【作者】 王久强；

【导师】 刘思当；

【作者基本信息】 山东农业大学 ， 基础兽医学， 2009， 硕士

【摘要】 艾滋病是由人免疫缺陷病毒(HIV)引起的人类免疫缺陷综合症。目前FDA批准的用于临床的抗HIV的药物只是逆转录酶抑制剂和蛋白酶抑制剂,这两类药物在艾滋病的治疗方面已经取得了巨大的进展。但由于病毒基因的变异,导致了耐药株的出现,导致常规治疗方案失效。因此研究新的抑制剂对于抑制HIV病毒在体内的复制具有重要意义。HR212是由本实验室构建的一种膜融合抑制剂,前期研究证实此蛋白能够有效的抑制HIV-1假病毒感染细胞,并且其结构稳定,能耐受蛋白酶酶切作用。此蛋白具有和T20相似的抗病毒活性,且能够对T20耐药株也具有良好的抑制效果。HR212除了能够抑制HIV-1外,还对HIV-2具有部分抑制效果。前期HR212是以融合表达的形式进行表达的,由于其氨基端存在非目的片段,故不能正确表明其膜融合性质。为向将来的药物临床应用提供资料,我们拟非融合表达了HR212,同时也检测HR212的免疫原性。将HR212基因连接于pBV220上,经诱导表达、纯化后免疫小鼠。经间接ELISA验证HR212按照传统方法免疫能够得到抗体滴度,而以10倍治疗剂量按照治疗程序多次注射时,却仅在1/1000时检测到抗体滴度,此结果为下一步降低其免疫原性的方法探讨具有重要意义。此外,为了研制治疗性疫苗,我们构建了HR212的腺病毒表达体系。通过将HR212连接到穿梭质粒pDC315上,然后与骨架质粒共转染239细胞,经过7天后获得具有复制缺陷的重组腺病毒rAd-TPA-HR212。经Western Blot验证,成功得到了13.3 kd的目的蛋白。经假病毒抑制试验证实,经腺病毒表达的HR212能有效的抑制假病毒介导的膜融合过程。为下一步研究奠定了基础。人工构建的五螺旋蛋白(5-helix)能够抑制HIV介导的膜融合过程中发卡三聚体的形成,从而抑制病毒感染靶细胞。但5-helix基因在原核细胞中直接表达时易形成包涵体,复性困难,给研究带来不便。通过同源建模,模拟了5-helix在pGEX-6P-1载体及pET44b载体上的融合蛋白形成的最有可能的两种构象。通过对比发现,其在pET44b载体中与NusA的融和蛋白的溶剂化能远大于在pGEX-6P-1中的GST蛋白,并且酶切位点位于蛋白表面。因此我们将HR212连接于pET44b上,构建重组载体pET44b-PSP-5Helix,经转化、诱导、纯化后,经HIV假病毒抑制试验证明,此蛋白具有明显的抑制HIV假病毒的活性。更多还原

【Abstract】 Acquired immune deficiency syndrome is human immudeficiency syndrome induced by human immunodeficiency virus. Currently, most approved anti-HIV drugs target two viral enzymes: reverse transcriptase and protease. Although clinically effective when used in combination, none of the existing drugs represent ideal therapies due to the long term toxic effects, and the ascendance of drug-resistant mutants. It is very important to research new inhibitors to inhibit virus reproduction in vivo.HR212 constructed by our laboratory is a kind of membrane fusion inhibitor , which can effectively inhibit HIV-1 pseudotype virus infecting cells and can endure the enzyme digestion because of its stable structure. Further studies indicates that it have similar antivirus activity with T20 and it has better effect to T20-persister virus. Otherwises, it can also inhibit HIV-2. Virus fusion inhibition experiments can not completely confirm its activity because there are no aim fragment in its N-terminus with fusion expression. In order to afford information for clinical utilization , we expressed HR212 with direct expression and detected its immunogenicity. Gene HR212 was linked to pBV220, and induced to express at 42℃. HR212 was then purified and immunized mice.Results of indirect ELISA show that it can get antibody titer at 1/4000 according to the traditional immunization method, whereas it can get can get antibody titer at 1/1000 when immunized at 10 times of therapy does ,which will lay the foundation to future study. In order to deplore remedial vaccine, we constructed adenovirus expression system of HR212. we linked HR212 to pDC315 to form pDC315-HR212, and then cotransfect cells with bone plasmid. After 7 days recombinant adenovirus rAd-TPA-HR212 was obverted. The result of Western blot shows that it successfully expresses 13.3 kd aim protein , and pseudotype virus inhibition research proved that HR212 expressed by adenovirus can effectively inhibit virus fusion , which will lay the foundation for further study.The artificial constructed 5-helix can inhibit the formation of trimer during the course of HIV-directed membrane fusion ,therefore it can inhibit virus infecting aim cells. However, 5-helix is apt to be expressed as inclusion body when directly expressed in prokaryocyte and is difficult to renature , which cause inconvenience to future research. By homology modeling ,we simulated the most possible two conformations of 5-Helix in vectors pGEX-6P-1 and pET44b. The contrast of conformations showed that the energy of salvation of its fusion protein with NusA in vector pET44b was higher than its fusion protein with glutathione-S-transferase(GST) in pGEX-6P-1 and its restriction site lay on the surface of its fusion protein in vector pET44b. Therefore HR212 was linked topET44b to construct recombinant vector pET44b-PSP-5Helix.When transformed ,induced and purified ,5-helix was used to detect its inhibition activity of HIV pseudotype virus .The results showed that it can obviously inhibit HIV pseudotype virus.更多还原