【作者】 蔺学芳；

【导师】 陈超；

【作者基本信息】 西北大学 ， 微生物， 2008， 硕士

【摘要】 人类免疫缺陷病毒(HIV)是引起艾滋病的病原体,分为两型:HIV-1和HIV-2,主要为HIV-1。它和一般的病毒有明显的差别,首先是其基因组相对复杂,编码更多的蛋白质来调控病毒潜伏、激活、转录与复制等过程,其次是它在宿主个体内变异迅速。鉴于HIV-1包膜糖蛋白gp120是HIV-1的主要抗原成分,是检测HIV抗体诊断试剂盒的重要组成成分,也是制备抗gp120单克隆抗体必需的免疫原,应用广泛,意义十分重大。本研究利用分子生物学手段,将gp120的部分编码基因(命名为gp120s)重组到大肠杆菌的表达载体中,并成功地进行了蛋白表达。外膜糖蛋白gp120s的编码基因的克隆和在大肠杆菌中的表达。用聚合酶链式反应PCR从HIV-1型国际标准株pHXB2-gp160的基因序列中扩增出糖蛋白片段基因537bp,插入质粒pET32a中构成重组表达质粒pET32a-gp120s,将其转化大肠杆菌BL21后获得了高效表达,经IPTG诱导表达后,SDS-PAGE和免疫印迹分析其表达产物证实蛋白得到表达且主要存在于包涵体中,凝胶扫描成像分析证实其表达量占菌体总蛋白的45.35%。在诱导4个小时蛋白表达量最大,以包涵体的形式表达。包涵体经过洗涤、溶解、复性和过镍柱后,其纯度达到85%,表明已获得高纯度的目的蛋白。用纯化的重组gp120蛋白作为包被抗原,间接ELISA对5份已知HIV阳性血清中的gp120抗体进行检测,结果表明该纯化蛋白具有一定的抗原活性,能特异性地与HIV抗体反应。本研究在大肠杆菌中成功表达了gp120s蛋白,为进一步发展HIV诊断试剂,研究AIDS亚单位疫苗奠定了一定的基础,为研究HIV主要抗原蛋白的性质提供可能,也为制备HIV-1的诊断抗原和基因工程重组疫苗打下基础。人细胞色素2A6作为细胞色素P450家族中重要的一员在许多药物的代谢过程中发挥着重要的作用。本研究利用体外重组技术,克隆及表达CYP2A6野生株,建立了一个研究CYP2A6酶的平台。通过对野生型进行酶动力学分析和药物抑制高通量筛选,测定酶动力学常数和药物抑制常数,所得数据可为以后研究其多态性等位基因在药物代谢中的作用和在新药开发早期进行新药评价提供重要的信息。通过定点突变法得到CYP2A6野生型的cDNA(1.5Kb),并将之克隆到酵母表达载体pYES2/CT上,通过测序得以证实;重组质粒转入已经整合了细胞色素P450氧化还原酶基因(POR)的酿酒酵母中,半乳糖诱导表达目的蛋白,利用SDS-PAGE和Western Blot分析表达产物,结果表明菌株得到表达,所得蛋白约为55Kd,与预期大小相同。大量诱导高表达菌株并制备微粒体蛋白,将所制备的微粒体用CO还原示差光谱法进行P450含量测定,在450nm处有吸收峰,含量为23.8 pmol/mg。用适量微粒体蛋白与2A6特异性荧光底物Coumarin进行酶学反应,所得数据用非线性回归分析法计算出Km值、Vmax值和内在清除率CLint值(Vmax/Km),结果表明:酶活很好,得到相应值均与相关报导相近。将底物浓度固定,与系列浓度的抑制剂共同孵育,用荧光检测技术测定已知抑制剂对重组CYP2A6野生型酶的抑制常数,结果表明:CYP2A6特异性抑制剂反苯环丙胺对重组CYP2A6野生型酶有特异性抑制,IC50约为0.05μM,与相关报道数据接近。本文同时用高效液相色谱法初步检测CYP4502A6的酶动力学,摸索条件,所得数据同荧光分析法计算其Km值、Vmax值和内在清除率CLint值(Vmax/Km),与荧光分析法比较,具有非常好的一致性,在说明这个酶稳定性好的同时,也说明这两种方法的科学一致性,使实验结果更加可信。本研究成功构建了CYP2A6野生型基因的酿酒酵母表达体系,所诱导制备的酵母微粒体蛋白活性良好而稳定,所建立的CYP2A6体外生化检测系统和药物高通量抑制筛选系统高效、灵敏、简便,从而为下一步研究与CYP2A6有关的药物-药物相互作用奠定基础,同时也有助于临床上的降低不良反应和提高药效。更多还原

【Abstract】 Human Immunodeficiency Virus ( HIV) is the pathogen that causes acquired immunodeficiency syndrome in human. It can be categorized into two types: HIV-1 and HIV-2. HIV-1 is the main type. It’s different from other viruses for its complicated genome related to the latency,activation,transcription and replication and for its variation in host cells.Envelope glycoproteins gpl20 is the main antigen of HIV-1 and important component of diagnosis kits for detecting HIV antibodies. It can be used to produce gp120 monoclonal antibodies. Considering its importance in applications, we have tried to choose part of the gpl20 gene (named gpl20s)to express in E. coli.DNA fragment 537bp was obtained by PCR from the plasmid pHXB2-gp160 and constructed into plasmid pET32a. The recombinant plasmid was transformed into E.coli BL21 (DE3) plys. After the induction by IPTG, the expressed protein was detected by SDS-PAGE and Western blot. It showed that the 40Kd fusion protein was mainly expressed in inclusion bodies with an amount that accounts to at least 43.35% of all E.coli proteins. We obtained very pure gp120s proteins (85%) by washing,dissolving and refolding the inclusion bodies. Primary applications of gp120 in detecting antibodies by indirect ELISA in HIV showed that it have good antigen.In this study we tried to express gp120s in E. coli, and succeeded in expressing gp120s in E.coli. This study has laid some ground work for further researches and development of HIV diagnosis reagents and subunit vaccines for AIDS. It’s also important for studying the properties of the main antigens of HIV. Human cytochrome P450 2A6 plays an important role in the metabolism of drugs and xenobiotics. PHarmacogenetic studies have shown that genetic polymorpHisms in this gene are important determinants of inter-individual and inter-ethnic variation in drug metabolism and toxicity. To develop a broadly applicable assay system for studying human CYP2A6 polymorpHic enzymes, we cloned and expressed the DNA of CYP2A6 wild-type(WT) in budding yeast Saccharomyces cerevisiaee, which was already integreted with CYP450 Oxidoreductase(POR). Using these recombinant enzymes in real-time biochemical assays and high throughput drug screening, the kinetic constants as well as inhibition constants for the test compounds were measured. The results we got can provid valuable information for studying the effects of polymorpHic genes on drug metabolism and evluating new drugs in the early pHase of drug discovery.Tempalte was obtained from DNA designed before.The wild-type DNA of CYP 2A6 was obtained by site-directed mutagenesis from the template . Transformed yeasts produced large quantities of microsome-bound 2A6 enzymes as determined by Western analysis, a 55 kDa protein was observed.The P450 content was assayed by reduced CO difference spectrum.There was a 450-nm peak observed in 2A6-WT, and itscontent respectively was 23.8pmol/mg.The isolated microsomes were used to measure the kinetic constants of 2A6 enzyme in real-time assays using a fluorogenic substrate Coumarin. The results showed that the enzyme possess robust activity, the Vmax value,Km value and Clint value were close with the related reports. The inhibition of recombinat CYP2A6-WT enzyme by known inhibitor drug was tested by serial titration of drugs in the fluorogenic assays. The results showed that: CYP2A6 was strongly inhibited by its known inhibitor tranylcypromine, IC50 was about0.05 mM. Used fluorogenic high throughput technique to detect the inhibition of 15 drugs on the active enzymes,the results showed :all of them can not inhibit 2A6. We also used HPLC to measure the kinetic constants of 2A6 enzyme using the fluorogenic substrate Coumarin. The results showed that the enzyme possess robust activity, the Vmax value,Km value and Clint value were close with real-time assays.It showed that 2A6 enzyme have robust stability and the two techniques have good consisitence.The experiments results can be convinced even more.For the first time,we successfully constructed the Saccharomyces cerevisiae expressing system for CYP2A6 gene; The catalytic activity of yeast microsome protein was robust and stable; Established an in vitro detection system for biochemical analysis and drug high throughput screening of CYP2A6 polymorpHic enzymes,which were efficient, sensitive and convenient. This work can guide the further study of CYP2A6 related drug-drug interaction,as well as can aid the future reducing adverse reaction and enhancing drug efficiency in vivo.更多还原