genes from the immune regulatory genes of KSHV. Methods: the open reading frame of 18 genes was amplified from KSHV genome and cloned into eukaryotic expression vector of PIRES2-EGFP by T-A cloning. The estimated transfection efficiency of Jurkat cells was observed 24 hours after transfection with recombinant plasmid electroporation. The total RNA, fluorescence quantitative PCR was used to detect the expression level of five anti-AIDS genes (A3GnA3F, CCL5, MX1,MX2) in the cells. Finally, 18 immunomodulatory genes of KSHV were screened out to activate the expression of host anti-AIDS genes. Results: the results of sequencing showed that the expression vectors of each gene were constructed correctly. Fluorescence microscope showed that the electrotransposable efficiency of each carrier was above 40%. The effect of recombinant vectors on the expression of anti-AIDS related genes in Jurkat cells was analyzed by quantitative PCR. Four of the 18 KSHV immunomodulatory genes (K4 / K6 / K13ORF4) were screened out to up-regulate the expression of host anti-AIDS-related genes. 鍏朵腑K4鍩哄洜浣縅urkat缁嗚優鍐匒3G,A3F,CCL5,MX1,MX2鍒嗗埆涓婅皟2.407,3.086,2.549,2.023,2.671鍊，
本文编号：2247605
本文链接：https://www.wllwen.com/xiyixuelunwen/2247605.html
最近更新
教材专著
