基于ccr5、pol和vif基因的三联miRNA慢病毒表达载体的构建及功能分析

发布时间：2018-06-03 12:51

  本文选题：I型人类免疫缺陷病毒 + 三联miRNA　； 参考：《南华大学》2013年硕士论文

【摘要】：背景及目的：RNAi技术在抗HIV-1研究方面表现出一定的优势和应用潜力。针对HIV-1病毒的高突变性，本实验室在前期研究中，以ccr5、pol和vif基因为靶点构建了稳转细胞株293T-CCR5、293T-POL和293T-VIF；针对3个靶基因各自设计和构建了4个miRNA表达载体，从中各筛选出一个沉默效果最好的，记为pccr5-1-4，ppol-3-4和pvif-2-1。在此基础上，本研究将3个miRNA串联到一个表达载体上，并利用Gatewan重组技术构建成慢病毒表达载体pLenti6.3-ccr5-pol-vif，理论上，该重组载体可同时作用于三个靶基因。将其转染进293T-CCR5细胞，293T-POL细胞和293T-VIF细胞，进行功能分析，为进一步开展抗HIV-1研究提供基础。方法：pcDNA6.2-GW/EmGFP载体上含有酶切位点BamH I，Xho I和Bgl II，且BamH I与Bgl II是同尾酶，利用这一特性将载体中目的片段miR-ccr5，miR-pol和miR-vif串联起来，，获得pcDNA6.2-GW/EmGFP-ccr5-pol-vif；Eag I单酶切载体pcDNA6.2-GW/EmGFP-ccr5-pol-vif后，利用Gatewan重组技术构建成慢病毒表达载体pLenti6.3-ccr5-pol-vif，重组载体经测序验证其正确性；通过qRT-PCR技术检测其对靶基因的抑制效率和对IFN-β表达的影响；Western Blot法检测其对CCR5、POL和VIF蛋白表达的影响；采用MTT法检测其对被转染细胞有无毒性伤害。结果：测序结果表明重组载体构建成功；qRT-PCR结果显示pLenti6.3-ccr5-pol-vif对ccr5、pol和vif基因都有一定抑制作用，抑制效率分别达到25%、35%和13%；Western Blot结果显示转染了pLenti6.3-ccr5-pol-vif的293T-CCR5、293T-POL和293T-VIF细胞中，靶蛋白表达水平与空白对照组相比分别降低18%、55%和65%(P0.01)；转染细胞中IFN-β的表达未明显增加； MTT实验证实转染pLenti6.3-ccr5-pol-vif不会对细胞的存活率造成影响。结论：1.成功构建了慢病毒表达载体pLenti6.3-ccr5-pol-vif；2. pLenti6.3-ccr5-pol-vif可同时靶向ccr5、pol和vif三个基因发挥抑制作用；3. pLenti6.3-ccr5-pol-vif不会引发转染细胞中的干扰素效应，也不会产生细胞毒性，为进一步开展抗HIV-1研究提供实验基础。
[Abstract]:Background and objective: RNAi technique has some advantages and potential applications in the study of anti-HIV-1. Aiming at the high mutation of HIV-1 virus, we constructed the stable cell lines 293T-CCR5293T-POL and 293T-VIFand designed and constructed four miRNA expression vectors for each of the three target genes in our laboratory, using ccr5Pol and vif as the target sites. One of the most effective silences was identified as pccr5-1-4 ppol-3-4 and pvif-2-1. On the basis of this, three miRNA were connected into one expression vector, and the lentivirus expression vector pLenti6.3-ccr5-pol-vif was constructed by using Gatewan recombination technique. Theoretically, the recombinant vector could act on three target genes simultaneously. It was transfected into 293T-CCR5 cells and 293T-VIF cells for functional analysis, which provided a basis for further research on anti-HIV-1. Methods: the plasmid pcDNA6.2-GW / EmGFP contained BamH I Xho I and Bgl II, and BamH I and Bgl II were the same tail enzyme. By using this characteristic, the target fragments miR-ccr5 miR-pol and miR-vif in the vector were connected together to obtain the single plasmid pcDNA6.2-GW/EmGFP-ccr5-pol-vif, which was digested by pcDNA6.2-GW / Emccccr5-pol-vifEag I. The lentivirus expression vector pLenti6.3-ccr5-pol-vifwas constructed by using Gatewan recombination technique, and its correctness was verified by sequencing, and its inhibition efficiency on target gene and the expression of IFN- 尾 were detected by qRT-PCR, and the expression of CCR5-POL and VIF protein was detected by Western Blot. MTT assay was used to detect its toxicity to transfected cells. Results: sequencing results showed that the recombinant vector was successfully constructed by qRT-PCR. The results showed that pLenti6.3-ccr5-pol-vif could inhibit both ccr5pol gene and vif gene, and the inhibition efficiency was 25% and 13% respectively. The results showed that the recombinant vector was transfected into 293T-CCR5293T-POL and 293T-VIF cells with pLenti6.3-ccr5-pol-vif. Compared with the control group, the expression level of target protein decreased by 18% and 65%, respectively, while the expression of IFN- 尾 in transfected cells was not significantly increased. MTT assay confirmed that transfection of pLenti6.3-ccr5-pol-vif had no effect on cell survival. Conclusion 1. The lentivirus expression vector pLenti6.3-ccr5-pol-vifan2.The pLenti6.3-ccr5-pol-vif can simultaneously target the three genes of ccr5Pol and vif to play an inhibitory role. PLenti6.3-ccr5-pol-vif will not induce interferon effect or cytotoxicity in transfected cells, which provides the experimental basis for further research on anti-HIV-1.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.91


本文编号：1972842