HIV-1 gp160多表位嵌合基因的构建及表达蛋白的免疫原性分析

HIV感染引起的AIDS已经成为严重影响人类健康和社会发展的全球性疾病。酶联免疫吸附试验和免疫印迹检验组合则被认为是HIV检测的“金标准”。因此本实验构建gp160的抗原多表位融合基因及在原核系统的高表达, 为HIV抗体测定提供特异、价廉的抗原。选定HIV-1 gp160基因中三个片段包含较多抗原表位的区域, 设计带有酶切位点的引物,用PCR的方法从HIV-1HXB2全基因扩增编码这三个片段的基因序列,通过质粒提取、酶切、测序方法鉴定基因片段的正确性, SDS-PAGE和Western Blot测定融合蛋白的抗原特异性。构建的HIV-1 gp160多表位嵌合基因的原核表达质粒,酶切和测序结果表明基因序列正确,基因全长969bp。在大肠杆菌BL21(DE3) 中高效表达的重组蛋白分子量为37kDa,以包涵体的形式存在。应用western blot测定10例正常人和12例HIV/AIDS病人血浆显示HIV-1 gp160多表位融合蛋白具有良好的抗原特异性。成功构建了高表达 HIV-1 gp160多表位蛋白的原核表达系统,纯化的融合蛋白有较强的抗原特异性。     

结核分枝杆菌HspX蛋白的原核高效可溶性表达及产物抗原性分析

目的:利用原核系统可溶性表达结核分枝杆菌HspX蛋白并进行纯化,通过免疫印迹反应初步鉴定重组蛋白的抗原性和特异性。方法:采用PCR方法,从结核分枝杆菌H37Rv基因组中扩增HspX核酸序列,克隆至原核表达载体pET-28a中,转入大肠杆菌BL21(DE3)进行诱导、表达和纯化,用Western印迹初步评价HspX的抗原性。结果:经IPTG诱导,HspX蛋白在原核系统内获得了可溶性表达,经镍柱亲和层析获得了纯度达95%以上的重组蛋白。Western印迹结果证明重组HspX蛋白与结核病患者血清标本呈强阳性反应,与健康人血清标本呈阴性反应。结论:重组蛋白HspX在大肠杆菌中以可溶性形式表达,高纯度的重组融合蛋白有可能成为结核病的血清学诊断抗原。     

HIV-1 C亚型密码子优化gp120基因重组腺病毒载体的构建及表达

目的构建含有HIV-1C亚型gp120基因重组腺病毒载体,并在293细胞中表达gp120蛋白。方法PCR扩增,获得HIV-1C亚型gp120片段,定向克隆人腺病毒转移载体pTrack-CMV,线性化后转化至含有腺病毒骨架载体pAd-easy-1的大肠埃希菌BJ5183,获得重组子prAd—gp120,PacI酶切纯化后转染293细胞,包装成复制缺陷型重组腺病毒vAd—gp120。结果经PCR、酶切及DNA测序,插入片段大小、方向正确,获得了具有感染力的vAd—gp120重组腺病毒;通过Western印迹检测,重组腺病毒在293细胞中表达出分子量为120kD的蛋白。结论成功构建了含有HIV-1C亚型gp120基因重组腺病毒载体,并获得该基因的表达。     

HIV-1 Gag多表位嵌合基因的构建及表达蛋白的抗体制备

旨在通过构建Gag的抗原多表位融合基因及在原核系统的高表达,为HIV诊断及可能的疫苗制备提供试验基础。选定HIV-1 Gag基因中3个片段包含较多抗原表位的区域,设计带有酶切位点的引物,用PCR的方法从HIV-1HXB2全基因扩增编码这3个片段的基因序列,通过质粒提取、酶切、测序方法鉴定基因片段的正确性,SDS-PAGE和Western blotting测定融合蛋白的表达,并免疫动物制备相应抗体。结果显示,构建的HIV-1 Gag多表位嵌合基因的原核表达质粒,酶切和测序结果表明基因序列正确,基因全长576bp。在大肠杆菌BL21(DE3)中高效表达的重组蛋白分子量为27kD,以包涵体的形式存在。纯化目的蛋白免疫家兔,制备多克隆抗体IgG。ELISA和免疫荧光方法检测显示制备的多克隆抗体能具有特异性反应。成功构建和高表达了HIV-1 Gag多表位融合蛋白,纯化蛋白制备的抗体与HIV-1Gag有特异性结合。为进一步研究HIV-1奠定了试验基础。     

人免疫缺陷病毒I型Vif蛋白的原核表达、纯化及单克隆抗体制备

目的：克隆、表达、纯化人免疫缺陷病毒I型（HIV-1）Vif蛋白,制备其单克隆抗体。方法：提取感染了HIV的细胞基因组DNA,PCR扩增vif基因,插入表达载体pET32a,转化大肠杆菌BL21（DE3）获得工程菌株,IPTG诱导蛋白表达,Western印迹鉴定目的蛋白,亲和层析纯化目的蛋白;免疫BALB／c小鼠,制备单克隆抗体。结果：构建了Vif蛋白的原核表达载体vif-pET32a,并在大肠杆菌中获得高表达,目的蛋白以包涵体形式存在;纯化获得高纯度的重组Vif蛋白,蛋白浓度可达0．56mg／mL;建立了抗Vif蛋白单克隆抗体细胞株,制备了腹水,滴度可达1：16×10^6,抗体纯化后保持了活性和特异性。结论：在原核表达系统中表达、纯化了重组Vif蛋白,制备了针对Vif蛋白的单克隆抗体,为研究Vif蛋白的功能和抗原性奠定了基础。     

HIV-1跨膜蛋白gp41螺旋结构的原核表达及功能研究

HIV-1跨膜蛋白gp41是HIV-1包膜与靶细胞膜的融合过程中的关键蛋白,是理想的HIV-1融合抑制剂靶点。为开展以gp41为靶点的抑制剂筛选,以HIV-1 B亚型病毒基因为模板,通过PCR、酶切、连接等方法构建得到gp41 5-helix与6-helix重组质粒,转入大肠杆菌BL21（DE3）进行表达,经变性和复性后亲和层析纯化蛋白。经SDS-PAGE鉴定,纯化后蛋白纯度较高。本研究还通过非变性凝胶电泳证明gp41 5-helix与C-多肽衍生物T-20存在相互作用,为下一步药物筛选模型的建立奠定了基础。     

HIV-1 Vpu蛋白的原核表达、鉴定和纯化

目的：克隆、表达、纯化人免疫缺陷病毒Ⅰ型（HIV-1）Vpu蛋白,为其功能及免疫学研究奠定基础。方法：PCR扩增Vpu基因,纯化、酶切后克隆到原核表达载体pET32a中,转化大肠杆菌BL21（DE3）菌株获得表达工程菌株,IPTG诱导蛋白表达,免疫印迹鉴定目的蛋白,亲和层析纯化蛋白。结果：构建了HIV-1Vpu蛋白的原核表达载体Vpu-pET32a,并在大肠杆菌中高效表达,目的蛋白呈可溶性形式存在,免疫印迹检测显示为目的蛋白,经Ni—NTAAgarose纯化获得了高纯度的目的蛋白。结论：在原核表达系统中表达了可溶性HIV-1Vpu蛋白,为进一步进行HIV-1Vpu蛋白的免疫原性和功能研究奠定了基础。     

禽多杀性巴氏杆菌P1059成熟黏附蛋白的原核表达、纯化和抗原性检测

目的：建立rCPM36在大肠杆菌中的表达体系,纯化表达产物并检测其抗原性。方法：运用PCR方法从禽多杀性巴氏杆菌国际标准株P1059基因组中扩增出编码36kDa成熟黏附蛋白的cpm36基因,构建原核表达载体pQE30-cpm36,转化到大肠杆菌M15中并诱导表达目的蛋白,用镍离子螯合层析柱纯化目的蛋白及制备其抗体,Western印迹分析其抗原性。结果：SDS-PAGE结果显示目标蛋白以可溶性形式表达在大肠杆菌M15细胞质中,其相对分子质量为37kDa,Western印迹结果表明表达蛋白具有良好的抗原性。结论：成功构建出原核表达载体并实现了目的蛋白表达,用镍离子螯合层析柱纯化得到具有抗原性的蛋白,为进一步开展禽多杀性巴氏杆菌黏附因子和保护性抗原的研究奠定基础。     

猪流行性腹泻病毒S基因片段的克隆及原核表达

为研究猪流行性腹泻病毒(porcine epidemic diarrhea virus PEDV)S基因片段的原核表达产物是否具有抗原性,分析S基因抗原位点后,用PCR技术扩增S蛋白主要抗原区的核酸序列,经克隆后将目的片段连接到原核表达载体pET-28a(+)中,成功构建了重组质粒pET-28a-PEDV-Sp,其重组菌于37℃、0.5 mmol/L IPTG诱导表达4 h后进行SDS-PAGE分析,在分子质量约为29 kDa处出现一新蛋白带,与预期相符。质谱鉴定表明,已成功表达了目的蛋白。纯化后的重组蛋白免疫兔制备多克隆抗体,抗体效价检测结果显示该蛋白具有良好的抗原性。该研究为猪流行性腹泻基因工程疫苗的研制奠定基础。     

携带HIV-1抗原的单纯疱疹病毒载体疫苗的构建及鉴定

利用细菌人工染色体技术将串联的HIV-1 gp160、gag和protease基因以及表达元件插入1型单纯疱疹病毒(Herpes simplex virus type 1,HSV-1)内部反向重复序列区,以获得携带HIV-1抗原的单纯疱疹病毒载体疫苗。首先将HIV-1 gp160(B型和C型)、gag和protease基因串联克隆入pc DNA3获得重组质粒pc DNA/g Bgp和pc DNA/g Cgp,重组质粒转染293FT细胞,Western blotting检测HIV抗原表达。继而将pc DNA/g Bgp和pc DNA/g Cgp中包括HIV-1抗原基因和表达元件的表达框克隆入p KO5/BN获得重组穿梭质粒p KO5/BN/g Bgp和p KO5/BN/g Cgp,穿梭质粒电转含BAC-HSV的大肠杆菌,筛选重组菌,提取重组DNA并转染Vero细胞,挑取病毒蚀斑纯化重组病毒,Southern blotting鉴定重组病毒DNA,Western blotting检测重组病毒感染细胞中HIV抗原表达,并分析病毒的增殖特性。结果表明,Western blotting在pc DNA/g Bgp和pc DNA/g Cgp转染的293FT细胞中检测到表达的gp160和gag蛋白。p KO5/BN/g Bgp和p KO5/BN/g Cgp分别电转获得重组菌,并从重组DNA转染的Vero细胞中纯化获得重组HSV,Southern blotting检测表明重组HSV基因组发生特异性重组,重组病毒感染细胞中检测到gp120和gp41,且重组HSV保留了在哺乳动物细胞中的复制能力。本研究获得携载HIV-1 gp160、gag和protease基因的重组HSV,并保留了在哺乳动物细胞中的复制能力,可作为HIV-1复制型病毒载体疫苗。     

基于gp41近膜端外部区域中和表位天然构象的人类免疫缺陷病毒1型疫苗的设计

疫苗一直被视为终结人类免疫缺陷病毒1型和2 型(human immunodeficiency virus type 1/2,HIV-1/2)最有力的武器。位于HIV-1 gp41胞外域C末端的近膜端外部区域(membrane-proximal external region, MPER)是一个重要的抗原位点,但糖蛋白41(glycoprotein 41,gp41)在野生型HIV-1的包膜蛋白中并未充分暴露,单独的gp41或MPER多肽无法模拟gp120/gp41包膜蛋白在病毒包膜上的天然状态。本研究以HIV-1 gp41 MPER为抗原进行疫苗设计,以期能诱导产生具有强效中和作用的MPER特异性抗体。通过在gp41的N末端七肽重复域(N-terminal heptad repeat, CHR)和C末端七肽重复域(C-terminal heptad repeat, NHR)引入突变,阻断二者相互作用形成6螺旋(6-helix bundle, 6-HB)构象;使用不同来源的流感病毒HA1亚基替代gp120,以防止机体产生大量针对HA1的抗体,并构建一系列包含不同HA1亚基的HA/gp41-1605嵌合DNA。结果发现,在细胞上表达的嵌合疫苗抗原能更好地展示MPER上的中和表位,但不显示gp41上免疫优势抗原表位。使用构建的HA/gp41嵌合DNA疫苗对新西兰大耳兔进行肌肉内序贯免疫,提示该疫苗策略的确不可诱生高滴度的HA抗体或gp41的loop及6-HB特异性抗体,而能诱生MPER特异性抗体。然而,该抗体对HIV-1不具有中和作用,说明该疫苗策略还有待进一步优化。     

Evidence for a stable interaction of gp41 with Pr55(Gag) in immature human immunodeficiency virus type 1 particles

Assembly of infectious human immunodeficiency virus type 1 (HIV-1) virions requires incorporation of the viral envelope glycoproteins gp41 and gp120. Several lines of evidence have suggested that the cytoplasmic tail of the transmembrane glycoprotein, gp41, associates with Pr55(Gag) in infected cells to facilitate the incorporation of HIV-1 envelope proteins into budding virions. However, direct evidence for an interaction between gp41 and Pr55(Gag) in HIV-1 particles has not been reported. To determine whether gp41 is associated with Pr55(Gag) in HIV-1 particles, viral cores were isolated from immature HIV-1 virions by sedimentation through detergent. The cores contained a major fraction of the gp41 that was present on untreated virions. Association of gp41 with cores required the presence of the gp41 cytoplasmic tail. In HIV-1 particles containing a functional protease, a mutation that prevents cleavage of Pr55(Gag) at the matrix-capsid junction was sufficient for the detergent-resistant association of gp41 with the isolated cores. In addition to gp41, a major fraction of virion-associated gp120 was also detected on immature HIV-1 cores. Isolation of cores under conditions known to disrupt lipid rafts resulted in the removal of a raft-associated protein incorporated into virions but not the HIV-1 envelope proteins. These results provide biochemical evidence for a stable interaction between Pr55(Gag) and the cytoplasmic tail of gp41 in immature HIV-1 particles. Moreover, findings in this study suggest that the interaction of Pr55(Gag) with gp41 may regulate the function of the envelope proteins during HIV-1 maturation.     

Humoral response to oligomeric human immunodeficiency virus type 1 envelope protein.

The humoral immune response to human immunodeficiency virus type 1 (HIV-1) is often studied by using monomeric or denatured envelope proteins (Env). However, native HIV-1 Env complexes that maintain quaternary structure elicit immune responses that are qualitatively distinct from those seen with monomeric or denatured Env. To more accurately assess the levels and types of antibodies elicited by HIV-1 infection, we developed an antigen capture enzyme-linked immunosorbent assay using a soluble, oligomeric form of HIV-1IIIB Env (gp140) that contains gp120 and the gp41 ectodomain. The gp140, captured by various monoclonal antibodies (MAbs), retained its native oligomeric structure: it bound CD4 and was recognized by MAbs to conformational epitopes in gp120 and gp41, including oligomer-specific epitopes in gp41. We compared the reactivities of clade B and clade E serum samples to captured Env preparations and found that while both reacted equally well with oligomeric gp140, clade B seras reacted more strongly with monomeric gp120 than did clade E samples. However, these differences were minimized when gp120 was captured by a V3 loop MAb, which may lead to increased exposure of the CD4 binding site. We also measured the ability of serum samples to block binding of MAbs to epitopes in gp120 and gp41. Clade B serum samples consistently blocked binding of oligomer-dependent MAbs to gp41 and, to a slightly lesser extent, MAbs to the CD4 binding site in gp120. Clade E serum samples showed equivalent or greater blocking of oligomer-dependent gp41 antibodies and considerably less blocking of CD4-binding-site MAbs. Finally, we found that < 5% of the antibodies in clade B sera bound to epitopes present only in monomeric gp120, 30% bound to epitopes present in both monomeric gp120 and oligomeric gp140, and 70% bound to epitopes present in oligomeric gp140, which includes gp41. Thus, captured oligomeric Env closely reflects the antigenic characteristics of Env protein on the surface of virions and infected cells, retains highly conserved epitopes that are recognized by antibodies raised against different clades, and makes it possible to detect a much greater fraction of total anti-HIV-1 Env activity in sera than does native monomeric gp120.     

The CH1α domain of mucosal gp41 IgA contributes to antibody specificity and antiviral functions in HIV-1 highly exposed Sero-Negative individuals

The antibody molecule comprises a variable domain conferring antigen specificity and affinity distinct from the heavy chain constant (CH) domains dictating effector functions. We here interrogate this paradigm by evaluating the unique influence of the CH1α domain on epitope specificity and functions using two mucosal gp41-specific Fab-IgAs (FabA) derived from HIV-1 highly-exposed but persistently seronegative individuals (HESN). These HESN develop selectively affinity-matured HIV-1-specific mucosal IgA that target the gp41 viral envelope and might provide protection although by unclear mechanisms. Isotype-switching FabAs into Fab-IgGs (FabGs) results in a >10-fold loss in affinity for HIV-1 clade A, B, and C gp41, together with reduced neutralization of HIV-1 cross-clade. The FabA conformational epitopes map selectively on gp41 in 6-Helix bundle and pre-fusion conformations cross-clade, unlike FabGs. Finally, we designed in silico, a 12 amino-acid peptide recapitulating one FabA conformational epitope that inhibits the FabA binding to gp41 cross-clade and its neutralizing activity. Altogether, our results reveal that the CH1α domain shapes the antibody paratope through an allosteric effect, thereby strengthening the antibody specificity and functional activities. Further, they clarify the mechanisms by which these HESN IgAs might confer protection against HIV-1-sexual acquisition. The IgA-specific epitope we characterized by reverse vaccinology could help designing a mucosal HIV-1 vaccine.     

Characterization of Humoral Immune Responses against Capsid Protein p24 and Transmembrane Glycoprotein gp41 of Human Immunodeficiency Virus Type 1 in China

The objective of this study was to extend our previous research and to further characterize the humoral immune responses against HIV-1 p24, gp41 and the specific peptides carrying the immunodominant epitopes (IDEs) that react with human serum samples from HIV-1-infected individuals in China. We found that the majority (90.45%, 180/199) of the samples did not react with any of the three HIV-1 p24 peptides carrying IDEs, but did react with the recombinant full-length p24, suggesting that these samples tested in China were primarily directed against the conformational epitopes of HIV-1 p24. In contrast, 84.54% (164/194) of the samples reacted with at least one HIV-1 linear gp41 peptide, in particular the gp41-p1 peptide (amino acids 560–616). Both recently and long-term HIV-1-infected individuals displayed similar humoral immune responses against the recombinant gp41. However, samples from long-term HIV-1-infected subjects but not from recently infected subjects, showed a very strong reaction against the gp41-p1 peptide. The different response patterns observed for the two groups against the gp41 and the peptide gp41-p1 were statistically significant (P<0.01, Chi-square test). These results have direct relevance and importance for design of improved HIV-1 p24 detection assays and the gp41- based immunoassay that can be used to reliably distinguish recent and long-term HIV-1 infection.     

Chimeric synthetic peptide as antigen for immunodiagnosis of HIV-1 infection

One chimeric peptide incorporating antigenic sequences from the gp41 transmembrane region (peptide H-18) and the gp120 envelope region (peptide H-15) corresponding to amino acids (587-617) on gp41 and (495-516) on gp120 of human immunodeficiency virus (HIV 1) was synthesized. Both sequences were separated by two glycine residues. This peptide was evaluated as antigen in an ultramicro-enzyme-linked immunosorbent assay (UMELISA) with samples derived from HIV-1 (n = 30) with different titers of antibodies and healthy blood donors (n = 30). The results were compared to plates coated with monomeric peptides and to plates coated with two monomeric peptides together. Results demonstrated that monomeric peptides gp41 (H-18) and gp120 (H-15) were good as antigens with samples that present antibodies to these regions. The chimeric peptide was the most antigenic. Those results may be related to the peptide structure, adsorption to the solid surface, and epitope accessibility to the antibodies. This chimeric peptide would be very useful for HIV-1 diagnostics.     

Single mutations in the transmembrane envelope protein abrogate the immunosuppressive property of HIV-1

ABSTRACT: BACKGROUND: The mechanism by which HIV-1 induces AIDS is still unknown. Previously, synthetic peptides corresponding to the conserved immunosuppressive (isu) domain in gp41 of HIV-1 had been shown to inhibit proliferation and to modulate cytokine expression of immune cells. The question is, whether the viral gp41 can do the same. RESULTS: We show for the first time that two trimeric forms of glycosylated gp41 released from transfected human cells modulated expression of cytokines and other genes in human PBMCs in the same manner, but at least seven hundred-fold stronger compared to that induced by the isu peptide. Single amino acid substitutions in the isu domain of gp41 introduced by site-directed mutagenesis abrogated this property. Furthermore, replication-competent HIV-1 with a mutation in the isu domain of gp41 did not modulate the cytokine expression, while wild-type virus did. Interestingly, most of the abrogating mutations were not reported in viral sequences derived from infected individuals, suggesting that mutated non-immunosuppressive viruses were eliminated by immune responses. Finally, immunisation of rats with gp41 mutated in the isu domain resulted in increased antibody responses compared with the non-mutated gp41. These results show that non-mutated gp41 is immunosuppressive in immunisation experiments, i.e. in vivo, and this has implications for the vaccine development. CONCLUSIONS: These findings indicate that the isu domain of gp41 modulates cytokine expression in vitro and suppresses antibody response in vivo and therefore may contribute to the virus induced immunodeficiency.     

HIV-1 glycoprotein 41 ectodomain induces activation of the CD74 protein-mediated extracellular signal-regulated kinase/mitogen-activated protein kinase pathway to enhance viral infection

Besides mediating the viral entry process, the human immunodeficiency virus (HIV-1) envelope protein gp41 can bind to many host cell components and regulate cell functions. Using a yeast two-hybrid system, we screened a human bone marrow cDNA library and identified a novel gp41-binding protein, CD74 (the MHC class II-associated invariant chain). Here, we report possible biological effects mediated by interaction between gp41 and CD74. We found that HIV-1 gp41 could bind directly to host CD74 in HIV-1-infected cells, and the peptide 6358 derived from gp41 loop region (aa 597-611) could effectively block the gp41-CD74 interaction. As a result of this binding, recombinant soluble gp41 and gp41 peptide 6358 activated the CD74-mediated ERK/MAPK pathway and significantly enhanced HIV-1 infection in vitro. Conversely, the enhancing effect could be suppressed by the recombinant CD74 extracellular domain. These results reveal a novel mechanism underlying gp41 mediation of HIV-1 infection and replication.     

The cytoplasmic domain of HIV-1 gp41 interacts with the carboxyl-terminal region of alpha-catenin.

To know the cellular protein interactions with the viral protein can give an insight into the molecular mechanisms of the virus life cycle. As the function of the cytoplasmic domain of human immunodeficiency virus type 1 (HIV-1) gp41 is not known clearly, we searched for a cellular protein that interacts with the cytoplasmic domain of the HIV-1 gp41 using the yeast two-hybrid assay system. Screening of HeLa cell cDNA library yielded alpha-catenin cDNA. The cytoplasmic domain of the HIV-1 gp41 and the simian immunodeficiency virus (SIV) gp41 were able to interact with the carboxyl-terminal region of alpha-catenin specifically. Mapping of the interaction sites revealed that the interaction between the domain containing the second helix structure from the carboxyl-terminus of HIV-1 gp41 and the carboxyl-terminal region of alpha-catenin was stronger than other domains of gp41.