乙脑病毒E—Hsp70与E-肽连接区对小鼠特异性免疫的比较

在本实验室已构建的原核表达载体(含乙脑疫苗株SA14-14-2株E蛋白基因主要抗原片段)的基础上用巴斯德毕赤酵母系统表达,该片段长1113bp,编码371个氨基酸残基,研究酵母表达的该乙脑病毒(Japaneseencephali-tisvirus)E蛋白主要抗原片段与结核杆菌热休克蛋白70(Hsp70)的融合蛋白以及该抗原肽与Hsp70上的一个功能域-肽连接区(Peptidebindingdomain,以下简称BD)融合形成的蛋白,用这三种蛋白分别免疫BALB/c小鼠,以酵母单独表达的E蛋白主要抗原片段免疫作为对照,比较它们对小鼠抗E蛋白主要抗原片段特异性的细胞免疫和体液免疫的影响。采用腹腔内注射蛋白的方法免疫小鼠,主要从IL-2的mRNA水平,淋巴细胞的增殖和抗体滴度这三个方面进行比较,最后我们得出E-BD融合蛋白在免疫效果方面比E-Hsp70略好一些,所以在本试验中肽连接区是完全可以代替Hsp70独立行使其佐剂功能。     

乙脑病毒E-Hsp70与E-肽连接区对小鼠特异性免疫的比较

在本实验室已构建的原核表达载体(含乙脑疫苗株SA14-14-2株E蛋白基因主要抗原片段)的基础上用巴斯德毕赤酵母系统表达,该片段长1113bp,编码371个按基酸残基,研究酵母表达的该乙脑病毒(Japanese encephali-tis uirus)E蛋白主经抗原片段与结核杆菌热休克蛋白70(Hsp70)的融合蛋白以及该抗原肽与Hsp70上的一个功能域-太连接区(Peptide binding domain,以下简称BD)融合形成的蛋白,用这三种蛋白分别免疫BALB/c小鼠,以酵母单独表达的E蛋白主要搞原惩一片段免疫作为对照,比较它们对小鼠抗E蛋白主要抗原片段特异性的细胞免疫和体液免疫的影响.采用腹腔内注射蛋白的方法免疫小鼠,主要从IL-2的mRNA水平,淋巴细胞的增殖和抗体滴度过这三个方面进行比较,最后我们得出E-BD融合蛋白在免疫效果方面比E-sp79略好一些,所以在本试验中肽连接区是完全可以区是完全可以代替Hsp70独立先例其佐剂功能.     

乙脑E蛋白与结核杆菌HSP70的融合蛋白对BALB/c小鼠特异性免疫的影响

研究酵母表达的乙脑病毒(JapaneseEncephalitisvirus)E蛋白主要抗原片段与结核杆菌热休克蛋白70 (hsp70 )形成的融合蛋白对小鼠细胞免疫和体液免疫的影响。采用腹膜内注射蛋白的方法免疫小鼠,以直接免疫E蛋白主要抗原片段和以E蛋白主要抗原片段和单独表达的hsp70两者均以5 0pmol的量进行混合后的蛋白免疫的小鼠作为对照,用半定量RT PCR检测细胞免疫因子IL 2mRNA的水平,IL 2是细胞介导的免疫反应和巨噬细胞激活中的关键分子,MTT法检测淋巴细胞的增殖情况以及通过ELISA检测抗体水平,从这3个方面来评价融合与未融合蛋白以及等量混合后的免疫效果。结果E蛋白主要抗原片段与结核杆菌hsp70重组后,mIL 2 ,淋巴细胞的增殖以及抗体水平均较重组前明显升高,因此,以乙脑E HSP70融合蛋白免疫能增强小鼠针对E蛋白主要抗原片段的细胞免疫和体液免疫     

基因Ⅰ型乙型脑炎病毒亚单位疫苗候选抗原的免疫原性比较

为了筛选出免疫原性最佳的基因Ⅰ型乙型脑炎病毒亚单位疫苗候选抗原,将基因Ⅰ型JEVGS株的prMEIII融合基因、polytope复合表位基因和prMEIII-polytope融合基因分别克隆构建到原核表达载体pET-30a上,经诱导表达纯化获得重组蛋白。将制备的重组蛋白免疫小鼠,通过ELISA监测体液免疫反应、通过噬斑减少中和试验滴定中和抗体滴度、通过细胞因子表达丰度和淋巴细胞增殖实验分析细胞介导的免疫反应,比较分析制备的乙型脑炎病毒亚单位疫苗候选抗原的免疫原性。结果表明:获得的分子量分别为35kDa(prMEIII)、28kDa(polytope复合表位抗原)和57 kDa (prMEIII-polytope)的重组蛋白均能诱导免疫小鼠产生较强的体液免疫和细胞免疫反应。与prMEIII-polytope和polytope重组蛋白免疫组相比,prMEIII蛋白可诱导免疫小鼠产生更高的IL-2和IFN-γ表达丰度和淋巴细胞增殖水平(P0.05)。prMEIII蛋白免疫小鼠诱导产生的中和抗体滴度接近于商品化乙脑减毒疫苗SA14-14-2 (P0.05)。上述研究结果表明,prMEIII重组蛋白可以作为乙型脑炎病毒亚单位疫苗的备选蛋白。     

乙型脑炎病毒SA14-14-2株E蛋白主要抗原片段的高效表达

在本实验室已构建的原核表达载体(含乙脑疫苗株SA14-14-2株E蛋白基因主要抗原片段)的基础上用巴斯德毕赤酵母系统表达,该片段长1113bp,编码371个氨基酸残基,将其亚克隆入酵母表达载体pPICZα-A,以电穿孔法转化酵母X-33,用Zeocin平板筛选重组子,经甲醇诱导表达后,SDS-PAGE和免疫印迹分析表达产物.由于糖基化不同,所表达产物有两种,其相对分子质量分别为44kDa和50kDa,表达量较高,约为290mg/L,经Western印迹验证,有较好的抗原性.在ELISA试验中,我们直接以PBS透析后的酵母上清包被,能够很明显地区分出乙脑阴阳性血清,与RT-PCR检测的相符率达95%,为制备JEV的诊断抗原和基因工程疫苗提供了依据.     

流行性乙型脑炎病毒E蛋白结构域Ⅲ的抗原表位鉴定与功能研究

流行性乙型脑炎病毒(Japanese encephalitis virus,JEV)是一种严重危害人畜健康的虫媒病毒.表面囊膜蛋白(E蛋白)是该病毒的主要结构蛋白.E蛋白在介导病毒与宿主细胞的吸附、融合,决定病毒的血凝活性、细胞嗜性以及决定病毒毒力和诱导宿主产生保护性免疫反应中起重要作用.E蛋白结构域Ⅲ(EⅢ)是诱导中和抗体的重要区域.为确定乙型脑炎EⅢ的抗原表位,实验首先克隆了JEV疫苗株SA14-14-2的EⅢ区域,并用pGEX-6P-1载体进行融合表达,免疫印迹分析表明,该融合蛋白能被抗JEV血清识别.为了进一步对该结构域进行抗原表位作图,设计了14个覆盖该区域且部分重叠的短肽.将各短肽与GST进行融合表达与纯化.短肽融合蛋白经JEV阳性血清免疫印迹和EUSA免疫反应性扫描分析,结果鉴定出,E39(306TEKFSFAKNPVDTGHG320)、EA5-l(355VTNPFVATSSA366)、FA8-1(377FGDSYIV384)和E49(385VGRGDKQINHHWHKAG400)4个线性抗原表位.分别将4个抗原表位融合蛋白免疫小鼠,制备各抗原表位单因子血清,结果经体外病毒中和试验表明,E39为具有病毒中和活性的抗原表位.试验结果为进一步分析JEVE蛋白结构与功能以及诊断试剂和表位疫苗的研究提供了重要工作基础.     

乙型脑炎病毒SA14—14—2株E蛋白主要抗原片段的高效表达

在本实验室已构建的原核表达载体(含乙脑疫苗株SA14-14-2株E蛋白基因主要抗原片段)的基础上用巴斯德毕赤酵母系统表达,该片段长1113bp,编码371个氨基酸残基,将其亚克隆入酵母表达载体pPICZα-A,以电穿孔法转化酵母X-33,用Zeocin平板筛选重组子,经甲醇诱导表达后,SDS-PAGE和免疫印迹分析表达产物.由于糖基化不同,所表达产物有两种,其相对分子质量分别为44kDa和50kDa,表达量较高,约为290mg/L,经Western印迹验证,有较好的抗原性.在ELISA试验中,我们直接以PBS透析后的酵母上清包被,能够很明显地区分出乙脑阴阳性血清,与RT-PCR检测的相符率达95%,为制备JEV的诊断抗原和基因工程疫苗提供了依据.     

SARS冠状病毒囊膜E蛋白的重组表达与单克隆抗体制备

从基因库中调取SARS冠状病毒囊膜E蛋白基因序列,用连接PCR方法合成完整片段。测序确认后与人免疫球蛋白（IgG）序列Fc段连接并克隆至原核表达载体pPRO EX Hta中。从SDS—PAGE凝胶中回收表达产物后免疫BALB／c小鼠,经融合、筛选制备特异性单克隆抗体（mAb）。连接PCR扩增出231bp的DNA,测序结果与GenBank公布的序列一致,与人IgG序列Fc段基因连接后克隆至原核表达载体,在大肠杆菌中获得较好表达。表达产物经SDS—PAGE后,在相对分子质量（Mr）约37000处可见1条明显的诱导表达条带。Western印迹的结果表明,该电泳带与SARS患者的恢复期血清呈特异性免疫反应,,从SDS—PAGE凝胶中回收表达产物并免疫BALB／c小鼠,制备出2株抗E蛋白的mAb。这些mAb与SDS—PAGE凝胶上Mr约为37000的蛋白带也呈现很强的免疫反应。所获得的重组表达E蛋白及特异性mAb为进一步建立SARS病毒感染早期诊断的方法奠定了基础。     

糖基因Glt8d2重组蛋白的表达、纯化及多克隆抗体的制备

目的构建新糖基转移酶基因Glt8d2的原核表达载体,诱导融合蛋白的表达,并对其进行纯化;制备兔抗GltSd2蛋白多克隆抗体并进行鉴定。方法应用RT—PCR技术,以HepG2细胞mRNA为模板,扩增Glt8d2目的基因片段,构建原核表达载体pET-32a（＋）-Glt8d2。转化大肠埃希菌B121,异丙基-D-半乳糖苷诱导并通过SDS—PAGE凝胶电泳分析、Western印迹分析、生物质谱分析证实Gh8d2重组蛋白表达正确。大量表达后利用Ni’亲和柱对表达蛋白进行纯化及柱上复性。纯化蛋白免疫新西兰大白兔,获得抗Gh8d2蛋白的多克隆抗体。以纯化的Glt8d2重组蛋白为抗原,分别以免疫前后的新西兰兔血清作为第一抗体,利用Western印迹和酶联免疫吸附法对多克隆抗体进行特异性分析及效价检测。结果扩增获得Glt8d2基因片段,成功表达了Gh8d2重组蛋白,经SDS—PAGE凝胶电泳和Western印迹分析得到证实。成功获得重组蛋白及兔抗Gh8d2多克隆抗体。ELISA检测证实多克隆抗体效价〉1：320000。免疫组织化学分析显示,该基因编码的糖基转移酶在肝实质细胞内呈胞质模式表达分布。结论利用大肠埃希菌B121能够成功表达Gh8d2重组蛋白,获得高特异性、高效价兔抗Glt8d2重组蛋白的多克隆抗体,为今后研究Glt8d2基因的生物学功能研究奠定了基础。     

抗HIV-1白喉免疫毒素的构建及原核表达

目的：构建重组DTA—hS120融合基因表达载体,并诱导其在大肠杆菌中表达。方法：通过PCR扩增,获得只含白喉毒素（DT）活性部位（DTA）的基因片段,将其克隆入原核表达载体pET-28a,转化大肠杆菌BL21（DE3）,IPTG诱导表达,经SDS—PAGE和Western印迹分析鉴定。结果：酶切及DNA序列测定鉴定质粒构建正确;SDS—PAGE和Western印迹分析证实,可表达出相对分子质量为54000的融合蛋白,与DTA—hS-20融合蛋白的分子量一致;经凝胶成像分析,其表达量约占菌体总蛋白的23％,表达形式主要为包涵体。结论：构建了DTA—hS-20重组表达质粒,并获得了高效表达,为进一步研究抗HIV-1治疗药物奠定了基础。     

Calculation of thermodynamic properties of protein in Escherichia coli K-12 grown on succinic acid, energy changes accompanying protein anabolism, and energetic role of ATP in protein synthesis

Using an average of the results from three methods of calculation, estimations are made of the thermodynamic properties of a unit carbon formula weight (UCFW) of Escherichia coli K-12 protein. These resulted in values fro DeltaG(f) of -38.09 kJ (-9.10 kcal)/ UCFW, for DeltaH(f) of -68.18 kJ (-16.29 kcal)/UCFW, and for DeltaS(f) of -94.2 J (-22.5 cal)/UCFW deg. The absolute entropy of one UCFW of E. coli K-12 protein is calculated to be 73.8 J/UCFW deg. Using these values, the corresponding changes in thermodynamic properties accompanying the anabolism of protein by this microorganism to from one UCFW of protein by this microorganism to from one UCFW of protein are calculated to be 1.97 kJ (0.47 kcal)/UCFW for DeltaG, 0.75 kJ (0.18 kcal)/UCFW for DeltaH, and -4.09 J (-0.98 cal)/UCFW deg for DeltaS. All these values are sufficiently close to zero that they may be considered to be so. The question is raised as to the quantity of ATP energy conserved within the substance of the protein as it is synthesized from succinic acid. It is calculated that only 3.8% of the total free energy available from ATP that is required during protein anabolism can have been conserved within the substance of the protein, there being a net conversion of the remaninder into heat and entropy.     

蛋白- 蛋白作用界面特征及界面预测研究进展

蛋白-蛋白界面与其余蛋白表面有明显的差别。本文对近年来国内外有关蛋白-蛋白界面几何学、物理学、化学、进化保守性等方面特征的研究概况及应用这些特征对单体中预测界面方法的研究进展于以综述。     

A 3-disulfide mutant of mouse prion protein expression, oxidative folding, reductive unfolding, conformational stability, aggregation and isomerization

The structure of wild-type mouse prion protein mPrP(23-231) consists of two distinctive segments with approximately equal size, a disordered and flexible N-terminal domain encompassing residues 23-124 and a largely structured C-terminal domain containing about 40% of helical structure and stabilized by one disulfide bond (Cys(178)-Cys(213)). We have expressed a mPrP mutant with 4 Ala/Ser-->Cys replacements, two each at the N-(Cys(36), Cys(112)) and C-(Cys(134), Cys(169)) domains. Our specific aims are to study the interaction between N- and C-domains of mPrP during the oxidative folding and to produce stabilized isomers of mPrP for further analysis. Oxidative folding of fully reduced mutant, mPrP(6C), generates one predominant 3-disulfide isomer, designated as N-mPrP(3SS), which comprises the native disulfide (Cys(178)-Cys(213)) and two non-native disulfide bonds (Cys(36)-Cys(134) and Cys(112)-Cys(169)) that covalently connect the N- and C-domains. In comparison to wild-type mPrP(23-231), N-mPrP(3SS) exhibits an indistinguishable CD spectra, a similar conformational stability in the absence of thiol and a reduced ability to aggregate. In the presence of thiol catalyst and denaturant, N-mPrP(3SS) unfolds and generates diverse isomers that are amenable to further isolation, structural and functional analysis.     

Reversible aggregation of mouse prion protein derivatives with PrPSC-like structural properties

Three carbamylated derivatives of reduced mouse prion protein (mPrP) were isolated during the aborted oxidative folding in the presence of urea. These three prion protein derivatives (mPrP-a, mPrP-b, and mPrP-c) exist as monomer in the acidic solution (pH < 2.0) and exhibit prevalent random coil structure. However, they undergo rapid aggregation and transformation to a predominant -sheet structure upon exposure to ionic buffer with pH greater than 3.0. The stability of aggregates of mPrP conformers is in part dependent upon the time that they were allowed to develop. The nascent aggregates comprise a significant fraction of loosely packed mPrP monomers that can be dissociated by treatment with strong acidic solution. Matured aggregates acquired through prolonged sample incubation contain more tightly packed mPrP monomers that cannot be dissociated by strong acid but can be disaggregated by denaturant. The properties of reversible aggregation of mPrP-a, mPrP-b, and mPrP-c bear a striking resemblance to that observed with aggregates of hamster PrP^SC.     

酪氨酸蛋白激酶和蛋白激酶C对N-乙酰氨基葡萄糖转移酶V的双重调控

在人肝癌细胞７７２１中研究了酪氨酸蛋白激酶（ＴＰＫ）和蛋白激酶Ｃ（ＰＫＣ）的激活剂［分别为表皮生长因子（ＥＧＦ）和佛波酯（ＰＭＡ）］和各种蛋白激酶抑制剂对Ｎ－乙酰氨基葡萄糖转移酶Ｖ（ＧｎＴ－Ｖ）活力的影响,以探讨ＴＰＫ和ＰＫＣ对ＧｎＴ－Ｖ的调节。结果发现,ＥＧＦ或ＰＭＡ处理细胞４８ｈ后,ＧｎＴ－Ｖ的活力明显增高;蛋白激酶的非特异性抑制剂槲皮素和染料木黄酮（ｇｅｎｉｓｔｅｉｎ）在抑制ＴＰＫ和ＰＫＣ的同时,抑制ＧｎＴ－Ｖ的基础活力,并完全阻断ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的增高作用;ＴＰＫ的特异性抑制剂Ｔｙｒｐｈｏｓｔｉｎ－２５和ＰＫＣ的特异性抑制剂Ｄ－鞘氨醇分别应用时,各自只能部分地取消ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的诱导。但当Ｔｙｒｐｈｏｓｔｉｎ－２５和Ｄ－鞘氨醇同时加入培养基中则可完全阻断ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的诱导激活。蛋白质合成抑制剂环己亚胺和蛋白激酶抑制剂作用相仿,不但可抑制ＧｎＴ－Ｖ的基础活力,也可完全消除ＥＧＦ或ＰＭＡ对ＧｎＴ－Ｖ的激活。以上结果提示ＥＧＦ或ＰＭＡ通过蛋白激酶调节ＧｎＴ－Ｖ的酶蛋白合成,并且ＧｎＴ－Ｖ受到膜性ＴＰＫ和ＰＫＣ的双重调节,其中ｍ－ＴＰＫ较ｍ－ＰＫＣ更为重要。     

蛋白质丰度调控及整体分布的规律性认识

蛋白质是生命的重要物质基础之一,也是生命活动的主要承担者.蛋白质丰度与其执行的生物学功能息息相关,受基因表达各个过程严格精密的调控.蛋白质丰度的直接影响因素包括相应mRNA初始量、蛋白质合成速率和降解速率.细胞对此3因素的调控将决定蛋白质最终的丰度.得益于定量蛋白质组学的飞速发展,规模化蛋白质丰度数据的产出,使得研究者可致力于发掘蛋白质丰度与其内在性质(如进化特征、结构特征、功能类型等)间规律性的相关性,这对于深入认识生命系统组成的基本原则具有重要意义.本文总结了蛋白质丰度调控及蛋白质丰度与其内在性质相关性的最新研究进展,及对这些规律性现象反映的生物学意义的解读.     

PDBcor: An automated correlation extraction calculator for multi-state protein structures

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Weak conservation of structural features in the interfaces of homologous transient protein–protein complexes

Residue types at the interface of protein–protein complexes (PPCs) are known to be reasonably well conserved. However, we show, using a dataset of known 3‐D structures of homologous transient PPCs, that the 3‐D location of interfacial residues and their interaction patterns are only moderately and poorly conserved, respectively. Another surprising observation is that a residue at the interface that is conserved is not necessarily in the interface in the homolog. Such differences in homologous complexes are manifested by substitution of the residues that are spatially proximal to the conserved residue and structural differences at the interfaces as well as differences in spatial orientations of the interacting proteins. Conservation of interface location and the interaction pattern at the core of the interfaces is higher than at the periphery of the interface patch. Extents of variability of various structural features reported here for homologous transient PPCs are higher than the variation in homologous permanent homomers. Our findings suggest that straightforward extrapolation of interfacial nature and inter‐residue interaction patterns from template to target could lead to serious errors in the modeled complex structure. Understanding the evolution of interfaces provides insights to improve comparative modeling of PPC structures.