HIV-1 Gag蛋白酵母表达产物的纯化及免疫原性检测

目的 纯化HIV-1Gag蛋白，并检测其免疫原性。方法 将表达HIV-1Gag蛋白的酵母工程菌GS115／pPICGAG接种到YPD培养基中多次传代后，用PCR检测其目的基因。该酵母工程菌在BMMY培养基中经甲醇诱导表达，上清液初步浓缩后上柱进行凝胶过滤层析，并将纯化蛋白免疫Balb／c小鼠，检测其血清抗体水平。结果 该酵母工程菌经多次传代后外源基因不丢失。Western blot检测结果表明纯化蛋白具有很好的反应原性，其纯度可达85％。纯化蛋白免疫小鼠后可诱导特异性抗体产生。结论 表达HIV-1Gag蛋白的酵母工程菌具有很好的遗传稳定性，其表达产物可诱导小鼠产生特异性体液免疫应答。     

HIV-1中国株CN54 Gag蛋白在毕赤酵母中的表达纯化和鉴定

目的　利用毕赤酵母表达系统高效表达HIV 1中国株CN5 4的Gag蛋白,初步确立发酵和纯化工艺。方法　PCR扩增CN5 4Gag基因,插入毕赤酵母表达载体pPS1 0 ,电转化毕赤酵母菌株GS115 ,G4 18筛选高表达工程菌,利用5L发酵罐进行高密发酵,通过SPSepharoseFF和DEAESeparoseFF柱层析,用HIV 1阳性血清和p2 4抗原检测试剂盒对纯化后的Gag蛋白进行免疫学性质的鉴定。结果　构建了高效表达CN5 4Gag蛋白的毕赤酵母工程菌株,发酵罐培养的菌体密度吸光度(A6 0 0 值)超过30 0 ,Gag蛋白表达量达到12 0mg L。纯化后的Gag蛋白纯度高于90 % ,p2 4检测强阳性并能够与HIV 1阳性血清发生特异的抗原抗体结合反应。结论　本研究在毕赤酵母中高效表达了HIV 1中国株CN5 4的Gag蛋白,并进行了纯化和鉴定,为针对我国新一代HIV蛋白亚单位疫苗的研制奠定了基础。     

HIV-1 p15(Gag)基因的克隆、蛋白表达及抗体制备

目的 为了研究HIV-1 p15(Gag)的生物学活性,制备HIV-1 p15(Gag)蛋白及其特异性抗体.方法 用PCR的方法扩增编码p15(Gag)基因序列,将其克隆到原核表达载体pET28a( )中表达HIV-1 p15蛋白,分别用His抗体和HIV阳性血清做western blot鉴定目的 蛋白.以纯化目的 蛋白为抗原免疫日本大耳白兔,制备多克隆抗体.通过酶联免疫吸附实验(ELISA),免疫细胞化学法检测抗体滴度及其特异性.结果 原核表达载体pET28 a( )-p15(Gag)成功构建,可在大肠杆菌BL21 (DE3)中诱导表达,得到相对分子质量约20000的p15(Gag)蛋白经western blot鉴定正确.纯化蛋白免疫家兔,制备的多克隆抗体具有较强免疫特异性.结论 得到纯化的HIV-1 p15蛋白,制备的多克隆抗体能够检测自然状态下病毒蛋白p15(Gag),为进一步研究HIV-1奠定了实验基础.     

口蹄疫病毒 VP1 蛋白在酵母中的表达及免疫原性分析

目的 :利用毕赤酵母表达系统表达牛O型口蹄疫外壳蛋白(FMDVVP1) ,并对表达的蛋白进行免疫原性鉴定。方法 :将FMDVvp1基因克隆到毕赤酵母Pichiapastoris分泌性表达载体pSuperY中 ,构建重组表达载体pSuperY/vp1,经测序证明vp1基因序列的正确性。将纯化的重组质粒经线性化酶切后 ,用电转化法将pSuperY/vp1导入毕赤酵母菌种SMD116 8H中。对表达产物用SDS PAGE和Westernblot进行分析 ,并用酵母表达的FMDVVP1蛋白免疫小鼠。结果 :以重组质粒pSuperY/vp1转化毕赤酵母菌后 ,能表达相对分子量 (Mr)为 6 6 0 0 0和4 30 0 0的FMDVVP1蛋白。动物免疫结果表明 ,FMDVVP1蛋白能诱导小鼠产生特异性的体液和细胞免疫应答。结论 :在毕赤酵母中成功地表达FMDVVP1蛋白 ,为研制新型FMDVVP1的基因工程疫苗奠定了基础     

中国HIV-1C亚型代表株候选疫苗免疫原性的研究

1981年发现艾滋病至今,艾滋病毒(ＨＩＶ)累积感染了近5000万人,造成近1400万人死亡,其主要感染人群是青壮年[1]。ＨＩＶ的蔓延给国家社会和经济的发展带来严重的挑战,并会对公共财政系统造成巨大的压力。我国ＨＩＶ1的流行自1994年以后进入快速增长期,ＨＩＶ1多种亚型(Ａ、Ｂ、Ｂ’、Ｃ、Ｄ、Ｅ、Ｆ)在我国流行[2],如果不尽快控制其流行,到2010年累积感染者有可能突破1000万人[1]。有效的疫苗是控制ＨＩＶ流行的根本手段。我国流行亚型主要以Ｂ’亚型和Ｃ亚型为主,大约占流行亚型的45%和36%。针对这一流行趋势,我们在杆状病毒系统表达了我国…     

HIV-1CN54 DNA疫苗滴鼻免疫小鼠诱发系统和黏膜免疫应答

目的：探讨重组痘苗病毒rVVsyngp120或rVVmCN54gp120候选疫苗是否增强HIV-1CN54合成gp120基因(syngp120)DNA疫苗的免疫原性。方法：第0、7、14、21天用DNA疫苗滴鼻免疫小鼠,第28、35、42天再滴鼻接种rVVsyngp120或rVVmCN54gp120。体外测脾和肠系膜淋巴结(MLN)淋巴细胞增殖应答与CD8^ CTL应答。测血清和黏膜洗液特异的IgG和IgA,并测其是否中和实验室适应株HIV-1SF33。结果：单纯DNA免疫后,脾和MLN淋巴细胞在体外发生增殖应答和CTL应答,且测出血清特异的IgG和黏膜洗液特异的IgA。重组痘苗病毒末次免疫后第2周(第56天),发现rVVmCN54gp120增强MLN淋巴细胞增殖应答和CTL应答,脾CTL应答也增强。rVVsyngp120则增强MLN CTL应答。同时发现：2组重组痘苗病毒免疫的动物其血清中特异IgG抗体滴度均有所增高,但黏膜(粪便和阴道)洗液特异IgA抗体滴度却未增高,未测出血清特异IgA和黏膜洗液特异IgG。免疫血清可中和HIV-1SF33,而阴道洗液却不能。结论：单纯DNA疫苗滴鼻免疫可诱发较弱的系统和黏膜体液免疫与细胞免疫,但维持时间短。重组痘苗病毒主要增强局部黏膜的细胞免疫应答,且稍增强系统体液免疫应答,未增强黏膜的IgA应答。免疫血清有中和作用。     

中国株HIV-1核心蛋白Gag核酸疫苗的构建与表达

目的 构建含HIV－1中国流行株B亚型核心蛋白Gag基因的真核表达质粒，并在体外进行表达和鉴定。方法 将Gag基因插入到核酸疫苗载体质粒pVAX1中，构建真核表达质粒pVAXGAG。用脂质体法。将重组质粒转染人Hela细胞后进行表达产物的检测。结果 间接免疫荧光检测显示，转染重组质粒的细胞表面有绿色荧光。Western blot和Dot ELISA分析显示，重组质粒转染细胞的裂解物中存在表达的Gag蛋白。结论 构建的核酸苗可在体外进行表达，且表达的蛋白具有特异性。     

HIV-1 Gag、Tat、Rev和Nef蛋白特异性的免疫应答

目的：探讨中国HIV／AIDS患者HIV—1 Gag、Tat、Rev和Nef蛋白特异性CTL应答的特征。方法：应用覆盖HIV-1 B、C亚型Gag、Tat、Rev和Nef蛋白的220个肽段作为抗原，通过ELISPOT方法俭测HIV／AIDS患者HIV特异性CTL应答。结果：无沦HIV—1 B亚型还是HIV-1C亚型所构建肽库的应答强度和频率，主要集中在Gag和Nef蛋白，Tat和Rev蛋白也有不同程度的应答。HIV—1 B、C亚型间应答比较，整体应答强度大致相同，但免疫优势区间存在着一定的差异，B亚型Gagp24亚蛋白的288～313氨基酸区应答最强，而C亚型Gagp24亚蛋白的155～181氨基酸区应答最强；两个亚型免疫优势区应答频率最高的都是Nef蛋白106～143氨基酸区(48．1％)。结论：中国人群CTL应答多集中在Gag和Nef蛋白，B、C业型间略有差异且存在交叉识别，这对设计针对中国人群的HIV疫苗是有重要的意义。     

HIV-1蛋白酶基因的克隆、蛋白表达及抗体制备

目的为了研究HIV-1蛋白酶的生物学活性,制备HIV-1 PR蛋白及其特异性抗体。方法用PCR方法扩增编码PR的基因序列,将其克隆到原核表达载体pET28a(+)中,并表达HIV-1 PR蛋白,用His抗体为一抗做Western blot鉴定目的蛋白。以纯化的目的蛋白为抗原免疫日本大耳白兔,制备多克隆抗体。通过酶联免疫吸附实验(ELISA),免疫细胞化学法检测抗体滴度及其特异性。结果原核表达载体pET28 a(+)-PR成功构建,并可在大肠杆菌BL21(DE3)中诱导表达,得到的PR蛋白经SDS-PAGE和Western blot鉴定正确。用纯化蛋白免疫家兔,制备的多克隆抗体具有较强免疫特异性。结论得到纯化的HIV-1PR蛋白,制备的多克隆抗体能够检测自然状态下病毒蛋白PR,为进一步研究HIV-1奠定了实验基础。     

FSH-occludin融合蛋白在毕赤酵母中的表达纯化及免疫原性检测

目的 纯化FSH-occludin融合蛋白,并检测其免疫原性.方法 将表达FSH-occludin 蛋白的酵母工程菌GS115/ pPIC9K接种到YPD培养基中多次传代后,用PCR 检测其目的 基因.该酵母工程菌在BMMY培养基中经甲醇诱导表达,上清液初步浓缩后上柱进行凝胶过滤层析,并将纯化蛋白免疫Balb/c 小鼠,检测其血清抗体水平 ,以免疫组化的方法分析各组织occludin的表达与定位.结果 该酵母工程菌经多次传代后外源基因不丢失,Western blot检测结果表明纯化蛋白具有很好的反应原性,免疫组化证实occludin在实验组高表达.结论 表达FSH-occludin蛋白的酵母工程菌具有很好的遗传稳定性,其表达产物可诱导小鼠产生特异性体液免疫应答.     

Evolutionary gamut of in vivo Gag substitutions during early HIV-1 subtype C infection

Two analyses of HIV-1 subtype C Gag quasispecies were performed in a prospective cohort of 42 acutely and recently infected individuals by SGA on viral RNA/proviral DNA templates. First, in vivo Gag substitutions were assessed in relation to the HIV-1C consensus sequence, which revealed that 29.3% of detected amino acid substitutions can be classified as reversions to subtype consensus, 61.3% as forward substitutions from subtype consensus, and 9.3% as polymorphisms not associated with the subtype consensus sequence. Second, the proportion, dynamics, and relationships within individual pools of viral quasispecies were analyzed. Among reverse substitutions, 16.1% were minor, 11.0% transient, 13.6% dominant, and 59.2% fixed. In contrast, 31.6% of forward substitutions were minor, 59.3% transient, 3.8% dominant, and 5.3% fixed. The distinct patterns in the spectrum and dynamics of reverse and forward Gag substitutions suggest that these differences should be considered in HIV-1 evolutionary studies and analyses of viral mutational pathways.     

HIV-1 B''亚型gag-env融合基因的DNA疫苗构建和免疫原性分析

目的 构建HIV-1 B′亚型中国流行株gag和env融合基因的DNA疫苗，对其免疫原性进行研究。方法 根据已报道的HIV-1 B′亚型RIA2分离株gag和env基因的氨基酸序列按哺乳动物密码子使用频率进行优化并人工合成基因，插入真核表达载体pDRVISV1．0中，构建表达RL42 gag-env，融合蛋白的DNA疫苗，pSVRL／GE。用Western blot和抗gag p55抗体细胞内染色的方法体外检测pSVRUGE的表达效率。DNA疫苗pSVRL/GE免疫BALB／c小鼠后，用ELISPOT检测小鼠的细胞免疫反应。结果 限制性内切酶鉴定表明融合基因已成功插入pDRVISV1．0载体中，Western blot证实融合基因可有效表达融合蛋白；细胞内染色结果表明，pSVRL/GE转染的293T细胞中49．8％表达gag p55，荧光强度均值为924；而空载体pDRVISV1．0转染的293T细胞中非特异背景染色只有0．5％。经免疫的小鼠脾细胞体外用H-2^d限制性表位肽AMQMIKET刺激后，ELISPOT检测显示，pSVRUGE免疫小鼠每10^6脾细胞形成226个斑点（SD=140），而空载对照组每1驴脾细胞形成29个斑点（SD=16）（P〈0．05）。结论 所构建的DNA疫苗pSVRL/GE可高效表达相应抗原蛋白，并可有效激活机体的细胞免疫反应。     

The Vpu-regulated endocytosis of HIV-1 Gag is clathrin-independent

Recent results by us and others have shown that the accessory protein Vpu determines plasma membrane versus endosomal accumulation of the HIV-1 core protein Gag and progeny virions in the HeLa model of HIV-1 infection, since Vpu suppresses endocytosis of cell surface-associated Gag. In this report, we used pulse-chase studies and subcellular fractionations to investigate endocytosis of newly synthesized Gag in HeLa H1 cells. The uptake of Gag in Delta Vpu-virus background was not blocked by inhibitors of clathrin-mediated endocytosis and macropinocytosis. The cholesterol-sequestering drug filipin inhibited the uptake, but only if the drug was applied before extensive multimerization of Gag had taken place. Thus, the uptake mechanism most likely is only indirectly dependent on cholesterol. Our results also indicated that targeting phenotype of Gag was different in confluent versus subconfluent cell cultures, which could perhaps explain some of the controversies in intracellular targeting of Gag.     

Interaction of HIV-1 Gag with the clathrin-associated adaptor AP-2

The capsid (CA) sequence of human immunodeficiency virus type 1 (HIV-1) Gag protein consists of two independently folded domains named the N-terminal domain (NTD) and C-terminal domain (CTD) that are connected by a flexible linker. Most of the CTD sequence adopts rigid structure except for the last 11 amino acids (positions 354 to 364) that are disordered even in the context of the downstream SP1 and nucleocapsid (NC) sequence. Although disordered, this short peptide region plays a crucial role in HIV-1 replication. In this study, we identified three second-site mutations within Gag named A238T, G358S, and N373K that rescued a deleterious mutation R362A located at the C-terminus of CA. A238T is located within the NTD of CA, G358S and N373K are positioned proximal to R362A. One of the mechanisms underlying this compensation event is correction of reduced packaging of viral RNA into the R362A mutated viruses, as shown by the results of RNase protection assays, native Northern blots experiments as well as filter-binding assays. These data suggest that one potential function for the C-terminal disordered sequence of CA in HIV-1 replication is to regulate HIV-1 RNA packaging.     

The capsid-spacer peptide 1 Gag processing intermediate is a dominant-negative inhibitor of HIV-1 maturation

The human immunodeficiency virus type 1 (HIV-1) maturation inhibitor bevirimat disrupts virus replication by inhibiting the cleavage of the capsid-spacer peptide 1 (CA-SP1) Gag processing intermediate to mature CA. The observation that bevirimat delays but does not completely block CA-SP1 processing suggests that the presence of uncleaved CA-SP1 may disrupt the maturation process in trans. In this study, we validate this hypothesis by using a genetic approach to demonstrate that a non-cleavable CA-SP1 mutant exerts a dominant-negative effect on maturation of wild-type HIV-1. In contrast, a mutant in which cleavage can occur internally within SP1 is significantly less potent as a dominant-negative inhibitor. We also show that bevirimat blocks processing at both the major CA-SP1 cleavage site and the internal site. These data underscore the importance of full CA-SP1 processing for HIV-1 maturation and highlight the therapeutic potential of inhibitors that target this Gag cleavage event.     

Characterization of Gag and Nef-specific ELISpot-based CTL responses in HIV-1 infected Indian individuals

Cytotoxic T lymphocyte (CTL) responses to Gag have been most frequently linked to control of viremia whereas CTL responses to Nef have direct relationship with viral load. IFN-γ ELISpot assay was used to screen CTL responses at single peptide level directed at HIV-1 subtype C Gag and Nef proteins in 30 antiretroviral therapy naive HIV-1 infected Indian individuals. PBMCs from 73.3% and 90% of the study population showed response to Gag and Nef antigens, respectively. The magnitude of Gag-specific CTL responses was inversely correlated with plasma viral load (r = −0.45, P = 0.001), whereas magnitude of Nef-specific responses was directly correlated (r = 0.115). Thirteen immunodominant regions (6 in Gag, 7 in Nef) were identified in the current study. The identification of Gag and Nef-specific responses across HIV-1 infected Indian population and targeting epitopes from multiple immunodominant regions may provide useful insight into the designing of new immunotherapy and vaccines.     

含HIV-1核心蛋白基因的重组质粒的构建及其免疫应答的研究

目的构建含有HIV-1核心蛋白gag基因的真核表达质粒,并研究其免疫应答反应。方法采用PCR方法从1例HIV感染者中扩增出gag基因,构建重组质粒pcDNA-GAG。接种小鼠后,检测其抗体及抗体亚类,并对小鼠的淋巴细胞亚群进行分析,采用3H-TdR法、ELISPOT方法和51Cr释放法分别检测T淋巴细胞的增殖反应性、特异性分泌IFN-γ的CD8+T淋巴细胞以及特异性CTL杀伤活性。结果重组质粒体外表达目的蛋白的相对分子质量(Mr)约为55×103。免疫小鼠后可诱导产生特异性抗体,其中IgG2a与IgG的比例显著高于IgG1与IgG的比例;T淋巴细胞体外经ConA刺激后SI达到160.67,显著高于对照组(14.04,P<0.05);产生IFN-γ的CD8+T淋巴细胞数目以及特异杀伤率均显著高于对照组小鼠(P<0.05)。结论重组质粒pcDNA-GAG可诱导小鼠产生特异性体液和细胞免疫应答反应,而且ELISPOT方法检测特异性细胞免疫应答反应的结果与51Cr释放法一致,提示可用此法对DNA疫苗诱导的细胞免疫进行评价。