本文選題：I型人類(lèi)免疫缺陷病毒 + 三聯(lián)miRNA��； 參考：《南華大學(xué)》2013年碩士論文

【摘要】：背景及目的：RNAi技術(shù)在抗HIV-1研究方面表現(xiàn)出一定的優(yōu)勢(shì)和應(yīng)用潛力。針對(duì)HIV-1病毒的高突變性，本實(shí)驗(yàn)室在前期研究中，以ccr5、pol和vif基因?yàn)榘悬c(diǎn)構(gòu)建了穩(wěn)轉(zhuǎn)細(xì)胞株293T-CCR5、293T-POL和293T-VIF；針對(duì)3個(gè)靶基因各自設(shè)計(jì)和構(gòu)建了4個(gè)miRNA表達(dá)載體，從中各篩選出一個(gè)沉默效果最好的，記為pccr5-1-4，ppol-3-4和pvif-2-1。在此基礎(chǔ)上，本研究將3個(gè)miRNA串聯(lián)到一個(gè)表達(dá)載體上，并利用Gatewan重組技術(shù)構(gòu)建成慢病毒表達(dá)載體pLenti6.3-ccr5-pol-vif，理論上，該重組載體可同時(shí)作用于三個(gè)靶基因。將其轉(zhuǎn)染進(jìn)293T-CCR5細(xì)胞，293T-POL細(xì)胞和293T-VIF細(xì)胞，進(jìn)行功能分析，為進(jìn)一步開(kāi)展抗HIV-1研究提供基礎(chǔ)。方法：pcDNA6.2-GW/EmGFP載體上含有酶切位點(diǎn)BamH I，Xho I和Bgl II，且BamH I與Bgl II是同尾酶，利用這一特性將載體中目的片段miR-ccr5，miR-pol和miR-vif串聯(lián)起來(lái)，，獲得pcDNA6.2-GW/EmGFP-ccr5-pol-vif；Eag I單酶切載體pcDNA6.2-GW/EmGFP-ccr5-pol-vif后，利用Gatewan重組技術(shù)構(gòu)建成慢病毒表達(dá)載體pLenti6.3-ccr5-pol-vif，重組載體經(jīng)測(cè)序驗(yàn)證其正確性；通過(guò)qRT-PCR技術(shù)檢測(cè)其對(duì)靶基因的抑制效率和對(duì)IFN-β表達(dá)的影響；Western Blot法檢測(cè)其對(duì)CCR5、POL和VIF蛋白表達(dá)的影響；采用MTT法檢測(cè)其對(duì)被轉(zhuǎn)染細(xì)胞有無(wú)毒性傷害。結(jié)果：測(cè)序結(jié)果表明重組載體構(gòu)建成功；qRT-PCR結(jié)果顯示pLenti6.3-ccr5-pol-vif對(duì)ccr5、pol和vif基因都有一定抑制作用，抑制效率分別達(dá)到25%、35%和13%；Western Blot結(jié)果顯示轉(zhuǎn)染了pLenti6.3-ccr5-pol-vif的293T-CCR5、293T-POL和293T-VIF細(xì)胞中，靶蛋白表達(dá)水平與空白對(duì)照組相比分別降低18%、55%和65%(P0.01)；轉(zhuǎn)染細(xì)胞中IFN-β的表達(dá)未明顯增加； MTT實(shí)驗(yàn)證實(shí)轉(zhuǎn)染pLenti6.3-ccr5-pol-vif不會(huì)對(duì)細(xì)胞的存活率造成影響。結(jié)論：1.成功構(gòu)建了慢病毒表達(dá)載體pLenti6.3-ccr5-pol-vif；2. pLenti6.3-ccr5-pol-vif可同時(shí)靶向ccr5、pol和vif三個(gè)基因發(fā)揮抑制作用；3. pLenti6.3-ccr5-pol-vif不會(huì)引發(fā)轉(zhuǎn)染細(xì)胞中的干擾素效應(yīng)，也不會(huì)產(chǎn)生細(xì)胞毒性，為進(jìn)一步開(kāi)展抗HIV-1研究提供實(shí)驗(yàn)基礎(chǔ)。
[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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R512.91


本文編號(hào)：1972842