APOBEC3G突变体抗HIV-1活性研究

发布时间：2018-05-17 00:40

本文选题：人类免疫缺陷病毒 + 载脂蛋白B　；参考：《北京工业大学》2012年硕士论文

【摘要】：艾滋病仍是目前严重威胁人类健康的一种重大疾病，尽管高效抗逆转录病毒疗法能有效控制HIV-1的复制，，然而高昂的治疗费用、病毒耐药性和药物毒性，阻碍了HIV-1的有效治疗，因此急需研发具有新靶点的新型抗HIV-1药物和新的治疗手段。载脂蛋白B mRNA编辑酶催化多肽样蛋白3G（Apolipoprotein BmRNA-editing Enzyme Catalytic Polypeptide-like3G，APOBEC3G，简称A3G）是机体固有的抗病毒因子，但HIV-1的病毒感染因子（Viral Infectivity Factor，Vif）经泛素-蛋白酶体途径介导A3G降解，从而拮抗其抗HIV-1活性。人A3G上第128位氨基酸与Vif拮抗A3G活性的种属特异性有关。本文利用重叠PCR技术将人A3G上第128位天冬氨酸分别突变为赖氨酸和精氨酸，构建了两个可与绿色荧光蛋白融合表达的pEGFP-A3G D128K和pEGFP-A3G D128R真核表达载体。将pEGFP-C3或真核表达载体转染239FT和HeLa细胞，激光扫描共聚焦显微镜观察到野生型和突变型GFP-A3G融合蛋白均定位于胞浆中。采用Western Blot和Real-time PCR技术检测发现，在蛋白水平上，突变保护了94.85%的GFP-D128K和97.09%的GFP-D128R不被降解；在mRNA水平上，Vif对野生型及突变型A3G mRNA拷贝数没有明显影响。采用HIV-1假病毒单轮感染实验发现，高剂量组野生型与突变型A3G（100ng和50ng）对HIV-1的抑制作用均较强，抑制率均在90%以上；中等剂量组野生型A3G（20ng、10ng和5ng）对HIV-1的抑制率在25%~38%之间，突变型A3G的抑制率在80%~90%之间；低剂量组野生型与突变型A3G（1ng和0.1ng）几乎没有抑制作用。研究显示突变型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.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373

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