SpltMNPV日本分离株gp41的克隆表达及gp41和ph的进化分析

从斜纹夜蛾核型多角体病毒(Spodopteralituramulticapsidnucleopolyhedrovirus,SpltMNPV)日本分离株(C3)基因组中克隆了gp41基因。该基因编码区含993bp核苷酸,编码分子量为36.9kDa的多肽。将该基因克隆至原核表达载体pET28a,经IPTG诱导后在大肠杆菌BL21(DE3)中获得了表达。应用CLUSTAL程序分析表明,SpltMNPV日本株(C3)gp41的核苷酸序列和氨基酸序列与SpltMNPV中国G2株相似性最高,均达99.9%。用MEGA分别构建了基于gp41和ph的聚类分析图和分子进化树,发现它们具有相似的拓扑结构。将这两个基因序列结合在一起构建进化树,该树的结构与基于gp41的进化树相似。突变率分析显示gp41的突变率高于ph,这意味着在杆状病毒进化过程中,gp41和ph面临不同的选择压力。     

杆状病毒SpltMNPV ORF124基因截短片段的克隆与表达

目的:克隆并表达斜纹夜蛾核多角体病毒(Spodotura litura mulfieapsid nucleopolyhedrovirus,SpltMNPV)ORF124基因的部分编码序列.方法:用PER方法扩增目的基因序列片段,并将其克隆至原核表达载体pQE-30上,转化到Escherichia.coli M15[pREP-4]中进行诱导表达.结果:构建了pQE-tr124原核表达质粒,含有该质粒的大肠杆菌经IPTG诱导表达了一个与预期理论值相符的约为33kDa的蛋白.以Ni2+-NTA偶连抗体检测证明所表达的蛋白为带有组氨酸的融合蛋白.结论:成功表达了SpltMNPV ORF124的部分编码序列,该融合蛋白的成功表达为进一步深入研究基因的功能奠定了基础.     

斜纹夜蛾核型多角体病毒p74基因的克隆和序列分析

用ＡｃＮＰＶｐ７４基因３’端的１．４ｋｂ片段,通过Ｓｏｕｔｈｅｒｎｂｌｏｔｔｉｎｇ将ＳｐｌｔＮＰＶ的ｐ７４基因定位于ＸｈｏＩ（４．９ｋｂ）、ＥｃｏＲＩ（４．４ｋｂ）、ＢａｍＨＩ（３．０ｋｂ）片段上。进一步用ＥｘｏⅢ和构建亚克隆测序法,对长２５４５ｂｐ的ｐ７４基因进行,其中１９７４ｂｐ的编码编码６５８个氨基酸,蛋白分子量约７５．８７ｋＤ,其编码蛋白与ＡｃＭＮＰＶ和ＣｆＭＮＰＶｐ７４蛋白的氨基酸序列同     

斜纹夜蛾核型多角体病毒不同分离株基因序列的同源性分析

研究SpltMNPV不同分离株及SpltMNPV分离株与SpliNPV间基因序列的同源性,为SpltMNPV分离株的利用提供理论基础。根据已发表的斜纹夜蛾核型多角体病毒（SpltMNPV）中国株（Zh）基因组全序列（AF527603）和海灰翅夜蛾核型多角体病毒（SpliNPV）Not I-D片段序列（AF527603）设计引物,PCR方法扩增得到SpltMNPV日本福冈株（Fu）、埃及株（Eg）和小笠原株（Og）的ORF39～ORF42和ORF119～ORF124编码区全序列。SpltMNPV不同分离株及SpltMNPV分离株与SpliNPV间基因序列的相似性比较, Zh株和Og株,Eg株、Fu株和SpliNPV的相似性高,而Zh株和Eg株、Fu株或SpliNPV,Og株和Eg株、Fu株或SpliNPV的相似性都比较低。亦即SpltMNPV 3种基因型,B型和C型的同源性高,A型与B型或C型的同源性比较低,但A型与SpliNPV的同源性高;同一基因型内不同分离株（Eg株和Fu株）的同源性高。ETG分子进化分析表明Eg株、Fu株和SpliNPV处于一个分支,而Eg株、Fu株和SpliNPV与Zh株和Og株则处于不同的分支。因此推断Eg株和Fu株为SpliNPV的分离株,而Og株为SpltMNPV的分离株。     

杆状病毒SpltMNPV SL136蛋白的功能

先前的研究发现斜纹夜蛾核多角体病毒 (Spodopteralituramulticapsidnucleopolyhedrovirus,SpltMNPV)基因组中Sl136表达产物具有膜融合功能。通过RT PCR检测了该基因的转录时相 ;制备该蛋白质的多克隆抗血清 ,SDS PAGE、Western印迹实验证明SL136蛋白质是芽生型病毒粒子特有的蛋白质。该基因在斜纹夜蛾离体细胞系中表达产物分子量为 86、6 5kD的两条带 ,后者与芽生型病毒粒子中检测到的一条蛋白质带分子量基本一致。另外 ,细胞酶联免疫吸附测定 (cellenzyme linkedimmunosorbantassay ,CELISA)实验证明SL136蛋白可分布于重组病毒Bac Sl136和野生型SpltMNPV分别感染的Hi5、Sl zsu 1细胞表面 ,并进行了定量分析。生物测定结果表明 ,弗林蛋白酶 (furin)抑制剂对于病毒感染力没有明显的影响 ,但是抑制病毒蛋白的糖基化却使病毒的滴度大为下降     

一种新的遍在蛋白融合基因Uba256的克隆及表达

斜纹夜蛾核多角体病毒 (Spodopteralituramulticapsidnucleopolyhedrovirus ,SpltMNPV)的Uba2 5 6基因是已知昆虫病毒基因组中惟一编码遍在蛋白与GP37融合蛋白的基因。通过PCR方法扩增得到该基因及其N端的遍在蛋白编码区与C端的GP37编码区。将这 3个基因片段分别克隆至质粒pBV2 2 0与pQE30 ,构建得到重组质粒pB VUBCP、pQEUB与pQECP。pBVUBCP转化大肠杆菌DH5α ,经热激诱导表达了一条 38kD的蛋白带 ,证明Uba2 5 6表达的蛋白在大肠杆菌中没有发生剪切。pQEUB、pQECP转化大肠杆菌M15 ,经IPTG诱导 ,Western印迹分析结果表明遍在蛋白与GP37蛋白获得高效表达。以Ni2 NTA偶联抗体检测证明所表达的蛋白质均为融合蛋白质。纯化的融合蛋白质免疫新西兰大白兔可诱导产生特异抗体。ELISA和Western印迹检测结果显示 ,SpltMNPV遍在蛋白抗体及GP37抗体均与表达的融合蛋白质呈阳性反应 ,表明所表达的融合蛋白质仍保持原有蛋白质的免疫原性。以获得的抗体对感染SpltMNPV 72h的Sl zsu 1细胞进行检测 ,发现仅有一条 34kD的蛋白质带与GP37抗体发生特异反应 ;而有 4条分子量分别为 14、2 4、33、5 1kD的蛋白质带与遍在蛋白抗体发生特异反应 ,表明Uba2 5 6基因表达的蛋白质在宿主细胞内发生了剪切。     

斜纹夜蛾核多角体病毒gp37及其邻近序列的克隆及分析

从斜纹夜蛾核多角体病毒(Spodoptera litura nucleopolyhedrovirus, SpltMNPV)基因组中克隆了gp37基因.分子生物学软件分析表明,在gp37基因内部存在糖基化位点.含有晚期表达基因的启动子序列(ATAAG),推测为晚期表达的病毒膜糖蛋白基因.通过GP37蛋白的同源性比较,绘制了以gp37基因为基础的杆状病毒进化树, 发现与以多角体蛋白基因（polyhedrin）为基础所绘制的杆状病毒分子进化树有较大差异.以polyhedrin基因为基础绘制的杆状病毒分子进化树将家蚕核多角体病毒（Bombyx mori nucleopolyhedrovirus, BmNPV）与杆状病毒代表种——苜蓿丫纹夜蛾核多角体病毒（Autographa californica multinucleocapsid nucleopolyhedrovirus, AcMNPV）分开,根据gp37基因分析则将二者归到同一分枝,这与以egt基因为基础绘制的杆状病毒分子进化树结果一致.     

甜菜夜蛾核型多角体病毒中国分离株的分离鉴定及毒力测定

本文对分离自中国的甜菜夜蛾病毒进行提纯、鉴定.生物测定的结果表明其对二龄、三龄甜菜夜蛾的LC50分别为6.6× 104,2.6×105PIB/mL.     

日本三株斜纹夜蛾核型多角体病毒的增殖特性及其多角体蛋白基因的序列分析

利用斜纹夜蛾Spodoptera litura培养细胞,对近年来在日本本州、九州和四国等地发现并筛选出的对斜纹夜蛾幼虫具有强烈杀虫活性的3株斜纹夜蛾核型多角体病毒(SpltMNPV)（K-3、G1-2和G10-3）进行了生物学活性和分子生物学的初步研究,克隆了多角体蛋白基因,并进行了序列分析和比较。结果表明:（1）SpltMNPV日本分离株K-3、G1-2和G10-3分别具有不同的特征性酶切图谱,分别属于3种基因型（A型、B型和C型）; （2）3个分离株的芽生型病毒（budded virus）产生能力和多角体产生能力有差异,免疫印迹分析表明,多角体蛋白的分子量也不同;（3）日本株SpltMNPV核型多角体蛋白结构基因由747个核苷酸编码序列（编码249个氨基酸）组成,其序列与中国株SpltMNPV的同源性为98.9%,与其他6种核型多角体病毒有较高的同源性（61.7%～74.2%）,但其5′端侧翼序列(nt-1～-100)与AcMNPV和BmNPV相比差异显著,在对该基因表达调控起决定性作用的8个高度保守核苷酸序列中（nt-44～-51）有2处发生自然突变。     

甜菜夜蛾核多角体病毒泛素基因的克隆及原核表达

甜菜夜蛾核多角体病毒（Spotoptera exigua multi-nucleopolyhedrovirus,SeMNPV)泛素基因ubiquitin被克隆和序列分析,该基因编码区全长243bp,编码80个氨基酸残基,预计蛋白质分子量为9.4kDa。将这一ubiquitin基因克隆到原核表达载体pET-28a上,转化至BL21（DE3）中,用IPTG进行诱导表达,对表达的条件进行优化,用异源的泛素单克隆抗体检测目的蛋白,Western blot 实验证明所表达的蛋白是泛素蛋白。同时,我们制备了特异性的抗体,为以后的研究工作做了基础,通过计算机软件Gendoc对不同来源的泛素进行分析,结果显示,病毒中的泛素与真核细胞中的泛素相比较,泛素的氨基酸序列有较大的变化,杆状病毒的深入素基因在分子进化上可能有比较独特的途径。     

HIV gp41基因分段低温诱导表达

Clone N3 and C from Human immunodeficiency virus(HIV) gp41 gene were expressed using the pET expression system. When induced by IPTG at 37℃, both two clones did not express in E.coli BL21(DE)3. Howerver, when induced at 16℃, the two clones were both overexpressed, and the amount of the product was about 20% of the total bacteria protein. In Western blotting test, the protein product could react with HIV-positive serum. After IPTG induction, E. coli cells had much higher death rate at 37℃ than at 16℃; [^3H]uridine release assay also showed that after IPTG induction, E. coli had a higher release at 37℃. The results suggested that overexpression of the two proteins was due to their decreased toxicity at lower temperature.     

HIV gp41基因分段低温诱导表达

人类免疫缺陷病毒(Human immunodeficiency virus,HIV)GP41跨膜蛋白由于具有特殊的穿膜拓扑结构,对宿主菌细胞膜产生毒性作用而使其难以在E.coli中有效表达[1].本室前期工作发现GP41蛋白中有三个区域对表达菌细胞具有毒性作用,其分别为:位于N端2/3区域(N3片段:nt7373-8006)的融合肽(aa512-527)和跨膜区(Aa 684-705),它们含有丰富的疏水性氨基酸;以及位于C端1/3区域(C片段:nt8007-8339)的慢病毒裂解肽LLP1(aa 826-854)和LLP2(aa 768-788),可形成2个两亲性α螺旋,从而对宿主菌产生较强的细胞膜毒性作用使细菌大量死亡,最终致使GP41蛋白难以获得有效表达[2].为此我们尝试在低温条件(16℃)下对HIV-gp41N端2/3和C端1/3区域在大肠杆菌BL21(DE)3中进行诱导表达,并对低温条件下GP41蛋白表达对细菌细胞膜的毒性作用特征进行初步探讨.     

Identification of membrane-active regions of the HIV-1 envelope glycoprotein gp41 using a 15-mer gp41-peptide scan

The identification of membrane-active regions of the ectodomain of the HIV-1 envelope glycoprotein gp41 has been made by determining the effect on membrane integrity of a 15-mer gp41-derived peptide library. By monitoring the effect of this peptide library on membrane leakage, we have identified three regions on the gp41 ectodomain with membrane-interacting capabilities: Region 1, which would roughly correspond to the polar sequence which follows the fusion domain and extends to the N-terminal heptad repeat region; Region 2, which would correspond to the immunodominant loop; and Region 3, which would correspond to the pre-transmembrane region of gp41. The identification of these three regions supports their direct role in membrane fusion as well as facilitating the future development of HIV-1 entry inhibitors.     

Molecular dynamics studies of the transmembrane domain of gp41 from HIV-1

Helix-helix interactions in the putative three-helix bundle formation of the gp41 transmembrane (TM) domain may contribute to the process of virus-cell membrane fusion in HIV-1 infection. In this study, molecular dynamics is used to analyze and compare the conformations of monomeric and trimeric forms of the TM domain in various solvent systems over the course of 4 to 23-ns simulations. The trimeric bundles of the TM domain were stable as helices and remained associated in a hydrated POPE lipid bilayer for the duration of the 23-ns simulation. Several stable inter-chain hydrogen bonds, mostly among the three deprotonated arginine residues located at the center of each of the three TM domains, formed in a right-handed bundle embedded in the lipid bilayer. No such bonds were observed when the bundle was left-handed or when the central arginine residue in each of the three TM helices was replaced with isoleucine (R_I mutant), suggesting that the central arginine residues may play an essential role in maintaining the integrity of the three-helix bundle. These observations suggest that formation of the three-helix bundle of the TM domain may play a role in the trimerization of gp41, thought to occur during the virus-cell membrane fusion process.     

HIV-1包膜蛋白gp41优势抗原的构建及可溶性表达

目的:筛选1型人免疫缺陷病毒(HIV-1)中国流行株中包膜蛋白gp41的优势抗原片段,构建具有区域流行代表性的HIV-1 gp41重组抗原,为改进现有HIV-1初筛试剂盒中使用的同类抗原奠定基础。方法:利用免疫斑点杂交和生物信息学方法,从收集自重庆、广州、上海的区域代表性150份HIV-1感染者血清标本中筛选gp41抗原性强的候选样本,利用RT-PCR及巢式PCR方法扩增包含重要抗原表位决定蔟的gp41基因片段,与原核表达载体pQE30连接,转化大肠杆菌M15构建gp41重组抗原表达菌株,表达后经亲和层析纯化、SDS-PAGE和Western印迹鉴定。结果:兔源HRP标记的gp41多抗能识别标本中gp41抗原性差异,得到候选样本,扩增包含gp41主要抗原表位片段;构建了包含gp41抗原表达簇的重组原核表达质粒,表达、纯化后经His标签抗体Western印迹鉴定为阳性。结论:高纯度的重组优势gp41抗原的构建和鉴定,为进一步改进现有HIV初筛诊断奠定了基础。     

Identification of the HIV-1 gp41 core-binding motif--HXXNPF

The HIV-1 gp41 core, a six-helix bundle formed between the N- and C-terminal heptad repeats, plays a critical role in fusion between the viral and target cell membranes. Using N36(L8)C34 as a model of the gp41 core to screen phage display peptide libraries, we identified a common motif, HXXNPF (X is any of the 20 natural amino acid residues). A selected positive phage clone L7.8 specifically bound to N36(L8)C34 and this binding could be blocked by a gp41 core-specific monoclonal antibody (NC-1). JCH-4, a peptide containing HXXNPF motif, effectively inhibited HIV-1 envelope glycoprotein-mediated syncytium-formation. The epitope of JCH-4 was proven to be linear and might locate in the NHR regions of the gp41 core. These data suggest that HXXNPF motif may be a gp41 core-binding sequence and HXXNPF motif-containing molecules can be used as probes for studying the role of the HIV-1 gp41 core in membrane fusion process.     

HIV-1跨膜蛋白gp41的截短及表达

将HIV-1跨膜蛋白gp41进行截短,在大肠杆菌中进行表达并纯化。PCR扩增gp41的部分编码基因,回收的PCR产物纯化后克隆到连接载体pGEM-T上,然后用EcoRI和Sal I切下目的基因,并构建到表达载体pGEX-4T3上,导入宿主细胞BL21(DE3),用IPTG诱导表达,表达产物用亲和层析进行纯化并作相应鉴定。截短的HIV-1跨膜蛋白gp41能直接在大肠杆菌内进行表达,利用亲和层析能方便地将目的蛋白进行纯化,为跨膜蛋白的进一步应用打下基础。     

诱发血管瘤型J亚群禽白血病病毒gp85基因的克隆与表达

2007年7月至11月,中国开产前后的商品海兰褐蛋鸡群大面积暴发血管瘤,造成巨大经济损失。将病料接种DF1细胞,通过PCR和间接免疫荧光(IFA)确定此次暴发的血管瘤为J亚群白血病病毒(ALV-J)感染引起。从患病鸡群中分离到5株ALV-J(前4株已经报道),将第5株病毒命名为WS0705。为研究该毒株抗原性的特点,用PCR方法扩增出gp85基因,并克隆进pMD18-T载体进行测序。氨基酸系统进化树分析显示WS0705与英国ALV-J原型毒株HPRS-103同源性最高。从已构建的质粒pMD18-T-WS0705gp85中酶切回收gp85基因,构建重组转移载体pFastBacH Tb-WS0705gp85。利用Bac-to-Bac表达系统获得了重组杆状病毒rBac-WS0705gp85。间接免疫荧光和Western blot检测WS0705gp85基因的表达产物。间接免疫荧光显示,构建的重组杆状病毒感染的Sf9细胞呈现明显的强阳性反应;Western blot分析,重组病毒感染的Sf9细胞蛋白显示出约35kD的阳性条带。结果表明,WS0705gp85基因在Sf9细胞中得到良好的表达,并且其编码产物完全可以被外源性ALV-J的特异性单抗JE9识别,进一步证明本次暴发血管瘤的病原为ALV-J,并为进一步开发ALV-J相关诊断产品奠定了基础。     

Fast folding of the HIV-1 and SIV gp41 six-helix bundles

Human (HIV-1) and simian (SIV) immunodeficiency virus fusion with the host cell is promoted by the receptor-triggered refolding of the gp41 envelope protein into a stable trimer-of-hairpins structure that brings viral and cellular membranes into close proximity. The core of this hairpin structure is a six-helix bundle in which an inner homotrimeric coiled coil is buttressed by three antiparallel outer HR2 helices. We have used stopped-flow circular dichroism spectroscopy to characterize the unfolding and refolding kinetics of the six-helix bundle using the HIV-1 and SIV N34(L6)C28 polypeptides. In each case, the time-course of ellipticity changes in refolding experiments is well described by a simple two-state model involving the native trimer and the unfolded monomers. The unfolding free energy of the HIV-1 and SIV trimers and their urea dependence calculated from kinetic data are in very good agreement with data measured directly by isothermal unfolding experiments. Thus, formation of the gp41 six-helix bundle structure involves no detectable population of stable, partly folded intermediates. Folding of HIV-1 N34(L6)C28 is five orders of magnitudes faster than folding of its SIV counterpart in aqueous buffer: k(on),(HIV-1)=1.3 x 10(15)M(-2)s(-1) versus k(on),(SIV)=1.1 x 10(10)M(-2)s(-1). The unfolding rates are similar: k(off),(HIV-1)=1.1 x 10(-5)s(-1) versus k(off),(SIV=)5.7 x 10(-4)s(-1). Kinetic m-values indicate that the transition state for folding of the HIV-1 protein is significantly more compact than the transition state of the SIV protein. Replacement of a single SIV threonine by isoleucine corresponding to position 573 in the HIV-1 sequence significantly stabilizes the protein and renders the folding rate close to that of the HIV-1 protein yet without making the transition state of the mutant as compact as that of the HIV-1 protein. Therefore, the overall reduction of surface exposure in the high-energy transition state seems not to account for different folding rates. While the available biological evidence suggests that refolding of the gp41 protein is slow, our study implies that structural elements outside the trimer-of-hairpins limit the rate of HIV-1 fusion kinetics.