人免疫缺陷病毒1型TAT蛋白点突变对其反式激活作用的影响

不同免疫缺陷病毒（ＨＩＶ－１,ＨＩＶ－２和ＳＩＶ）的Ｔａｔ均有３个高度保守的结构域：Ｃｙｓ富集域、核心域和碱性氨基酸富集域,用ＰＣＲ定点突变法在ＨＩＶ－１Ｔａｔ蛋白的这些区域引入单氨基酸或多氨基酸突变;构建了以ＨＩＶ－１ＬＴＲ－１５８到＋８Ｏ区域为启动子,含有不同突变点的突变Ｔａｔ基因表达质粒;以荧光酶基因为报告基因,瞬时共转染Ｊｕｒｋａｔ细胞：分析不同氨基酸突变对Ｔａｔ的反式激活作用的影响。结果发现,突变后的Ｔａｔ蛋白的活性均极大地降低或丧失,表明这些序列内的氨基酸的确定性对Ｔａｔ的活性至关重要。     

辛德毕斯病毒非结构蛋白nsP2在细胞内的分布

应用免疫电镜技术,直观地显示出辛德毕斯病毒的nsP2蛋白存在于细胞核中以及它在核内的分布.将含有nsP2蛋白的一段SbV的cDNA转染细胞,结果表明,单独表达的nsP2仍可进入细胞核中.     

新型基因重组白细胞介素-2的研究开发

本文根据IL- 2基因定点突变研究所提供的有关IL- 2结构和功能重要信息 ,特别是某些关键氨基酸残基的改变对IL- 2生物活性影响 ,探讨了IL- 2和IL- 2受体相互作用关系、新型IL- 2的研制方法和变异IL -2作用机制 ,并提出设计新型天然IL -2单位点到多位点突变的策略 ,从而指导研制更有效或毒副作用降低的新型IL- 2类似物。     

烟草在PVYN病毒与烟蚜作用下对高CO2浓度的响应

以CO2浓度为主处理因子,研究了加倍CO2浓度和对照大气CO2浓度条件下,烟蚜、马铃薯Y病毒N株(PVYN)以及二者共同作用下烟草各指标的响应。结果表明,在当前CO2浓度条件下,PVYN、烟蚜及两者联合作用对烟草生物量影响不显著;而在未来高CO2浓度条件下,PVYN、烟蚜及两者联合作用对烟草生物量影响很大。CO2浓度升高后,PVYN和蚜虫二者联合作用显著降低烟草产量,危害加重,高CO2的"肥料"作用被极大地削弱。在有烟蚜、PVYN以及两者共同作用时烟草的化学物质及主要的次生代谢物烟碱的含量对CO2浓度升高的响应也发生一定的变化,表现在:高CO2浓度条件下,蚜虫、蚜虫与PVYN共同作用显著增加了烟草的含氮量;显著减少了烟叶含糖量;PVYN及其与蚜虫共同作用显著升高叶片可溶性蛋白含量;当高CO2浓度下,各处理的烟草烟碱含量均显著下降,而且PVYN感染的烟叶烟碱含量无论在哪一种CO2浓度条件下,都比无毒无虫的对照烟叶烟碱含量升高。结果显示,烟蚜和马铃薯Y病毒N株(PVYN)对烟草的产量、营养物质及防御物质都有影响;CO2浓度升高对烟草的生长有促进作用,增加了烟草的产量,但蚜虫的危害和PVYN感染使烟草产量下降,在高CO2浓度条件下,烟蚜和PVYN共同作用相对于目前CO2浓度对烟草产量的危害加重。     

血清总胆酸酶法分析及其应用

总胆汁酸(TBA)是胆甾醇在肝内分解以及在肠肝循环中胆甾酸代谢产物的总称。又分为初级胆汁酸(胆酸、鹅脱氧胆酸)和二级胆汁酸(脱氧胆酸、石胆酸、熊脱氧胆酸)。血清胆汁酸水平反映肝实质性损伤,尤其在急性肝炎,慢性活动性肝炎、酒精肝损伤和肝硬化时有较灵敏的改变,是肝病实验诊断的一项重要指征。     

SARS-CoV S蛋白受体结合区的重组表达及免疫原性分析

为了表达SARS-CoV的S蛋白的受体结合区并对其免疫原性进行分析,用PCR方法扩增S蛋白的受体结合区基因片段,克隆至原核表达质粒pET-F32a＋并在大肠杆菌中表达,应用Western—blot鉴定表达的目的蛋白,而后以该蛋白作为诊断抗原包被酶联卡反来检测20份SARS病人血清和28份健康人血清,结果原核表达的S蛋白能够和所用的SARS病人血清反应。这提示表达的S重组蛋白具有良好的抗原性。将变性纯化的重组蛋白和复性蛋白分别皮下免疫小鼠,第三次免疫一周后收集抗血清,用ELISA测定抗体和同时测定中和抗体活性。用变性的抗原免疫的小鼠血清均无中和活性;而用复性的蛋白免疫的小鼠产生了中和抗体。实验表明,S蛋白受体结合区无线性中和表位,中和抗体的产生是由构象表位诱导的。提示该蛋白有可能应用于亚单位疫苗的研究。     

鸡白细胞介素2增强传染性法氏囊病病毒多聚蛋白DNA疫苗免疫原性的研究

鸡白细胞介素 2(IL-2)基因是新近被确定的非哺乳类IL-2基因。将鸡白细胞介素2(IL-2)基因和传染性法氏囊病病毒 (IBDV)多聚蛋白基因 (VP2/VP4/VP3)分别插入真核表达载体pCI的CMV启动子下游 ,制备DNA疫苗 ,免疫 14日龄SPF鸡 ,14d后二免 ,二免后 3d攻击标准强毒株。结果表明共注射鸡IL 2质粒能明显增强DNA疫苗对强毒攻击 ,保护率达 80 % ;能增强DNA疫苗诱导的中和抗体效价 (P<0.05 ) ;能显著促进鸡胸腺、脾脏和外周血液T淋巴细胞及法氏囊B淋巴细胞增殖反应(P<0.05)。这些结果提示鸡IL 2能明显增强IBDV多聚蛋白DNA疫苗的免疫原性 ,是一种优良的禽类DNA疫苗佐剂。     

同时表达麻疹病毒L4株HA和F蛋白及人白细胞介素2(IL2)的重组痘苗病毒疫苗株的构建

构建了同时表达麻疹病毒ＬＡ株ＨＡ和Ｆ基因及人白细胞介素２（ＩＬ２）的重组痘苗病毒疫苗株ＲＶＪＭＬＨＡＦＫＩＬ２。Ｗｅｓｔｅｍｂｌｏｔ结果显示,ＨＡ、Ｆ和人ＩＬ２基因均在痘苗病毒中稳定有效表达,且产物与天然蛋白相近。ＨＡ分子有糖化的７９ｋＤ和非糖化的田ｋＤ两种形式;Ｆ分子以６０ｋＤ的前体Ｆ０、４３ｋＤ的Ｆ１和１８ｋＤ的Ｆ２三种形式存在。表达产物的血凝效价为１：８,血溶活性ＯＤ５４０的测定结果是Ｏ３７。该重组病毒免疫新西兰白兔及Ｃ５７小鼠,可以产生抗麻诊病毒ＨＡ和Ｆ蛋白的特异性ＥＬＩＳＡ（１：６４４～１６００）、血凝抑制（１：２５６～５１２）、血溶抑制（１：８０～１６０）和ＮＴ（１：６４０）抗体。接种兔及裸鼠后的毒力反应,明显低于只表达ＩＬ２的重组痘苗病毒疫苗株ＲＶＪ１２３,表现为兔皮肤红肿范围小,持续时间短,皮肤无坏死;裸鼠毒力的结果,表现出ＲＶＪＭＬＨＡＦＫＩＬ２病毒只存留于接种局部,而且滴度大大降低,未发现该病毒向其它脏器播散和增殖。麻疹重组痘苗病毒疫苗株的构建为该疫苗株的人体免疫观察奠定了基础。     

Gln49-磷脂酶A2基因工程定点突变及酶活性分析

为了确认49位谷氨酰胺磷脂酶A2（Glutamine 49 phospholipase A2, Gln49-PLA2）酶活性缺失与氨基酸序列的相关性,对Gln49-PLA2编码基因第49位氨基酸进行PCR定点突变,利用pET32a+质粒载体在大肠杆菌中表达Gln49-磷脂酶A2的突变体--天冬氨酸磷脂酶A2（Aspartic acid 49 phospholipase A2, Asp49-PLA2--Q49D-PLA2）。将表达的包涵体蛋白变性,采用固定化金属离子亲和层析进行柱上复性、纯化获得突变体融合蛋白（fusion Q49D-PLA2--fQ49D-PLA2）;突变体融合蛋白经蛋白水解酶Factor Xa酶切后,采用Hitrap SP阳离子交换层析和Superdex 75凝胶层析进一步纯化,得到突变体蛋白Q49D-PLA2,得率为1.3%,比酶活为72U/mg。从而证实Gln49-PLA2酶活性缺失的关键原因是49位氨基酸为谷氨酰胺。     

中国长白山乌苏里蝮蛇金属蛋白酶解整合蛋白Ussurin基因克隆和序列分析

采用Clontech链转换建库试剂盒,建立了中国长白山乌苏里蝮蛇毒腺cDNA文库,从中克隆了金属蛋白酶/解整合蛋白Ussurin,并进行了序列分析。结果显示,Ussurin开框读码序列由1434bp组成,编码478个氨基酸。由核苷酸顺序推导的氨基酸序列可以看出,Ussurin最初的翻译产物是酶原前体;依次含有18氨基酸组成的信号肽,171氨基酸组成的酶原区和由289氨基酸组成的Ussurin（200氨基酸组成的金属蛋白酶结构域、16氨基酸组成的间隔区和73氨基酸组成的解整合蛋白结构域）。Ussurin的金属蛋白酶结构域含有3对二硫键;解整合蛋白结构域含有6对二硫键和特征性RGD(精氨酸甘氨酸天冬氨酸)结构。其基因序列和结构域组成与GenBank中蛇毒金属蛋白酶/解整合蛋白呈现高度同源性属于P-Ⅱ。氨基酸序列blast比对发现,酶原区和解整链蛋白结构域呈现极高的同源性,而金属蛋白酶结构域却出现了极高的变异,推测这些变异结构区是为了适应不同的底物、不同受体或同一受体的不同结构域。     

Self-cleavage of fusion protein in vivo using TEV protease to yield native protein

Overproduction of proteins from cloned genes using fusion protein expression vectors in Escherichia coli and eukaryotic cells has increased the quantity of protein produced. This approach has been widely used in producing soluble recombinant proteins for structural and functional analysis. One major disadvantage, however, of applying this approach for clinical or bioindustrial uses is that proteolytic removal of the fusion carrier is tedious, expensive, and often results in products with additional amino acid residues than the native proteins. Here we describe a new method for productions of native proteins with original amino termini in vivo via intracellular self-cleavage of the fusion protein using tobacco etch virus (TEV) protease. Our design allows one to simultaneously clone any gene into multiple fusion protein vectors using two unique cloning sites (i.e., SnaBI and XhoI) without restriction digestion, and then rapidly identifies those constructs producing soluble native proteins. This method will make the fusion protein approach more feasible for protein drug research.     

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.     

膜蛋白的拓扑学

膜蛋白的拓扑学是研究膜蛋白三维结构的出发点．利用融合蛋白和化学修饰等实验技术已确定了很多膜蛋白的拓扑学．对膜蛋白的转运与插膜的研究确定可能存在两类插膜元件．对已知拓扑学的膜蛋白的统计分析以及蛋白质工程的研究表明存在膜蛋白拓扑学的内正规则．目前已形成预测膜蛋白的拓扑学的比较可靠的策略,这在反向生物学上具有重要意义.但要进行三维结构的预测还有许多路要走．     

Membrane topology of transmembrane proteins: determinants and experimental tools

Membrane topology refers to the two-dimensional structural information of a membrane protein that indicates the number of transmembrane (TM) segments and the orientation of soluble domains relative to the plane of the membrane. Since membrane proteins are co-translationally translocated across and inserted into the membrane, the TM segments orient themselves properly in an early stage of membrane protein biogenesis. Each membrane protein must contain some topogenic signals, but the translocation components and the membrane environment also influence the membrane topology of proteins. We discuss the factors that affect membrane protein orientation and have listed available experimental tools that can be used in determining membrane protein topology.     

Tobacco Etch Virus Protease: Crystal Structure of the Active Enzyme and Its Inactive Mutant

Tobacco Etch Virus Protease (TEV protease) is widely used as a tool for separation of recombinant target proteins from their fusion partners. The crystal structures of two mutants of TEV protease, the active autolysis-resistant mutant TEV-S219D in complex with the proteolysis product, and the inactive mutant TEV-C151A in complex with a substrate, have been determined at 1.8 and 2.2 Å resolution, respectively. The active sites of both mutants, including their oxyanion holes, have identical structures. The C-terminal residues 217–221 of the enzyme are involved in formation of the binding pockets S ₃–S ₆. This indicates that the autolysis of the peptide bond Met218–Ser219 exerts a strong effect on the fine-tuning of the substrate in the enzyme active site, which results in a considerable decrease in the enzymatic activity.     

Structural determinants of tobacco vein mottling virus protease substrate specificity

Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac‐RETVRFQSD) at 1.7‐Å resolution. As observed in several crystal structures of TEV protease, the C‐terminus (～20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by ～10‐fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1′ position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k_cat and K_m for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.     

水稻OsRacD蛋白G15V、T20N点突变对其细胞定位的影响及其与靶蛋白的相互作用

OsRacD是水稻小GTP结合蛋白Rho家族成员,功能之一是作为“分子开关",通过控制花粉管的延伸生长,参与光敏核不育水稻光周期育性转换过程. 为研究该蛋白的作用机制,采用重叠延伸PCR方法,分别在其GTPase结构域中引入G15V、T20N点突变,模拟GTP和GDP结合形式的OsRacD. 进一步构建了受控于CaMV35S的与绿色荧光蛋白融合表达的双元植物表达载体;采用农杆菌介导法转化洋葱表皮细胞,在荧光显微镜下观察蛋白在活细胞内定位的特点. 结果显示,野生型蛋白在细胞质和细胞膜都有分布,组成型激活的蛋白主要分布在细胞膜上,而失活型蛋白则大都集中到细胞核周围. 蛋白相互作用的酵母双杂交体系分析显示,OsRacD及其2个突变体具有不同的靶蛋白结合特性. 研究证实,水稻OsRacD蛋白G15V和T20N点突变不仅影响其在活细胞内的定位,而且也影响了与靶蛋白的相互作用.说明处于不同鸟苷酸结合状态的OsRacD具有不同的胞内定位方式,可能通过结合不同的靶蛋白,引发不同的细胞应答事件.     

Mapping the antigenic determinants and reducing the immunogenicity of trichosanthin by site-directed mutagenesis

Summary Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) possessing multiple pharmacological properties. One of its interesting properties is to inhibit human immunodeficiency virus (HIV) replication but its strong immunogenicity has limited the repeated clinical administration. To map the antigenic determinants and reduce the immunogenicity of TCS, two potential antigenic sites (YFF81–83 and KR173–174) were identified by computer modeling, and then three TCS mutants namely TCS_{YFF81–83ACS}, TCS_{KR173–174CG}, and TCS_YFF-KR were constructed by site-directed mutagenesis. The RI activity and DNase-like activity of the three constructed TCS mutants were similar to natural TCS but with much lower immunogenicity. Results suggested that the two selected sites are all located at or near the antigenic determinants of TCS. In toxicity studies, the LD₅₀ of the three TCS mutants was not different from natural TCS. These findings would be useful in designing a better therapeutic agent for AIDS.Qunxing An and Sanhua Wei equally contribute to this work.