重组刺桐胰蛋白酶抑制剂a在大肠杆菌中的表达和纯化

为了大量制备重组刺桐胰蛋白酶抑制剂a(rETIa) ,对构建的基因工程菌株E .coliBL2 1(DE3)pET2 2b mETIa进行了表达条件的优化 .用摇瓶培养 ,rETIa蛋白占菌体总蛋白 4 0 %以上 .经破碎菌体 洗涤包涵体 溶解包涵体 复性初步纯化后 ,再经二步柱层析纯化获得电泳纯的rETIa蛋白 .测定了rETIa对胰蛋白酶、胰凝乳蛋白酶、组织型纤溶酶原激活因子缺失突变体 (NTA)的抑制活性 .     

丝状支原体丝状亚种SC型脂蛋白Q N末端基因的表达、纯化与抗原性分析

为了表达丝状支原体丝状亚种SC型(MmmSC)中国分离株HVRIⅩ脂蛋白Q(LppQ)N末端基因,将该基因经PCR扩增后克隆至原核表达载体pET32a中,经酶切、PCR、测序证实获得了重组表达质粒,转化Escherichia coliBL21(DE3)菌,经IPTG诱导后获得可溶性融合蛋白,表达量占菌体总蛋白的53.7%,用Ni-NTAHis.Bind纯化试剂盒纯化后,蛋白纯度达95%以上。表达蛋白经Western blot检测其抗原活性,结果表明纯化蛋白可与CBPP标准阳性血清发生强烈的反应,而与阴性血清不发生反应。     

人呼吸道合胞病毒截短F1重组蛋白包涵体的制备、纯化和复性

目的将前期在大肠埃希杆菌中获得表达的A型人呼吸道合胞病毒兰州分离株截短的F1重组蛋白进行纯化和复性,为后期动物免疫制备抗原。方法 37℃诱导重组菌体p ET-42b-F1J/Rossata,诱导完毕后离心收集菌体,高压破碎菌体并收集包涵体后用不同浓度的Triton X-100(细胞裂解液)洗涤包涵体3次。洗涤的包涵体用8 mol/L尿素进行溶解并用镍离子亲和层析方法进行初步纯化,用阳离子交换层析方法对初步纯化蛋白进行最终的纯化。亲和层析纯化蛋白用3种不同的复性液进行了稀释复性。结果 37℃诱导5 000 m L重组菌p ET-42b-F1J/Rossata共收获37 g湿菌体,经过不同浓度Triton X-100洗涤包涵体后纯度可达75%。包涵体用8 mol/L尿素溶解后经镍离子亲和层析纯化纯度约为40%,再用阳离子交换层析介质SP HP进一步纯化样品后纯度可达90%。纯化蛋白以3种不同的复性液都能得到复性,其中复性液3的复性效果相对较好。结论实验中探索了人呼吸道合胞病毒截短F1重组蛋白包涵体的纯化方法及步骤,为后期的蛋白制备及动物免疫奠定了基础。     

重组刺桐胰蛋白酶抑制剂a的制备及其在tPA突变体纯化中的应用

将基因工程菌株E.coliBL21(DE3) pET22b-mETIa高密度发酵,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导,重组刺桐胰蛋白酶抑制剂a(rETIa)蛋白在E.coli中得到较高水平表达,表达量占菌体总蛋白的40%以上.经菌体破碎、包涵体变性、复性,二步柱层析纯化得到电泳纯的rETIa蛋白.测得rETIa对t-PA突变体(NTA)的抑制平衡常数K_i为8.72×10^－8 mol/L.据此利用纯化的rETIa蛋白制备rETIa-Sepharose 4B亲和层析柱.直接一步纯化NTA复性液,纯化的NTA纯度达90 %以上,收率为96.2 %,纯化倍数为13.2,比活为(565.7±71.3) U/μg.     

重组人胰岛新生相关蛋白的高水平表达与纯化

通过RT-PCR体外扩增目的基因胰岛新生相关蛋白(islet neogenesis associated protein,INGAP),并将其克隆入原核表达载体pET22b(+),在大肠杆菌BL21(DE3)中诱导表达.表达的目标蛋白主要以包涵体形式存在,洗涤杂蛋白后用尿素溶解,经Heparin Agrose亲合柱层析分离后,再用Superdex75凝胶过滤层析进一步纯化.纯化后的INGAP皮下注射免疫家兔,制备兔抗INGAP血清,采用免疫双扩、ELISA评价INGAP的免疫活性.结果显示INGAP表达量高达菌体总蛋白的40%左右,经HPLC测定,分离纯化后的目标蛋白纯度达到98.81%,且具有良好的免疫原性.     

重组人胰岛新生相关蛋白的表达、纯化及免疫活性研究

摘要 目的：对胰岛新生相关蛋白（Islet neogenesis associated protein ,INGAP）进行表达、纯化,并检测其免疫活性。方法： INGAP基因片段插入表达载体pET22b(+),在E.coli BL21(DE3)中表达。包涵体经洗涤并用8M尿素溶解,Heparin Agrose亲合柱层析为第一步纯化,Superdex75凝胶过滤层析作为第二步精细纯化,HPLC测定INGAP蛋白的浓度,将纯化的INGAP蛋白经注射途径免疫家兔,制备兔抗INGAP血清,采用免疫双扩、ELISA及Western Blot分析INGAP的免疫活性。结果INGAP以包涵体形式表达,表达产量高达总菌体蛋白的40%左右,经Heparin Agrose亲合柱层析和凝胶过滤层析二步组合纯化目的蛋白,经HPLC测定目的蛋白的最终纯度为98.81%,表达及纯化的INGAP具有良好的免疫活性。     

重组人角质细胞生长因子-2基因克隆、表达、纯化与活性分析

 从人胚肺二倍体细胞KMB17中抽提总RNA ,经RT PCR扩增获得编码人角质细胞生长因子 2 (keratinocytegrowthfactor 2 ,KGF 2 )的cDNA .克隆于硫氧环蛋白表达载体pThioHisA ,序列分析表明与文献报道一致 .经IPTG诱导 ,在大肠杆菌BL2 1中实现高效表达 ,表达量可达菌体总蛋白 10 %～15 % .菌体超声破碎 ,上清经CM SepharoseFF阳离子交换 ,Heparin Sepharose亲和层析 ,Superdex 75凝胶过滤层析纯化得到重组人KGF 2 ,纯度高于 95 % .生物活性分析表明 ,它能够促进成纤维细胞NIH 3T3的增殖 ,诱导鸡胚背根神经结神经轴突的生长 ,促进鸡胚尿囊膜血管生成 .研究结果表明 ,获得了 95 %纯度的具有生物学活性的重组人KGF 2 ,为进一步的基础与应用研究提供了基础     

猪丹毒丝菌SpaA蛋白的表达与纯化

[目的]在大肠杆菌中表达猪丹毒丝菌spaA基因并纯化重组蛋白。[方法]利用PCR扩增猪丹毒丝菌临床分离株spaA基因,构建重组质粒p GEX-4T-1-spaA,转化大肠杆菌BL21(DE3)进行诱导表达融合蛋白GST-SpaA,并优化表达条件。最后采用GST琼脂糖纯化树脂纯化,SDS-PAGE和Western Blotting检测。[结果]成功扩增spaA基因,获得重组表达菌株BL21(DE3)/p GEX-4T-1-spaA;在菌体OD_(600)为0. 9时,加入IPTG至终浓度0. 1 mmol/L,34℃诱导6 h的条件下表达效果最好。经SDS-PAGE检测和纯化后得到大小为97 kDa的GST-SpaA; Western Blotting检测结果表明,GST-SpaA具有良好的免疫原性。[结论]成功在大肠杆菌中表达了SpaA蛋白,经纯化得到具有免疫原性的重组蛋白,为后续研制猪丹毒丝菌SpaA蛋白亚单位疫苗奠定基础。     

HIV-1 p24蛋白在大肠肝菌中的高效可溶性表达、一步纯化及抗原性分析

合成引物扩增HIV-1 p24基因,并将其克隆到pQE-30质粒中,使其在大肠杆菌E.coli M15中以IPTG诱导高效表达,经SDS-PAGE分析,该表达产物约占菌体总蛋白20%,并且以可溶蛋白的形式存在于细菌裂解液上清之中.经镍离子柱亲和层析一步纯化,洗脱产物中p24蛋白纯度达95%.ELISA分析表明,该蛋白可与HIV感染者血清发生特异性免疫反应.以此蛋白交联Sepharose 4B,亲和层析纯化HIV感染者血清中的抗体,用所得抗体与HIV确认试剂反应,发现该纯化抗体仅与确认试剂中的p24蛋白反应.上述结果表明在大肠杆菌中已经高效表达了可溶性HIV-1 p24蛋白,该蛋白具有良好的抗原性.     

丙型肝炎病毒ns3区全长基因在大肠杆菌中的高效表达及纯化

利用PCR技术扩增HCV ns3基因,经BamHⅠ和HindⅢ双酶切后与原核表达质粒pProEX—HTb连接,转化感受态细胞Ecoli DH50α,酶切鉴定得阳性重组质粒pProEX—HTb—ns3并测序;pProEX—HTb—ns3转化宿主细胞获得工程菌,用IPTG诱导,获得NS3蛋白的高效表达,薄层扫描显示其占菌体总蛋白的35％;目的蛋白在变性条件下经Ni^2 -NTA凝胶亲和层析纯化,透析并浓缩后用丙型肝炎患阳性血清做为一抗行Western—Blot证实特异性和抗原性。结果成功表明,诱导表达产物主要以包涵体形式存在;6His—NTA纯化后获得目的蛋白,Western—blot结果显示纯化蛋白具有良好的抗原性。HCVNS3蛋白的高效表达及纯化,为利用NS3蛋白作为诊断抗原及制备单克隆抗体奠定了基础。     

Analysis of the axial filaments of Treponema hyodysenteriae by SDS-PAGE and immunoblotting

Purified axial filaments from eight serotypes of Treponema hyodysenteriae and two non-pathogenic intestinal spirochaetes were characterized by SDS-PAGE and Western blotting. Axial filaments of all ten strains had similar SDS-PAGE profiles; five major axial filament polypeptides were identified, with molecular masses of 43.8, 38, 34.8, 32.8 and 29.4 kDa. Hyperimmune gnotobiotic pig serum raised against purified axial filaments of strain P18A (serotype 4) cross-reacted with all other serotypes and with the non-pathogens, and convalescent serum taken from a pig with persistent swine dysentery also showed a strong response to the axial filament polypeptides. Hyperimmune gnotobiotic pig serum raised against axial filaments failed to agglutinate viable organisms and did not inhibit growth in vitro. Hence, the axial filaments of T. hyodysenteriae have been identified as major immunodominant antigens, although the role that antibodies to these antigens play in protection has yet to be established.     

Immobilization effects of anticell and antiaxial filament sera on Treponema zuelzerae

Rabbit antisera were produced against whole cells and against highly purified axial filaments of Treponema zuelzerae. Both types of sera react with axial filaments. Antisera against whole cells immobilize the organism; antisera against axial filaments do not. The immobilizing activity of anticell sera could be neutralized by preabsorption with whole cells but not by preabsorption with axial filaments. Preabsorption with axial filaments did, however, abolish the ability of anticell sera to react with axial filaments. Anticell sera also react with the outer cell envelope; this ability was not affected by preabsorption with axial filaments. The data show that antibody directed at something other than axial filaments causes immobilization of T. zuelzerae. The results do not exclude the possibility that the axial filament is the organelle of motility. Titration of the immobilizing activity of anticell sera by adding a constant amount of motile cells to serial dilutions of serum gave two zones of maximum immobilization, one with undiluted serum and one at higher dilutions. For a fixed amount of antiserum, increasing the numbers of cells in the titration almost, but not quite, abolished the zone phenomenon. This phenomenon appears to result from the presence of two kinds of immobilizing antibodies in anticell serum. One of these belongs to the IgG class of immunoglobulins. The other has not been identified but is present in a serum globulin fraction which contains IgM. At intermediate concentrations, the "IgM fraction" inhibits the immobilizing activity of IgG without itself causing immobilization.     

Chemistry of axial filaments of Treponema zuezerae

Highly purified axial filaments have been prepared from the spirochete Treponema zuelzerae, which possess a fine structure similar to the "beaded" form of bacterial flagella. The preparations consist largely of protein but also contain small amounts of hexose (less than 1%). The buoyant density of these filaments is 1.29 g/cm(3). At pH 4.3, in the presence of 4 m urea and 10(-3)m ethylenediaminetetraacetic acid, filament protein migrates as a single band in acrylamide gel electrophoresis. Filaments dissociate to subunits in acid, alkali, urea, guanidine or with heating, indicating that these subunits are not covalently bonded in the organized structure. This is consistent with amino acid analysis which reveals that, like bacterial flagella, the filaments are completely lacking in half-cystine. Sedimentation equilibrium measurements on dissociated axial filaments in 6 m guanidine show that the subunits are homogeneous with respect to molecular weight. A weight-average molecular weight of 37,000 +/- 1,600 daltons is obtained from these measurements. The amino acid composition of axial filaments is similar to that of various types of flagellin molecules, but the filament protein is somewhat richer in tyrosine, phenylalanine, and proline than flagellin. Tryptic peptide maps of axial filaments are consistent with the amino acid composition calculated for a molecular weight of 37,000 daltons. No amino terminal end group could be detected by the dansyl chloride method, suggesting that this end group might be blocked in the axial filament protein. The results obtained show that the axial filaments of T. zuelzerae are similar chemically to bacterial flagella and suggest that they are composed of aggregates of a single species of protein subunit.     

Structure of the peripheral domains of neurofilaments revealed by low angle rotary shadowing

The structure of the peripheral domains of neurofilaments (NFs) was revealed by rotary shadowing electron microscopy. NFs were isolated from bovine spinal cords by Sepharose CL-4B gel filtration and examined by low angle rotary shadowing. The peripheral domains appeared as thin, flexible, filamentous structures projecting from the intermediate filament core, with a constant density along their entire length. The average length of the projections was approximately 85 nm and the width about 4 nm. These projections appeared from regularly distributed sites, at 22 nm spacing, which seemed to correspond to the typical repeat of the alpha-helix-rich rod domain of the core filament. The density of the projections was found to be 4.1 (+/- 0.6) per 22 nm. We performed reconstitution experiments using purified NF polypeptides to confirm that the projection was indeed the NF peripheral domain. Individual components of the NF triplet, i.e. NF-L, NF-M and NF-H, were purified by DE-52 and Mono-Q anion exchange chromatographies in the presence of 6 M-urea and were assembled in various combinations into filaments. Reassembled filaments were somewhat more slender than the isolated NFs and exhibited a distinct 22 nm axial periodicity. While prominent projections were not observed in the filaments assembled from NF-L alone, reconstructed filaments containing NF-L plus either NF-M or NF-H revealed many projections. The average length of the projections in the filaments reconstructed from NF-L and NF-H was about 63 nm. The projections of reconstructed filaments from NF-L and NF-M were about 55 nm in length. The difference in the lengths of the projections might reflect the difference in the length of the carboxy-terminal tail domain between NF-M and NF-H. The results are interpreted to show that the carboxy-terminal tail domains of NFs project in a regular pattern from the core filament, which is consistent with a half-staggered organization of the tetrameric subunits.     

Purification and Characterization of Axial Filaments from Treponema phagedenis Biotype reiterii (the Reiter Treponeme)

Axial filaments have been purified from Treponema phagedenis biotype reiterii (the Reiter treponeme) and partially characterized chemically. The preparations consist largely of protein but also contain small amounts of hexose (3%). Filaments dissociate to subunits in acid, alkali, urea, guanidine, and various detergents. Amino acid analyses show an overall resemblance to other spirochetal axial filaments and to bacterial flagella. Dissociated filaments migrate as a single band upon acrylamide gel electrophoresis at pH 4.3 (in 4 M urea and 10 (3) M ethylenediaminetetraacetate) and at pH 12, but in sodium dodecyl sulfate gels, three bands are obtained under a wide variety of conditions. Two of these bands migrate very close together, with molecular weights of 33,000 +/- 500. The other band has a molecular weight of 36,500 +/- 500. Analysis of axial filaments by the dansyl chloride method yields both methionine and glutamic acid as amino terminal end groups. Sedimentation equilibrium measurements on dissociated axial filaments in 7 M guanidine hydrochloride yield plots of log C against varkappa(2) which vary with the speed and initial protein concentration used. Molecular weight values calculated from these plots are consistent with a model in which axial filament subunits are heterogeneous with respect to molecular weight in the approximate range of 32,000 to 36,000.     

The uvsX protein of bacteriophage T4 arranges single-stranded and double-stranded DNA into similar helical nucleoprotein filaments

The bacteriophage T4 uvsX gene codes for a DNA-binding protein that is important for genetic recombination in T4-infected cells. This protein is a DNA-dependent ATPase that resembles the Escherichia coli recA protein in many of its properties. We have examined the binding of purified uvsX protein to single-stranded DNA (ssDNA) and to double-stranded DNA (dsDNA) using electron microscopy to visualize the complexes that are formed and double label analysis to measure their protein content. We find that the uvsX protein binds cooperatively to dsDNA, forming filaments 14 nm in diameter with an apparently helical axial repeat of 12 nm. Each repeat contains about 42 base pairs and 9-12 uvsX protein monomers. In solutions containing Mg2+, the uvsX protein also binds cooperatively to ssDNA. The filaments that result are 14 nm in diameter, show a 12-nm axial repeat, and they are nearly identical in appearance to the filaments that contain dsDNA. In the filaments formed along ssDNA, each axial repeat contains about 49 DNA bases and 9-12 uvsX monomers. Both the filaments formed on the ssDNA and dsDNA show a strong tendency to align side-by-side. T4 gene 32 protein also binds cooperatively to ssDNA and interacts both physically and functionally with uvsX protein. However, when gene 32 and uvsX proteins were added to ssDNA together, no interaction between the two proteins was detected.     

Assembly of vimentin in vitro and its implications concerning the structure of intermediate filaments

After dialysis against 10 mM-Tris-acetate (pH 8.5), vimentin that has been purified in the presence of urea is present in the form of tetrameric 2 to 3 nm X 48 nm rods known as protofilaments. These building blocks in turn polymerize into intermediate filaments (10 to 12 nm diameter) when they are dialyzed against a solution of physiological ionic strength and pH. By varying the ionic conditions under which polymerization takes place, we have identified two classes of assembly intermediates whose structures provide clues as to how an intermediate filament may be constructed. The structure of the first class, seen when assembly takes place at 10 to 20 mM-salt at pH 8.5, strongly suggests that one of the initial steps of filament assembly is the association of protofilaments into pairs with a half-unit axial stagger. Increasing the ionic strength of the assembly buffer leads to the emergence of short, full-width intermediate filaments at approximately 50 mM-salt at pH 8.5. In the presence of additional protofilaments, these short filaments elongate to many micrometers when the ionic strength and pH are further adjusted to physiological levels. The electron microscope images of the assembly intermediates suggest that vimentin-containing intermediate filaments are made up of eight protofilaments, assembled such that there is an approximately 22 nm axial stagger between neighboring protofilaments. We propose that this half-unit staggering of protofilaments is a fundamental feature of intermediate filament structure and assembly, and that it could account for the 20 to 22 nm axial repeat seen in all intermediate filaments examined so far.     

株纯化轴丝在梅毒血清学中初步应用

本试验以纯化的Reiter株轴丝为抗原,建立了AF—ELISA试验方法,用该方法检测了35份自家免疫病人血清、285份非梅毒病人血清及78份各期梅毒病人血清共398份血清,并同时与FTA-ABS等进行比较,结果AF-ELISA特异性为99.3%,FTA—ABS为99.7%,两者敏感性为100%.     

Three-dimensional structure of the Z band in a normal mammalian skeletal muscle

The three-dimensional structure of the vertebrate skeletal muscle Z band reflects its function as the muscle component essential for tension transmission between successive sarcomeres. We have investigated this structure as well as that of the nearby I band in a normal, unstimulated mammalian skeletal muscle by tomographic three- dimensional reconstruction from electron micrograph tilt series of sectioned tissue. The three-dimensional Z band structure consists of interdigitating axial filaments from opposite sarcomeres connected every 18 +/- 12 nm (mean +/- SD) to one to four cross-connecting Z- filaments are observed to meet the axial filaments in a fourfold symmetric arrangement. The substantial variation in the spacing between cross-connecting Z-filament to axial filament connection points suggests that the structure of the Z band is not determined solely by the arrangement of alpha-actinin to actin-binding sites along the axial filament. The cross-connecting filaments bind to or form a "relaxed interconnecting body" halfway between the axial filaments. This filamentous body is parallel to the Z band axial filaments and is observed to play an essential role in generating the small square lattice pattern seen in electron micrographs of unstimulated muscle cross sections. This structure is absent in cross section of the Z band from muscles fixed in rigor or in tetanus, suggesting that the Z band lattice must undergo dynamic rearrangement concomitant with crossbridge binding in the A band.     

The assembly of keratins from higher plant cells

Summary In vitro assembly and morphological characteristics of purified 58 kDa, 52 kDa, 50 kDa, and 45 kDa polypeptides in the leaves and the cotyledons of the cabbage (Brassica pekinensis Rupt.) were investigated by electron microscopy and scanning tunneling microscopy. The three or four purified intermediate filament (IF) polypeptides can spontaneously assemble into intermediate filaments in vitro with a 23–24 nm axial repeat, which indicates that keratin IFs in higher plant cells have the same molecular arrangement as in animal cells. STM images suggest that the plant keratin filaments display a pronounced structural polymorphism, which can be composed of 3 nm, 4.5 nm, or 6 nm wide keratin protofilaments.Abbreviation IF intermediate filament - STM scanning tunneling microscopy - SDS sodium dodecyl sulfate - BCIP 5-bromo-4-chloro-3-indolyl phosphate-toluidine - NBC p-nitroblue tetrazolium chloride - PMSF phenylmethyl sulfonylfluoride - HOPG high oriented pyrolytic graphite