【作者】 蒋卫民；

【导师】 翁心华；

【作者基本信息】 复旦大学 ， 内科学， 2004， 博士

【摘要】 背景:艾滋病(AIDS)是一种严重危害人类健康的传染病[1],目前国际上多采用高效抗逆转录病毒治疗(HAART)该疾病,HAART能在短期内将病毒载量控制在很低的水平,病人的免疫功能很快恢复,从而可以延长病人的生命[2]。但病人体内的病毒尚不能完全清除,一旦条件成熟,这些病毒又会释放到血液中,并且复制繁殖,对免疫系统进行破坏[3]。且HAART费用昂贵,有一定的副作用,并易产生耐药[4]。而控制HIV感染的根本措施在于疫苗的研制[5]。多肽疫苗是用人工合成产生的蛋白稳定片段在黑猩猩体内诱导抗HIV的中和抗体[6]。多肽疫苗与核酸疫苗一样是目前HIV疫苗研究较受重视的领域之一[7]。1999年美国NIH公布了两种HIV-1病毒多肽疫苗,对人体进行的临床实验结果证实两种多肽能刺激机体产生特异性抗体和特异性细胞免疫,并有较好的安全性[8]。但是目前单一的多肽疫苗抗原性较差,产生的中和抗体较弱,无保护及治疗作用,而复合性多肽疫苗具有较强的抗原性,必将成为新的候选疫苗[9]。通过对我国主要流行区28名HIV-1感染者的基因亚型分析发现,我国的HIV-1以B亚型为主,DNA直接测序发现p24基因和gp41基因序列相对保守。通过软件分析,p24-gp41融合蛋白表达后,其各自的空间构象无改变,结合位点仍然存在,为线性中和表位。因此,用基因重组技术将p24和gp41基因重组构建质粒,并在大肠杆菌中高效表达融合蛋白p24-gp41[10],为进一步动物试验,研制治疗性疫苗提供基础。本课题共分三部分。第一部分 HIV-1p24蛋白编码区基因变异性的研究以及p24蛋白和gp41蛋白编码区基因片段的获得目的:用RT-PCR方法获得HIV-1p24蛋白编码区基因片段以及gp41截短体蛋白编码区基因片段。了解我国HIV感染者p24蛋白编码区的基因变异情况。方法:抽提HIV感染者血浆中总的RNA,反转录成互补的DNA(cDNA),设计带有酶切位点的引物,用巢式PCR方法扩增出所需要的目的基因片段,经琼脂糖<WP=5>凝胶电泳验证,并进行PCR产物的纯化。使用DNA测序仪直接测序;应用CLUSTAL W、PHYLIP等软件对序列进行比对及进化树分析。结果:28份p24样本中25份为B亚型,3份为A亚型;与共享序列相比,25例B亚型的p24编码区中发生核苷酸改变的位点比例为0.4%～4.8%,平均为1.4%,其中A->G占20.5%,G->A占17.3%,3例A亚型发生核苷酸改变的位点比例平均为0.24%,在所有变异位点中均未发现G->A的过度突变;B亚型内的基因离散率平均为2.9%,A亚型内的基因离散率平均为0.58%,A亚型与B亚型之间的基因离散率平均为11.1%。25例B亚型根据基因序列所推测的p24蛋白发生氨基酸改变的比例为0.4%～5.2%,平均为2.2%;进化树分析表明在我国河南地区HIV感染B亚型多为与泰国株相近的B’亚型。HIV-1p24编码区仍相对保守。而根据文献报道,gp41编码区则高度保守。琼脂糖电泳显示p24和gp41基因片段PCR产物条带与所要扩增片段相吻合。第二部分 p24-gp41基因的连接以及表达载体的构建目的:用基因重组技术将HIV-1p24基因以及gp41基因的截短体重组连接,构建重组表达质粒。方法:将PCR扩增后得到的p24和gp41的截短体两个基因分别连接到pGEM-T easy、pMD18—T载体中,测序验证后,挑选出含有目标基因的正确克隆。然后将p24片段连接到pGEM-T easy载体中,gp41截短体基因片段连接到pMD18—T载体中,p24片段经酶切后连接到gp41截短体基因所在的pMD18—T载体中,再将连接后的两个基因酶切,重新连接到pET21a表达载体中,经酶切验证和测序验证均证明两个基因已经正确连接到终载体中,并且保证了读码框的正确。结果:p24-gp41融合蛋白的表达载体pET21a 构建成功,经酶切验证和测序验证正确。第三部分p24-gp41 融合蛋白的表达及鉴定(Western-Blot鉴定)<WP=6>目的:在大肠杆菌中表达融合蛋白p24-gp41,并经Western blot验证。方法:将终载体转化大肠杆菌,经IPTG(异丙基-β-硫代半乳糖吡喃糖苷)诱导,在大肠杆菌中得到表达。并经Western blot验证表达正确。结果:融合蛋白p24-gp41在大肠杆菌中表达,经Western blot验证确实。结论:融合蛋白p24-gp41可以在pET21a表达载体中表达,为治疗性疫苗的研制打下基础。更多还原

【Abstract】 Background: AIDS (acquired immunodeficiency syndrome) is a devastating disease. Since 1980s, there has been more than 68 million people infected with human immunodeficiency-1 (HIV-1) and more than 28 million people have died. Highly active antiretroviral therapy (HAART) is an effective therapy against HIV, and has led to profound decreases in morbidity and mortality rates in HIV-1-infected persons. Many infected persons have plasma levels of HIV-1 RNA that are less than the limits of detection of most clinical assays due to combination antiretroviral therapy. Nonetheless, HIV-1 has not been eradicated by HAART. Therefore, vaccine is a fundamental method to get rid of HIV-1.Currently, candidates of recombinant HIV gp120 protein vaccine are unable to generate antibodies capable of neutralizing infectivity of primary isolates from patients. Here, “fusion-competent HIV vaccine” immunogenes were generated to capture the transient envelop-CD4-coreceptor structures that arise during HIV binding and fusion. Development of these fusion-dependent immunogens may lead to a wide-range effective HIV vaccine. Construction of vector of HIV-1 p24 and gp41 fusion gene and expression, purification of p24-gp41 fusion protein in E.coli lays ground for therapeutic vaccine.Part one Sequence variation of p24 coding region in gag gene of human immunodeficiency virus type 1,Acquisition of HIV-1 p24 protein region encoding gene and gp41 protein region encoding gene <WP=8>Objective To acquire HIV-1 p24 protein region encoding gene and gp41 protein region encoding gene using by RT-PCR. To comprehend the variation of p24 coding region in p24 protein of HIV-1 in China.Method Total RNA in plasma of HIV-1 infected human were extracted,and RNA were retranscripted into cDNA,the primer with restriction endonuclease site were designed ,target gene segment were amplified by nest PCR.The sequence of p24 region,680 nucleotides were determined,then phylogenetic analyses were performed.Result Of 28 specimens,25 were B subtype,and 3 were A subtype. Comparing to the consensus sequence, nucleotide variation in B subtype was 0.4%~4.8%, with an average of 1.4%. The transition changes of A-to-G and G-to-A were 20.5% and 17.3% respectively. While the G-to-A hypermutation was not observed. The intrasubtype distance for B subtype was 2.9%, and the substitution for predicted amino acid was 0.4%~5.2%. Because of the small number, the variation in A subtype was much lower than that of B subtype. Phylogenetic analyses implied that most of HIV-1 B subtype infections in Henan province were close to Thailand isolate. Acquisition of gene fragment of HIV-1 region encoding p24 protein and region encoding gp41 protein is corresponding to p24 and gp41 of HXB2. The changes of p24 coding region of HIV-1 patients in this study was still relatively conservative. And gp41 coding region of HIV-1 was highly conservative.Part two Ligation of p24 and gp41 gene and construction of expression vectorObjective To ligate p24 and gp41 gene and to construct expression vector of p24 -gp41 fusion protein. <WP=9>Method Both of p24 and gp41 gene were ligated into pGEM-T easy vector and pMD18—T vector. The target DNA fragment amplified from p24 and gp41 gene were sequenced after T-A cloning. The post-linked gene were cleaved and linked into pET21a vector. The correct sequence was proved by enzyme incision and sequence test and open reading frame was correct.Result Expression vector pET21a of p24-gp41 fusion protein were constructed successfully. The correct sequence was proved by enzyme incision and sequence test and open reading frame was correct.Part three Expression of p24-gp41 fusion protein and proven by Western blottingObjective To express p24-gp41 fusion protein in E.coli and then proved by Western-Blot.Method The vector pET21a was transformed into E.coli.p24-gp41 fusion protein induced by IPTG (isopropyl-beta-thiogalactopyranoside) was examined by SDS-PAGE. That the expressed product could react with serum of 6×his antibody were proved by Weste更多还原