含抗HIV广谱中和抗体2F5、4E10靶基因载体的构建及鉴定

目的:构建含HIV gp120,gp41序列中广谱中和抗体2F5,4E10作用靶基因的载体并进行鉴定,为后期重组载体表达产物诱导产生中和抗体及抗HIV亚单位疫苗的研究奠定基础.方法:根据NCBI中HIV gp120,gp41基因序列中可与2F5、4E10结合的区域设计引物并进行PCR反应,将PCR得到的目的片段插入到载体pET28a中,对重组载体进行PCR鉴定、酶切鉴定及DNA测序.结果:PCR鉴定、酶切鉴定及DNA测序结果证实重组载体构建成功.结论:成功构建了含HIV gp120,gp41序列中广谱中和抗体2F5,4E10作用靶基因的载体.     

原核表达HIV-1抗原蛋白的ELISA活性检测

研究了HIV-1 gp120s和gp41在大肠杆菌中表达产物的抗原特异性.首先构建表达质粒p120s和pT41,并转化到BL21和M15中.宿主细胞经IPTG诱导表达重组蛋白,SDS-PAGE和Western blot表征分析蛋白产物.总蛋白中gp120s占17.9%,为可溶性表达;gp41占24.6%,为包涵体形式.亲和层析法检测蛋白纯度分别为63.1%和89%.酶联免疫吸附(ELISA)实验说明重组蛋白有良好的抗原特异性.     

影响HIV-GP41N端1/2在E.coli中表达的两段氨基酸序列界定

GP41蛋白二级结构预测显示 ,前 1 /2片段的 N端 ( 4～ 2 6位氨基酸 )和 C端 ( 1 67～ 1 89位氨基酸 )各有一个富含疏水氨基酸的穿膜螺旋 (可能性 >0 .9) ,分别从 N1 (前 1 /2片段无 C端穿膜螺旋 )的 N端和 N6(前1 /2片段无 N端穿膜螺旋 )的 C端进行缺失构建一系列缺失突变体 ,只有 p ET-N2 ,p ET-N3 ,p ET-N4,p ET-N5在大肠杆菌中获得高效表达 ,其表达量占菌体总蛋白的 2 0 % ,Western blot显示表达蛋白与 HIV阳性血清有良好的反应性 .而 p ET-N1 ,p ET-N6在大肠杆菌中表达量很低或不表达 .从而证明 1～ 6位 ,1 84～ 2 0 1位氨基酸是影响 gp41基因 N端 1 /2片段表达的主要因素     

HIV-1外膜蛋白gp120在酵母Pichia pastoris中的表达

目的 用巴斯德毕赤酵母系统表达HIV外膜糖蛋白gp120.方法 将从HIV-1型国际标准株pHXB2-gp160的基因序列中扩增出的gp 120分子的长片段基因(1 419 bp)和短片段基因(417bp)分别克隆入真核表达载体pPICZαA与pPICZB中,以电穿孔法转化酵母GS 115.用YPDS-zeo平板筛选重组子、PCR方法检测整合到酵母菌GS 115基因组中的gp 120片段基因,经甲醇诱导表达后,SDS-PAGE和免疫印迹分析表达产物.结果 诱导后gp 120短片段多肽在GS 115中少量表达,在诱导表达24 h重组蛋白表达量及抗原性达到最高,表达产物被降解,具有良好的抗原特异性.诱导后gp 120长片段多肽未被GS 115所表达.结论 在巴斯德毕赤酵母中成功表达gp 120分子长片段需要优化gp 120基因,为制备HIV-1的诊断抗原和基因工程重组疫苗打下基础.     

中国流行株HIV—1gp120及其与IFNα基因共表达核酸疫苗质粒的构建及表达

目的 研究pGP和pGPIFNα在真核细胞中的表达。方法 以表达中国流行株HIV－1gp120基因的核酸疫苗质粒pGP及其表达中国流行株HIV－1gp120基因与IFNα基因与IFNα基因的核酸疫苗质粒pGPIFNα。转染BHK－21细胞，以间接免疫荧光鉴定其表达产物。结果 荧光显微镜下，pGP和pGPIFNα转染细胞可见绿色荧光，而HK－21细胞和空质粒则不见绿色荧光。结论 pGP和pGPIFNα可在真核细胞中表达目的蛋白gp120。     

人纤溶酶原K1—3区基因在大肠杆菌中的表达

将人纤溶酶原Krigle 1-3(K1-3)基因插入融合表达载体pET-17b，获得重组质粒pET-K13,转化E.coli BI21(DE3),在IPTG诱导下，人纤溶酶原K1－3基因在E.coli BI21(DE3,pET-K13)中获得高效融合表达，表达量占菌体总蛋白的24％，表达产物以包函体存在，Western blot证明重组 蛋白对人纤溶酶原抗血清有特异免疫原性。     

HIV-1CRF07-BC gp41 HR2高突变区氨基酸结构和功能分析

HIV-1包膜蛋白ENV介导病毒进入靶细胞,这个过程可以作为设计抗艾滋病药物的一个重要靶点,进入抑制剂能够抑制HIV进入靶细胞,从而在感染的最初环节抑制病毒的传播.为了更好地筛选针对中国株CRF07-BC的进入抑制剂,建立了细胞-细胞融合系统.通过比较CRF07-BC gp41和B亚型gp41的序列同源性和融合能力,gp41 HR2存在着一个7个氨基酸的高突变区.通过结构生物学对gp41 HR2高突变区进行分析发现,高突变区内氨基酸可以在功能上互补,保持了gp41的融合能力.这种功能互补性的研究可以为HIV gp41进化提供理论参考,为HIV-1进入抑制剂的设计提供更准确的靶点.     

糖多孢红霉菌酮还原酶域在羰基还原中的应用

为研究糖多孢红霉菌聚酮合成酶模块1的酮还原酶域,催化羰基还原的底物特异性和立体选择性,PCR扩增了该酶域的编码基因(eryKR1),并将其克隆到表达载体pET-28a,得到重组质粒pET-eryKR1,转化到Escherichia coli BL21(DE3)后,获得了重组菌株E.coli BL21(pET-eryKR1).将E.coli BL21(pET-eryKR1)和异源表达枯草芽孢杆菌葡萄糖脱氢酶基因的重组大肠杆菌E.coli BL21(pET-gdh1)进行双重组菌耦合,对4-氯乙酰乙酸乙酯、苯乙酮、2-辛酮和环己酮4种底物进行转化还原,利用气相色谱分析转化液,结果显示E.coli BL21(pET-eryKR1)对环己酮的还原效果较好,产物环己醇的得率最高可达93.24%,而对其他3种底物几乎没有还原能力.利用E.coli BL21(pET-eryKR1)2催化2-甲基环己酮不对称还原,产物主要为顺式-2-甲基环己醇,产率可达41.87%,产物的对映体过量值为74.81%.     

Biological function of HIV-1 transmembrane protein gp41──A study on a putative cellular receptor of gp41

Since 1992, the study of biological functions of HIV-1 gp41 has made great progress. Experimental evidence from several research groups demonstrated that gp41 has a putative cellular receptor. A recombinant soluble gp41 (aa539-684) and gp41 immunosuppressive peptide (aa583-599) could bind to human B lymphocytes and monocytes, but weakly bind to T lymphocytes. It was found that gp41 contains two cellular binding sites (aa583-599 and 641-675). GP41 could selectively inhibit cell proliferation of human T, B lymphocytes and monocytes, enhance human MHC class Ⅰ, Ⅱ and ICAM-1 molecule expression on cell surface. Gp41 binding proteins and a monoclonal antibody against the first binding site could inhibit this modulation effect. Amino acid sequence homology exists between gp41 and human type Ⅰ interferons, and the homologous region is located in the first binding site on gp41 and in the receptor binding site on type Ⅰ interferons. Studies in other groups indicate that both binding sites in gp41 may be associated with HIV infection of cells. Peptides containing two binding sites could respectively inhibit HIV infection of cells. A monoclonal antibody recognizing the second binding site could neutralize lab-strains and recently separated strains of HIV-1. Besides, antibodies against two regions (homologous with gp41 binding sites) of SIV transmembrane protein gp32 could protect macaques from SIV infection. These results suggest that the study of gp41 binding sites and cellular receptor could contribute to understanding the mechanism of HIV infection and to developing HIV vaccine and anti-HIV drugs.     

Prevention of HIV-1 glycoprotein transport by soluble CD4 retained in the endoplasmic reticulum

The envelope glycoprotein (gp120/41) of the human immunodeficiency virus (HIV-1) attaches the virus to the cellular CD4 receptor and mediates virus entry into the cytoplasm. In addition to being required for formation of infectious HIV, expression of gp120/41 at the plasma membrane causes the cytopathic fusion of cells carrying the CD4 antigen. The expression of gp120/41 is therefore an ideal target for therapeutic strategies designed to combat AIDS. Here we show that expression of a soluble CD4 molecule, mutated to contain a specific retention signal for the endoplasmic reticulum, blocks secretion of gp120 and surface expression of gp120/41, but does not interfere with transport of wild-type CD4. By blocking transport of the HIV glycoprotein, this retained CD4 molecule prevents the fusion of CD4 cells that is normally caused by the HIV glycoprotein. Expression of the retained CD4 molecule in human T cells might therefore be useful in the intracellular immunization procedure suggested by Baltimore.     

Biological function of H1V-1 transmembrane protein gp41

Since 1992, the study of biological functions of HIV-1 gp41 has made great progress. Experimental evidence from several research groups demonstrated that gp41 has a putative cellular receptor. A recombinant soluble gp41 (aa539–684) and gp41 immunosuppressive peptide (aa583–599) could bind to human B lymphocytes and monocytes, but weakly bind to T lymphocytes. It was found that gp41 contains two cellular binding sites (aa583–599 and 641–675). GP41 could selectively inhibit cell proliferation of human T, B lymphocytes and monocytes, enhance human MHC class I, II and ICAM-1 molecule expression on cell surface. Gp41 binding proteins and a monoclonal antibody against the first binding site could inhibit this modulation effect. Amino acid sequence homology exists between gp41 and human type I interferons, and the homologous region is located in the first binding site on gp41 and in the receptor binding site on type I interferons. Studies in other groups indicate that both binding sites in gp41 may be associated with HIV infection of cells. Peptides containing two binding sites could respectively inhibit HIV infection of cells. A monoclonal antibody recognizing the second binding site could neutralize lab-strains and recently separated strains of HIV-1. Besides, antibodies against two regions (homologous with gp41 binding sites) of SIV transmembrane protein gp32 could protect macaques from SIV infection. These results suggest that the study of gp41 binding sites and cellular receptor could contribute to understanding the mechanism of HIV infection and to developing HIV vaccine and anti-HIV drugs.     

盘基网柄菌野生型KAx-3和突变型AK127细胞mRNA差异显示分析

为研究gp150蛋白调控盘基网柄菌发育相关基因的功能,用mRNA差异显示技术比较分析盘基网柄菌发育14 h的KAx-3细胞和AK127细胞.结果显示两种类型细胞基因表达有明显差异,突变细胞不能表达275 bp片段.对其测序分析后发现差异片断与编码组氨酸激酶、STATc蛋白及同源框蛋白等的基因中的一段序列有很高的相似性.推测gp150蛋白的缺失引起某些调控因子表达异常,从而使突变细胞的基因表达不正常,最终导致细胞不能完成多细胞发育.     

Structure of an unliganded simian immunodeficiency virus gp120 core

Envelope glycoproteins of human and simian immunodeficiency virus (HIV and SIV) undergo a series of conformational changes when they interact with receptor (CD4) and co-receptor on the surface of a potential host cell, leading ultimately to fusion of viral and cellular membranes. Structures of fragments of gp120 and gp41 from the envelope protein are known, in conformations corresponding to their post-attachment and postfusion states, respectively. We report the crystal structure, at 4 A resolution, of a fully glycosylated SIV gp120 core, in a conformation representing its prefusion state, before interaction with CD4. Parts of the protein have a markedly different organization than they do in the CD4-bound state. Comparison of the unliganded and CD4-bound structures leads to a model for events that accompany receptor engagement of an envelope glycoprotein trimer. The two conformations of gp120 also present distinct antigenic surfaces. We identify the binding site for a compound that inhibits viral entry.     

用杆状病毒表达系统表达的HIV-2 gp105和gp36的反应原性与抗原特异性分析

用ELISA方法分析由杆状病毒/昆虫细胞表达系统表达的HIV-2外膜蛋白gp105和跨膜蛋白gp36的反应原性和特异性. 结果表明, 二者均具有很好的反应原性和抗原特异性, 可作为免疫抗原和检测抗原加以应用.     

新型呼肠病毒S1基因与宿主细胞相互作用的初步研究

摘要 目的 筛选出新型呼肠病毒S1基因和宿主相互作用的关键区段,为后续的宿主受体蛋白筛选工作提供可靠的基础。 方法 将编码全长S1基因,以及N端和C端阅读框分别克隆入真核表达载体pIRSE2-EGFP,转染敏感细胞株鼠成纤维细胞（L929）和人宫颈癌细胞（Hela）后,通过荧光报告基因EGFP的表达分析,筛选出S1基因与宿主细胞相互作用时起决定性影响的区段。 结果 同等量的3种真核表达质粒在单独转染L929时,pGSN质粒转染后荧光表达量最大,pGSC质粒转染后荧光表达量最小。 不同比例混合的pGS与pGSN进行共转染时,pGSN在转染质粒中的比例越高,荧光表达量也越高。 在Hela细胞中的转染结果与L929相同。 结论 新型呼肠病毒R4株的S1基因N端阅读框编码区（62-406bp）在S1基因转染时起关键作用。     

An engineered poliovirus chimaera elicits broadly reactive HIV-1 neutralizing antibodies

The Sabin type 1 vaccine strain of poliovirus is probably the safest and most successful live-attenuated vaccine virus used in humans. Its widespread use since the early 1960s has contributed significantly to the virtual eradication of poliomyelitis in developed countries. We have reported previously the construction of an intertypic antigen chimaera of poliovirus, based on the Sabin 1 strain, and proposed that this virus could be modified to express on its surface antigenic determinants from other pathogens. We describe here the construction and characterization of a poliovirus antigen chimaera containing an epitope from the transmembrane glycoprotein (gp41) of human immunodeficiency virus type 1 (HIV-1). In antibody absorption experiments, the virus chimaera inhibited neutralization of HIV-1 by antipeptide monoclonal antibodies specific for the gp41 epitope and significantly reduced the group specific neutralizing activity of HIV-1-positive human sera. Rabbit antisera raised by subcutaneous injection of the polio/HIV chimaera in adjuvant was shown to be specific for HIV-1 gp41 in peptide-binding assays and by western blotting. Moreover, the antisera neutralized a wide range of American and African HIV-1 isolates and also inhibited virus-induced cell fusion. Monoclonal antibodies against the HIV-1 derived regions of the chimaera also neutralized HIV-1. These results establish the potential of using poliovirus for the presentation of foreign antigens and suggest that Sabin 1 poliovirus/HIV chimaeras could offer an approach to the development of an HIV vaccine.