重组HIV-1壳体蛋白在转基因枸杞根系中的分泌表达

P24壳体蛋白（capsid, CA）是HIV¬-1早期感染的一个重要标志.用含有植物表达载体pCAMBIA 1305.2- MA4-CA（包含GRP信号肽和MA4-CA融合基因）的农杆菌菌株侵染枸杞,将转有MA4-CA融合基因的转化株诱导生根，并进行毛状根的培养; western blot证实根系及培养液中的MA4-CA融合蛋白以二聚体的形式存在，分子量为50 kDa；免疫组织化学显示，CA定位在细胞浆、细胞壁和细胞间隙中，充分证实了利用GRP信号肽可以引导重组蛋白分泌表达。建立枸杞中HIV-1壳体蛋白的根分泌表达系统，为研究植物HIV-1 CA-病毒样颗粒（VLPs）疫苗奠定基础。     

黑曲霉N-2植酸酶phy4基因的克隆及其重组酵母表达载体的构建

根据已发表的植酸酶phyA基因序列设计并合成1对引物,应用PCR技术,以黑曲霉N-2总DNA为模板,扩增出不包含假定信号肽序列的phyA基因,将其克隆到pMD18-T载体中,测定其核苷酸序列,并推导其氨基酸序列。该基因全长为1350bp,与已发表的黑曲霉NRRL3135的phyA基因的同源性为92．4％(不计内含子),编码1个含449个氨基酸残基的蛋白质,推导的氨基酸序列同源性为95．1％。将该基因与分泌型载体pPIC9K连接,构建了植酸酶基因的重组酵母表达载体pPIC9K／phyA。     

copGFP和PuroR双标记基因慢病毒融合表达载体的构建及应用*

目的: 构建具有绿色荧光蛋白(copGFP)和嘌呤霉素抗性基因(PuroR)融合表达双筛选标记的慢病毒过表达载体,检测其嘌呤霉素抗性和绿色荧光蛋白表达的特性。方法: 从pCDH-CMV-MCS-copGFP载体中扩增copGFP编码区DNA序列,从pLKO.1载体中扩增Puro^R编码区DNA序列,运用重组PCR方法,扩增copGFP与Puro^R基因融合编码序列并克隆至经BamH Ⅰ+Sal Ⅰ双酶切的pCDH-CMV-MCS-copGFP载体片段中,构建含copGFP和Puro^R融合表达双筛选标记的慢病毒过表达载体;载体的融合标签序列进行测序确证;将该载体用辅助包装质粒PLP1、PLP2、VSVG在293T细胞中包装成慢病毒后感染肝癌细胞MHCC97H,检测感染细胞对嘌呤霉素的抵抗作用以及绿色荧光蛋白的表达情况;为验证该载体表达外源目的基因的有效性,将Sp1编码区DNA序列插入该载体中包装成慢病毒,用对照及表达Sp1的慢病毒感染肝癌细胞MHCC97H,感染细胞经1 mg/ml嘌呤霉素筛选7 d后获得稳定感染细胞株,提取稳定感染细胞的总RNA及总蛋白,分别运用RT-qPCR和Western blot方法检测Sp1在对照及表达Sp1的慢病毒感染的肝癌MHCC97H细胞中的mRNA和蛋白表达水平的差异。结果: 成功构建含copGFP和Puro^R融合表达双筛选标记的慢病毒过表达载体;该载体与辅助质粒包装出的慢病毒感染肝癌细胞后,感染细胞同时具有嘌呤霉素抗性和表达绿色荧光蛋白特性;将Sp1编码序列插入该载体,包装慢病毒并肝癌细胞,Sp1 的mRNA水平对照细胞相比分别升高3.3倍,蛋白水平升高2.2倍(P＜0.01)。 结论: 成功构建含copGFP和Puro^R融合表达双筛选标记的慢病毒过表达载体,该载体编码的融合双标记基因具有嘌呤霉素抗性和绿色荧光蛋白表达的特性,可高水平表达长片段的目的基因。     

一个抗真菌蛋白在绿色木霉中的分泌表达

AFP(antifungalprotein)是在丝状真菌巨大曲霉 (AspergillusgiganteusMDH18894 )中分泌的一个抗真菌蛋白。其mRNA含长度为 4 30bp的开放阅读框 ,编码 94个氨基酸的AFP前体 ,而成熟的AFP为 5 1个氨基酸的多肽。根据推测 ,在巨大曲霉中 ,AFP前体可能经两步剪切去除前导序列 (4 3个氨基酸 ) ,并最终形成具有抗真菌活性的成熟AFP ,已有报道证实 ,在另一种丝状真菌绿色木霉 (Trichodermaviride)基因组中存在一个类似AFP基因但不表达的序列 ,该序列与没有内含子的AFPcDNA序列完全一样。为了解巨大曲霉AFP基因可否在绿色木霉中表达 ,将AFP基因开放阅读框插入真菌表达载体trpC基因的启动子和终止子之间 ,并成功的转化了绿色木霉。SDS PAGE和Western印迹分析表明 ,绿色木霉转化子分泌表达了具有抗真菌活性的成熟AFP。为研究在绿色木霉中分泌表达具有重要应用价值的异源真核蛋白质打下了基础。     

猪干扰素-α基因的克隆及其植物表达载体的构建

目的:克隆与分析猪干扰素-α(INF-α)基因,构建猪干扰素基因的高效植物表达载体。方法:根据NCBI中DQ248997序列设计引物,以猪的总DNA为模板,PCR扩增出猪的INF-α基因,克隆至pBS-T载体后进行序列分析,构建猪干扰素基因的植物表达载体。结果:实验所克隆序列经Blastn比对,98%的核酸序列相同,98%的蛋白质序列相同,3个非功能性氨基酸与基因库中序列不一致,推测为猪INF-α的一个亚型。构建的2个植物表达载体经BamHⅠ/SacⅠ限制性内切酶消化,均可得到570bp的目的基因。结论:成功克隆了猪的INF-α基因,并构建出含猪INF-α基因的高效植物表达载体pBI121/INF和pCAMBIA1301/INF。     

白鹅催乳素基因的克隆及诱导表达条件的优化

摘要: 运用RT-PCR方法, 从白鹅脑垂体总RNA中扩增得到了催乳素(Prolactin, PRL)基因编码区序列cDNA, 并将其克隆到pMD18-T载体上。DNA序列分析表明, PRL cDNA包括终止密码子在内的长度为690 bp,编码230个氨基酸残基的蛋白质, 与皖西白鹅的有所差异, 二者碱基同源性在99.57%, 氨基酸同源性达99.56%。将PRL基因编码区序列cDNA定向克隆到表达载体pET-32a (+)中, 构建表达质粒pET-32a(+)-PRL。该质粒的BL21 (DE3)转化菌在IPTG的诱导下可表达PRL基因融合蛋白, IPTG终浓度1 mmol/L, 37℃, 诱导4 h表达量最高, 表达量约占菌体总蛋白的28.96%。     

分蘖洋葱查尔酮异构酶基因的克隆及表达载体的构建

旨在得到分蘖洋葱查尔酮异构酶基因,探明该基因的功能。克隆查尔酮异构酶基因并构建了其超表达和干扰载体,以期得到用于研究查尔酮异构酶基因功能的表达载体。从分蘖洋葱叶片中克隆得到查尔酮异构酶基因cDNA全长743 bp,GenBank登录号为KJ489062。结果显示,该基因633 bp的开放读码框编码210个氨基酸的多肽序列。将PCR扩增克隆得到的分蘖洋葱查尔酮异构酶基因片段连接到干扰载体pCAMBIA1 301和超表达载体SOL2095中,成功构建了35S启动子控制的植物表达双元载体pCAMBIA1301-CHI和SOL2095-CHI。     

青杨脊虎天牛CYP4G2基因片段的克隆、序列分析与表达

根据报道的十几种昆虫CYP4家族基因的氨基酸序列保守区域设计一对引物,利用RT-PCR技术扩增编码青杨脊虎天牛Xylotechus rusticus中肠细胞色素氧化酶CYP4G2蛋白的cDNA片段,构建原核表达载体pET-CYP4G2,将其转化入大肠杆菌Escherichia coli JM109中表达。序列分析结果表明,该基因(CYP4G2,GenBank登录号为EF429250)保守区域阅读框全长387 bp,编码129个氨基酸残基,预测分子量和等电点分别为16.9 kD和5.75;推导的氨基酸序列与已报道的昆虫CYP4家族氨基酸序列一致性较高（63%～86%）,且具有细胞色素氧化酶的典型特征。IPTG诱导后,SDS-PAGE电泳检测到一条22 kD大小的外源蛋白,与预测融合蛋白的分子量大小相应。CO差光谱分析证明重组菌表达了有活性的pET-CYP4G2。     

烟草环斑病毒外壳蛋白基因的原核表达及抗血清的制备

烟草环斑病毒(Tobacco ringspot virus,TRSV)是我国二类进境检疫危险性有害生物,对农业生产危害较大.本研究依据TRSV外壳蛋白基因cp序列设计合成了2条引物,通过RT PCR扩增得到长约1500bp的目的片段.将目的片段与质粒pET-22b(+)连接,构建了含TRSV cp基因的融合蛋白原核表达载体pETRSV-CP.序列分析表明,TRSV-SD1的cp基因全长1548bp,编码515个氨基酸与GenBank中其它TRSV分离物cp基因相比,核苷酸及推导的氨基酸序列同源性为90.7%～94.6%.将pETRSV-CP转入大肠杆菌,诱导表达.SDS-PAGE结果显示,表达的TRSV CP融合蛋白的相对分子质量约为58kDa.以此融合蛋白制备的抗血清的效价为1/1024,抗血清与TRSV具有良好的特异性反应.     

Tropic1808基因的原核表达及其表达产物的生物活性

Tropic180 8基因是新近获得的一个鼠源性的cDNA .Tropic180 8基因开放阅读框架片段通过PCR方法从质粒中扩增后 ,重组入表达载体pET 2 1a中 ,转化大肠杆菌BL2 1(DE3 ) .用IPTG诱导目的蛋白的表达 ,SDS PAGE并凝胶图象分析确定目的蛋白表达水平占细菌总蛋白的 14%以上 .表达蛋白在N端融合有 16个氨基酸 ,将表达蛋白电转移至PVDF膜 .氨基酸序列分析表明 ,其N端第 17～ 2 5位氨基酸序列与Tropic180 8基因编码序列一致 .利用融合部分含T7·Tag ,通过亲和层析纯化表达蛋白 ,经Westernblot检测为目的蛋白 ,加入到无血清培养的新生SD大鼠背根神经节 (DRG)中 ,观察到表达蛋白对DRG具有促进存活和促进突起生长的作用 .     

HIV-1衣壳蛋白在转基因枸杞中表达的免疫组织化学定位

目的:研究转基因植物中重组蛋白的细胞定位,有助于进一步了解转基因枸杞中HIV-1衣壳(CA)蛋白融合蛋白的分泌表达途径。方法:利用含有MA4-CA融合基因的农杆菌转化枸杞,转化植株获得再生。采用免疫组织化学方法对转基因枸杞表达的CA融合蛋白进行初步定位。结果:免疫组织化学定位表明,在转基因枸杞愈伤组织中,HIV-1CA主要在细胞浆、细胞壁和细胞间隙中表达。结论:免疫组织化学结果初步证明了CA融合蛋白在转基因枸杞中的表达分布。     

Humoral, mucosal, and cellular immunity in response to a human immunodeficiency virus type 1 immunogen expressed by a Venezuelan equine encephalitis virus vaccine vector.

A molecularly cloned attenuated strain of Venezuelan equine encephalitis virus (VEE) has been genetically configured as a replication-competent vaccine vector for the expression of heterologous viral proteins (N. L. Davis, K. W. Brown, and R. E. Johnston, J. Virol. 70:3781-3787, 1996). The matrix/capsid (MA/CA) coding domain of human immunodeficiency virus type 1 (HIV-1) was cloned into the VEE vector to determine the ability of a VEE vector to stimulate an anti-HIV immune response in mice. The VEE-MA/CA vector replicated rapidly in the cytoplasm of baby hamster kidney (BHK) cells and expressed large quantities of antigenically identifiable MA/CA protein. When injected subcutaneously into BALB/c mice, the vector invaded and replicated in the draining lymphoid tissues, expressing HIV-1 MA/CA at a site of potent immune activity. Anti-MA/CA immunoglobulin G (IgG) and IgA antibodies were present in serum of all immunized mice, and titers increased after a second booster inoculation. IgA antibodies specific for MA/CA were detected in vaginal washes of mice that received two subcutaneous immunizations. Cytotoxic T-lymphocyte responses specific for MA/CA were detected following immunization with the MA/CA-expressing VEE vector. These findings demonstrate the ability of a VEE-based vaccine vector system to stimulate a comprehensive humoral and cellular immune response. The multifaceted nature of this response makes VEE an attractive vaccine for immunization against virus infections such as HIV-1, for which the correlates of protective immunity remain unclear, but may include multiple components of the immune system.     

人类免疫缺陷病毒Ⅰ型核心蛋白(p24)在大肠杆菌中的表达、纯化及鉴定

在大肠杆菌中,利用新构建的含T7g-10L RBS以及λ-PR启动子的新型原核表达载体,通过表达gag-pol基因片段,获得了具有天然序列的人类免疫缺陷病毒1型(HIV-1)核心蛋白p24的高效表达。克隆的gag-pol基因片段在其阅读框架移位区域插入了4bp碱基,其表达的病毒蛋白酶在阅读框架上与gag一致,从而实现了对gag-pol融合蛋白的有效加工,产生成熟的核心蛋白p24及其它产物。重组p24以可溶形式存在,可以被抗p24的单克隆抗体特异识别。测定的N端8个氨基酸序列与从病毒纯化的p24完全一致。在使用硫酸铵沉淀后,采用两步离子柱层析,可将重组蛋白纯化到95％以上的纯度。结果表明,纯化的p24可以作为特异性很强的试剂而用于HIV感染的诊断及病情的预后,并可用于p24的生化及结构分析。     

Capsid is a dominant determinant of retrovirus infectivity in nondividing cells

A major difference between lentiviruses such as human immunodeficiency virus (HIV) and most other retroviruses is their ability to productively infect nondividing cells. We present here genetic evidence for involvement of the capsid protein (CA) in the infectious phenotype in nondividing cells. A chimeric HIV type 1 (HIV-1) in which the MA and CA of HIV-1 are replaced with the MA, p12, and CA encoding sequences from murine leukemia virus (MLV) loses the ability to efficiently infect nondividing cells. Analysis of the accumulation of two-long-terminal-repeat circles implies that the impairment of nuclear transport of preintegration complexes is responsible for the restricted infection of this chimeric virus in nondividing cells. Incorporation of MLV MA and MLV p12 into HIV virions alone does not exert any adverse effects on viral infection in interphase cells. These results suggest that CA is the dominant determinant for the difference between HIV and MLV in the ability to transduce nondividing cells.     

Immunogenic comparison of chimeric adenovirus 5/35 vector carrying optimized human immunodeficiency virus clade C genes and various promoters

Adenovirus vector-based vaccine is a promising approach to protect HIV infection. However, a recent phase IIb clinical trial using the vector did not show its protective efficacy against HIV infection. To improve the vaccine, we explored the transgene protein expression and its immunogenicity using optimized codon usage, promoters and adaptors. We compared protein expression and immunogenicity of adenovirus vector vaccines carrying native or codon usage-optimized HIV-1 clade C gag and env genes expression cassettes driven by different promoters (CMV, CMVi, and CA promoters) and adapters (IRES and F2A). The adenovirus vector vaccine containing optimized gag gene produced higher Gag protein expression and induced higher immune responses than the vector containing native gag gene in mice. Furthermore, CA promoter generated higher transgene expression and elicited higher immune responses than other two popularly used promoters (CMV and CMVi). The second gene expression using F2A adaptor resulted in higher protein expression and immunity than that of using IRES and direct fusion protein. Taken together, the adenovirus vector containing the expression cassette with CA promoter, optimized HIV-1 clade C gene and an F2A adaptor produced the best protein expression and elicited the highest transgene-specific immune responses. This finding would be promising for vaccine design and gene therapy.     

Nucleoporin NUP153 Phenylalanine-Glycine Motifs Engage a Common Binding Pocket within the HIV-1 Capsid Protein to Mediate Lentiviral Infectivity

Lentiviruses can infect non-dividing cells, and various cellular transport proteins provide crucial functions for lentiviral nuclear entry and integration. We previously showed that the viral capsid (CA) protein mediated the dependency on cellular nucleoporin (NUP) 153 during HIV-1 infection, and now demonstrate a direct interaction between the CA N-terminal domain and the phenylalanine-glycine (FG)-repeat enriched NUP153 C-terminal domain (NUP153_C). NUP153_C fused to the effector domains of the rhesus Trim5α restriction factor (Trim-NUP153_C) potently restricted HIV-1, providing an intracellular readout for the NUP153_C-CA interaction during retroviral infection. Primate lentiviruses and equine infectious anemia virus (EIAV) bound NUP153_C under these conditions, results that correlated with direct binding between purified proteins in vitro. These binding phenotypes moreover correlated with the requirement for endogenous NUP153 protein during virus infection. Mutagenesis experiments concordantly identified NUP153_C and CA residues important for binding and lentiviral infectivity. Different FG motifs within NUP153_C mediated binding to HIV-1 versus EIAV capsids. HIV-1 CA binding mapped to residues that line the common alpha helix 3/4 hydrophobic pocket that also mediates binding to the small molecule PF-3450074 (PF74) inhibitor and cleavage and polyadenylation specific factor 6 (CPSF6) protein, with Asn57 (Asp58 in EIAV) playing a particularly important role. PF74 and CPSF6 accordingly each competed with NUP153_C for binding to the HIV-1 CA pocket, and significantly higher concentrations of PF74 were needed to inhibit HIV-1 infection in the face of Trim-NUP153_C expression or NUP153 knockdown. Correlation between CA mutant viral cell cycle and NUP153 dependencies moreover indicates that the NUP153_C-CA interaction underlies the ability of HIV-1 to infect non-dividing cells. Our results highlight similar mechanisms of binding for disparate host factors to the same region of HIV-1 CA during viral ingress. We conclude that a subset of lentiviral CA proteins directly engage FG-motifs present on NUP153 to affect viral nuclear import.     

Structure of the N-terminal 283-residue fragment of the immature HIV-1 Gag polyprotein

The capsid protein (CA) of the mature human immunodeficiency virus (HIV) contains an N-terminal beta-hairpin that is essential for formation of the capsid core particle. CA is generated by proteolytic cleavage of the Gag precursor polyprotein during viral maturation. We have determined the NMR structure of a 283-residue N-terminal fragment of immature HIV-1 Gag (Gag(283)), which includes the intact matrix (MA) and N-terminal capsid (CA(N)) domains. The beta-hairpin is unfolded in Gag(283), consistent with the proposal that hairpin formation occurs subsequent to proteolytic cleavage of Gag, triggering capsid assembly. Comparison of the immature and mature CA(N) structures reveals that beta-hairpin formation induces a approximately 2 A displacement of helix 6 and a concomitant displacement of the cyclophylin-A (CypA)-binding loop, suggesting a possible allosteric mechanism for CypA-mediated destabilization of the capsid particle during infectivity.     

Human immunodeficiency virus type 1 matrix protein assembles on membranes as a hexamer

The membrane-binding matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) structural precursor Gag (PrGag) protein oligomerizes in solution as a trimer and crystallizes in three dimensions as a trimer unit. A number of models have been proposed to explain how MA trimers might align with respect to PrGag capsid (CA) N-terminal domains (NTDs), which assemble hexagonal lattices. We have examined the binding of naturally myristoylated HIV-1 matrix (MyrMA) and matrix plus capsid (MyrMACA) proteins on membranes in vitro. Unexpectedly, MyrMA and MyrMACA proteins both assembled hexagonal cage lattices on phosphatidylserine-cholesterol membranes. Membrane-bound MyrMA proteins did not organize into trimer units but, rather, organized into hexamer rings. Our results yield a model in which MA domains stack directly above NTD hexamers in immature particles, and they have implications for HIV assembly and interactions between MA and the viral membrane glycoproteins.     

Tracking and quantitation of fluorescent HIV during cell-to-cell transmission

The green fluorescent protein (GFP) is a powerful genetic marking tool that has enabled virologists to monitor and track viral proteins during HIV infection. Expression-optimized Gag-GFP constructs have been used to study virus-like particle (VLP) assembly and localization in cell types that are easily transfected. The development of HIV-1 variants carrying GFP within the context of the viral genome has facilitated the study of infection and has been particularly useful in monitoring the transfer of virus between cells following virological synapse formation. HIV Gag-iGFP, a viral clone that contains GFP inserted between the matrix (MA) and capsid (CA) domains of Gag, is the first replication competent molecular clone that generates fluorescent infectious particles. Here, we discuss some methods that exploit HIV Gag-iGFP to quantify cell-to-cell transmission of virus by flow cytometry and to track the proteins during assembly and transmission using live-cell imaging.