本文選題：人類免疫缺陷病毒 + 載脂蛋白B��； 參考：《北京工業(yè)大學(xué)》2012年碩士論文

【摘要】：艾滋病仍是目前嚴(yán)重威脅人類健康的一種重大疾病，盡管高效抗逆轉(zhuǎn)錄病毒療法能有效控制HIV-1的復(fù)制，，然而高昂的治療費(fèi)用、病毒耐藥性和藥物毒性，阻礙了HIV-1的有效治療，因此急需研發(fā)具有新靶點(diǎn)的新型抗HIV-1藥物和新的治療手段。 載脂蛋白B mRNA編輯酶催化多肽樣蛋白3G（Apolipoprotein BmRNA-editing Enzyme Catalytic Polypeptide-like3G，APOBEC3G，簡(jiǎn)稱A3G）是機(jī)體固有的抗病毒因子，但HIV-1的病毒感染因子（Viral Infectivity Factor，Vif）經(jīng)泛素-蛋白酶體途徑介導(dǎo)A3G降解，從而拮抗其抗HIV-1活性。人A3G上第128位氨基酸與Vif拮抗A3G活性的種屬特異性有關(guān)。 本文利用重疊PCR技術(shù)將人A3G上第128位天冬氨酸分別突變?yōu)橘嚢彼岷途彼�，�?gòu)建了兩個(gè)可與綠色熒光蛋白融合表達(dá)的pEGFP-A3G D128K和pEGFP-A3G D128R真核表達(dá)載體。將pEGFP-C3或真核表達(dá)載體轉(zhuǎn)染239FT和HeLa細(xì)胞，激光掃描共聚焦顯微鏡觀察到野生型和突變型GFP-A3G融合蛋白均定位于胞漿中。采用Western Blot和Real-time PCR技術(shù)檢測(cè)發(fā)現(xiàn)，在蛋白水平上，突變保護(hù)了94.85%的GFP-D128K和97.09%的GFP-D128R不被降解；在mRNA水平上，Vif對(duì)野生型及突變型A3G mRNA拷貝數(shù)沒(méi)有明顯影響。采用HIV-1假病毒單輪感染實(shí)驗(yàn)發(fā)現(xiàn)，高劑量組野生型與突變型A3G（100ng和50ng）對(duì)HIV-1的抑制作用均較強(qiáng)，抑制率均在90%以上；中等劑量組野生型A3G（20ng、10ng和5ng）對(duì)HIV-1的抑制率在25%~38%之間，突變型A3G的抑制率在80%~90%之間；低劑量組野生型與突變型A3G（1ng和0.1ng）幾乎沒(méi)有抑制作用。研究顯示突變型A3G可以抵抗Vif的降解作用，抑制HIV-1病毒的感染。
[Abstract]:AIDS is still a serious threat to human health. Although highly effective antiretroviral therapy can effectively control the replication of HIV-1, the high cost of treatment, drug resistance and drug toxicity hinder the effective treatment of HIV-1. Therefore, it is urgent to develop new anti-HIV-1 drugs and new treatment methods with new targets. Apolipoprotein B mRNA editing enzyme catalyzes the degradation of 3G(Apolipoprotein BmRNA-editing Enzyme Catalytic polypeptide-like 3GG (APOBEC3G), which is an inherent antiviral factor in the body, but the viral infection factor of HIV-1, virus Infectivity factor vif, is mediated by ubiquitin proteasome pathway to degrade A3G, thus antagonizing its anti-HIV-1 activity. The 128th amino acid on human A3G is related to the species specificity of Vif antagonizing A3G activity. In this paper, two eukaryotic expression vectors of pEGFP-A3G D128K and pEGFP-A3G D128R, which can be expressed with green fluorescent protein, were constructed by mutating aspartic acid at 128th position on human A3G into lysine and arginine by overlapping PCR technique. PEGFP-C3 or eukaryotic expression vector was transfected into 239FT and HeLa cells. Both wild-type and mutant GFP-A3G fusion proteins were detected by confocal laser scanning microscopy. Western Blot and Real-time PCR techniques showed that 94.85% of GFP-D128K and 97.09% of GFP-D128R were protected from degradation at protein level, while at mRNA level, VIF had no significant effect on the copy number of wild and mutant A3G mRNA. The results of HIV-1 pseudovirus single rotavirus infection showed that the inhibition rates of wild type and mutant type A3G(100ng and 50ng on HIV-1 in high dose group were all more than 90%, and the inhibition rate on HIV-1 was between 25% and 38% in middle dose group with wild type A _ 3G _ (20) G ~ (2 +) (10 ng and 5 ng). The inhibitory rate of mutant A3G was between 80% and 90%, while wild type and mutant type A3G(1ng and 0.1 ng in low dose group had little inhibitory effect. Studies have shown that mutant A3G can resist the degradation of Vif and inhibit the infection of HIV-1 virus.
【學(xué)位授予單位】：北京工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R373

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 楊怡姝;李嵐;李澤琳;曾毅;;激光掃描共聚焦顯微鏡研究APOBEC3G蛋白的亞細(xì)胞定位[J];病毒學(xué)報(bào);2007年01期

本文編號(hào)：1899141