【摘要】：目的：從KSHV的免疫調(diào)節(jié)基因中篩選能夠激活宿主抗艾滋病基因表達(dá)的特異基因。方法：以KSHV基因組為模板,PCR擴(kuò)增待篩選的18條基因的開放閱讀框,通過T-A克隆的方法將其分別克隆至PIRES2-EGFP真核表達(dá)載體。重組質(zhì)粒電穿孔轉(zhuǎn)染Jurkat細(xì)胞,24h后觀察細(xì)胞熒光估計(jì)轉(zhuǎn)染效率。36h后收集細(xì)胞,抽提細(xì)胞總RNA,熒光定量PCR檢測(cè)細(xì)胞內(nèi)五條抗艾滋病基因(A3G、A3F、 CCL5、 MX1、MX2)的表達(dá)水平,并最終從KSHV的18條免疫調(diào)節(jié)基因中篩選出具有激活宿主抗艾滋病基因表達(dá)的特異基因。結(jié)果：測(cè)序結(jié)果顯示正確構(gòu)建了各基因的表達(dá)載體。熒光顯微鏡顯示各載體的電轉(zhuǎn)效率均在40%以上。通過對(duì)各重組載體轉(zhuǎn)染Jurkat細(xì)胞后對(duì)細(xì)胞內(nèi)抗艾滋病相關(guān)基因表達(dá)影響的定量PCR結(jié)果分析,從18條KSHV免疫調(diào)節(jié)基因中共篩選出4條(K4,K6,K13,ORF4)能較強(qiáng)地上調(diào)宿主抗艾滋病相關(guān)基因表達(dá)的病毒序列。其中K4基因使Jurkat細(xì)胞內(nèi)A3G、A3F、CCL5、MX1、MX2分別上調(diào)2.407、3.086、2.549、2.023、2.671倍。K6基因使Jurkat細(xì)胞內(nèi)A3G、A3F、CCL5、MX1分別上調(diào)1.580、2.420、7.367、2.064倍。K13基因使Jurkat細(xì)胞內(nèi)A3G、A3F、CCL5、MX2分別上調(diào)2.199、1.518、6.713、21.782倍。ORF4基因使Jurkat細(xì)胞內(nèi)A3G、A3F、CCL5、MX1、MX2分別上調(diào)1.843、1.236、4.382、2.772、1.415倍。 結(jié)論：從KSHV免疫調(diào)節(jié)基因中共篩選出K4,K6,K13,ORF4四條能較強(qiáng)地激活Jurkat細(xì)胞A3G、A3F、CCL5等抗艾滋病相關(guān)基因表達(dá)的特異基因。
[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緇嗚優(yōu)鍐匒3G,A3F,CCL5,MX1,MX2鍒嗗埆涓婅皟2.407,3.086,2.549,2.023,2.671鍊，

本文編號(hào)：2247605