中国明对虾线粒体MnSOD在大肠杆菌中的重组表达、产物纯化及活性测定

采用PCR方法扩增编码中国明对虾线粒体锰超氧化物歧化酶(MnSOD)成熟肽的基因,并克隆到大肠杆菌表达载体pCRR T7/NT TOPOR TA中进行体外重组表达.重组质粒转化大肠杆菌BL21 (DE3) pLysS后,经IPTG诱导表达产生包涵体形式的目的蛋白.对重组蛋白进行 LC-ESI-MS 分析的结果表明,融合蛋白的三个肽段与中国明对虾线粒体MnSOD 相应肽段完全一致.将重组蛋白通过金属螯合柱进行纯化,进而透析、复性,最后获得了活性高达2373U/mg的重组蛋白.中国明对虾线粒体MnSOD的成功表达,为深入研究其在中国明对虾免疫反应和抗氧化胁迫中的作用奠定了基础.     

中国明对虾凝血栓蛋白基因全长cDNA的克隆及其表达特征

获得了中国明对虾凝血栓蛋白(TSP)基因全长cDNA,该基因由2868个碱基组成,具有一个2763bp的开放阅读框,编码920个氨基酸,其中包含21个氨基酸组成的信号肽.中国明对虾TSP由四类不同的结构域组成,从N端开始顺序为几丁质结合结构域,EGF-like结构域,Type Ⅲ repeat和TSP carboxyl-terminal domain(TSP-C结构域).结构域分析发现,中国明对虾的N端不具有TSP N-terminal domain(TSPN结构域),但有一个几丁质结合结构域,其C端的Type Ⅲ和TSP-C结构域非常保守.该基因在弧菌感染后的对虾淋巴器官和肝胰脏中的表达量显著增加,并具有不同的时空表达趋势,提示中国明对虾TSP基因在免疫反应中具有重要作用.     

牙鲆生长激素基因的克隆及其在大肠杆菌中的融合表达

采用逆转录聚合酶链式反应(RT-PCR)方法,从牙鲆(Paralichthys olivaceus)脑垂体总RNA中扩增出编码牙鲆生长素(GH)成熟肽基因序列。重组至融合表达载体pGEX-4T-3中,构建成牙鲆GH基因融合表达载体pGEX-gh,转化大肠杆菌BL21(DF3),筛选阳性克隆,IVIG诱导表达。经SDS-PAGE电泳显示在45kD处有特异的蛋白条带出现,该蛋白的表达量随诱导时间的延长而增加,3h达最高值,达到细胞总蛋白的18．3％。该融合蛋白在胞内主要以包涵体状态存在,经优化表达条件,成功地获得了可溶性的融合蛋白,经Glutathione Sepharase 4B凝胶纯化后用Western-blotting检测表明其为牙鲆生长激素,并通过酶联免疫吸附受体法证实其具有生物学活性。     

藻蓝蛋白对Hela细胞CD59基因表达调控作用的研究

探讨了钝顶螺旋藻藻蓝蛋白(PC)对Hela细胞CD59基因表达的调控作用.以正常人CD59cDNA基因为模板,经PCR扩增后重组入真核表达质粒载体pALTER-MAX,然后利用阳离子脂质体(Lipfectamine-2000)将重组质粒和PcDNA共转染人子宫颈癌细胞(Hela)和对照用正常中国仓鼠卵巢细胞(Chinese hamster ovary,CHO)进行表达.用不同浓度的钝顶螺旋藻藻蓝蛋白作用于转染细胞,通过核酸分子杂交技术、免疫荧光标记法和ELISA法对细胞中CD59分子的表达进行检测.结果表明:成功构建了重组质粒pALTER-MAX-CD59,并将其导入真核细胞(Hela,CHO),经G418筛选获得了CD59分子高效表达的细胞克隆.用藻蓝蛋白作用于筛选出的转基因细胞,证实藻蓝蛋白可促进Hela细胞表面CD59蛋白的表达并抑制Hela细胞的增殖,而对于正常CHO细胞无明显作用.     

植物选择标记基因hpt在大肠杆菌中的融合表达、纯化及活性测定

为对hpt基因编码蛋白进行的安全性评价，建立一种简便、高效的体外微生物表达体系，以获得大量的HPT蛋白，将用于植物转化的hpt基因的全编码序列克隆到原核表达载体pGEX4T-3上，在E．coli BL21中进行诱导表达，所得融合蛋白主要以包涵体的形式存在，且与Glutathione Sephrose 4B纯化介质的结合率不高。后经优化诱导表达的各种条件，成功地获得了可溶性的融合蛋白，该蛋白具有明显的生物活性。经Glutathione Sephrose 4B亲和层析，所得蛋白纯度＞95％。动物实验显示，融合蛋白还具有良好的免疫原性，免疫家兔后可诱导产生高滴度的抗体(＞1：10000)。ELISA及Western blotting检测进一步证实了所获纯化蛋白及其抗体的特性。这为深入进行HPT蛋白的安全性评价打下了良好的基础。     

人硒蛋白SelK在大肠杆菌中的高效表达

目的:构建硒蛋白SelK的重组表达载体pGEX-6P-1-SelK-GFP.方法:利用PCR、酶切和连接酶连接等技术将硒蛋白selk连接到质粒pGEX-6P-1上,通过酶切、序列测定进行鉴定.通过IPTG诱导重组载体在BL21大肠杆菌中表达,并筛选最适诱导剂浓度和最适诱导时间.结果:成功在大肠杆菌中高效表达带有GST的SelK-GFP融合蛋白,占菌体总蛋白的15%,主要以包涵体形式表达.结论:成功构建了pGEX-6P-1-SelK-GFP重组表达质粒,为进一步研究硒蛋白SelK的功能、抗体制各打下了基础.     

真鲷肿瘤坏死因子(TNFα)基因的体外重组与纯化研究

将真鲷肿瘤坏死因子(TNFα)基因的成熟肽区域克隆到表达载体pQE30中,并转化到大肠杆菌XL-blue中进行表达。转化子经载体和基因特异性引物进行PCR双向筛选,并将筛选到的阳性克隆经质粒纯化后,进行序列测定。序列测定结果显示,该基因结构正确,且准确插入到表达载体启动子的下游,获得了结构和组成正确的重组子。重、组子经IPTG诱导后,以SDS-PAGE电泳鉴定,电泳结果表明,该基因在大肠杆菌XL-blue中实现了表达。通过对诱导条件的调整,在体外获得了高效表达的重组蛋白,高效表达的重组蛋白约占菌体蛋白的25％-30％。不同诱导时间对重组蛋白影响实验显示,随诱导时间的增加,重组蛋白产量逐渐增高,经4h诱导,其表达量可以达到平台期,过长时间的诱导,对表达产量没有影响。重组蛋白经镍金属亲和层析纯化,获得了纯度较高的重组蛋白。采用交换缓冲液的方法,用Sephadex G150,对重组蛋白进行脱盐复性,使重组蛋白得到了进一步纯化。     

人肥胖基因在大肠杆菌中的表达

肥胖基因编码的瘦蛋白可以反映机体脂肪含量信息,在发育、繁殖、造血及体重调节等方面具有重要功能。本文利用PCR方法扩增去除信号肽的人肥胖基因cDNA序列,并将位于基因5’端的CCC转变为大肠杆菌常用密码子CCG,扩增片段经测序证实后,克隆原核表达载体pT7-7,重组质粒转化大肠杆菌BL21(DE3),经温度诱导,SDS-PAGE电泳可见分子量约为：16kD的特异蛋白表达带,表达量最高占菌体蛋白总含量的45％以上。表达重组蛋白经纯化,注射昆明白小白鼠,小白鼠体重明显下降,说明纯化的重组蛋白具有生物学活性。     

中国明对虾乙酰基专一性凝集素分离纯化方法的研究

为研究凝集素在中国明对虾天然免疫中的作用,利用3种不同物质--N-乙酰葡萄糖胺(GlcNAc)、胎球蛋白(Fetuin)和小牛黏液素(BSM)为配基,分别与3种柱材料结合进行亲和层析,从中国明对虾血淋巴中纯化凝集素.这3种亲和层析均得到了一种相同的凝集素FCL-1,经SDS-PAGE证明FCL-1的分子量为168kDa.利用新鲜小鼠血细胞进行分离纯化同样获得了这种凝集素.FCL-1与乙酰基糖类和唾液酸类糖蛋白均具有较强的结合特性.     

WSSV-VAP1基因克隆及在毕赤酵母中的表达

根据WSSV-黏附蛋白(VAP1)基因序列设计了一对引物,在5‘引物和3‘引物中分别引入EcoRI和XbaI酶切位点。经PCR扩增,将VAP1基因克隆至穿梭载体pGAPZαA之GAP启动子下游位点,构建成含α-factor信号肽的重组表达载体,获得的重组质粒pGAPZαA-VAP1经线性化后转入毕赤氏酵母SMD1168菌株,构建成分泌型表达VAP1的酵母工程菌株。SDS-PAGE和Western-blot及结合活性检测分析表明,VAP1基因在该体系中得到成功表达,表达产物与对虾细胞膜具明显的结合活性。     

Determination of recombinant human proinsulin fusion protein produced in Escherichia coli using oxidative sulfitolysis and two-dimensional HPLC.

A method for the sample preparation and determination of a human proinsulin fusion protein (ChPI) expressed in recombinant Escherichia coli samples is described. The method is applicable to samples containing whole cells or isolated inclusion bodies. The procedure involves the rapid sulfitolysis of samples in 7 M guanidine hydrochloride and analysis with a column-switch method using size exclusion and weak anion exchange HPLC. The response of the method was linear for ChPI-S-sulfonate concentrations up to 4.4 mg/mL. Recovery of standard added to samples was greater than 95% in all cases. The specificity of the method was demonstrated by the analysis of E. coli cells containing a negative control plasmid. The reproducibility of the method was good on a daily (% RSD = 1.618; n = 18) and a day-to-day (% RSD = 3.346; n = 26 days) basis. The general applicability of this approach was suggested by quantitating recombinant trypsinogen (methionyltrypsinogen expressed in E. coli), as the S-sulfonate, using size exclusion and cation exchange HPLC.     

八肋游仆虫一种富含谷氨酸的蛋白GARP的表达与纯化

为研究游仆虫中含有三核苷酸重复序列的GARP基因编码的GARP蛋白在细胞中的功能,利用定点突变技术,将GARP基因中的TGA突变为通用半胱氨酸密码子TGT.突变后的GARP基因构建于原核表达载体pRSETc中,得到重组质粒pRSETc-GARP,将pRSETc-GARP质粒转化至大肠杆菌BL21(DE3)中,经IPTG诱导,带有纯化标签的重组蛋白His6-GARP获得可溶表达,表达产物与抗His抗体在约26 kDa处有很强的交叉反应.His6-GARP蛋白在不同pH条件下通过两次阴离子交换层析和一次凝胶层析三步纯化达到电泳纯.     

Biosensor for asparagine using a thermostable recombinant asparaginase from Archaeoglobus fulgidus

Asparaginase from the hyperthermophilic microorganism Archaeoglobus fulgidus was cloned and expressed in Escherichia coli as a fusion protein with a polyhistidine tail. After heat treatment to denature most of the native E. coli proteins, the enzyme was purified by an immobilized metal ion affinity chromatography method. The activity of the enzyme was determined by monitoring the change in ammonium concentration in solution. It was found that the enzyme is thermostable at temperatures as high as 85 degrees C. The KM for L-asparagine was 8 x 10(-5) and 5 x 10(-6) M at 37 and 70 degrees C, respectively. The catalytic activity for L-asparagine was 5-fold higher than for D-asparagine. The enzyme was immobilized in front of an ammonium-selective electrode and used to develop a biosensor for asparagine. The biosensor had a detection limit of 6 x 10(-5) M for L-asparagine. Unlike a sensor based on asparaginase from E. coli, the biosensor based on recombinant asparaginase from A. fulgidus demonstrated higher stability.     

人重组血管生成素在大肠杆菌的表达与鉴定

利用ＰＣＲ技术从人肝细胞文库中扩增出血管生成素的ｃＤＮＡ，将其连入大肠杆菌表达载体后，在大肠杆菌成功地表达出人重组的Ａｎｓ融合蛋白和Ａｎｇ非融合蛋白，表达量占体蛋白的２５％，有明显的促进新生血管生成作用和ＲＮＡ酶及Ｌ９２９细胞粘附活性。     

Fabrication of a reversible protein array directly from cell lysate using a stimuli-responsive polypeptide

We report a new method to reversibly bind proteins to a surface in a functionally active orientation directly from cell lysate by exploiting a thermodynamically reversible hydrophilic-hydrophobic lower critical solution temperature (LCST) transition exhibited by a recombinant, stimuli-responsive elastin-like polypeptide (ELP). An ELP is covalently micropatterned on a glass surface against an inert BSA background. The ELP-patterned surface is incubated with the soluble fraction of E. coli lysate containing an expressed ELP fusion protein, which is appended with the same ELP as on the surface. The LCST transition of the grafted ELP and the ELP fusion protein is simultaneously triggered by an external stimulus. The LCST transition results in capture of the ELP fusion protein from solution onto the immobilized ELP by hydrophobic interactions between the grafted ELP and the ELP fusion protein. The captured ELP fusion protein is oriented such that the fusion partner is accessible to binding of its target from solution. We also demonstrate that TRAP is reversible; the bound protein-ligand complex is released from the surface by reversing the LCST transition. The triggered control of interfacial properties provided by an immobilized stimuli-responsive polypeptide at the solid-water interface is an enabling technology that allows reversible and functional presentation of ELP fusion proteins on a surface directly from cell lysate without the necessity of intermediate purification steps and subsequent recovery of the protein-ligand complex for downstream analysis by other analytical techniques. TRAP has application in lab-on-a-chip bioanalytical devices as well as in the fabrication of peptide and protein arrays.     

A genetically engineered fusion protein with horseradish peroxidase as a marker enzyme for use in competitive immunoassays

Horseradish peroxidase is one of the most widely used marker enzymes in immunoassays. Several disadvantages are encountered upon chemical conjugation of peroxidase with antibodies or antigens, as are low reproducibility and undefined stoichiometry. We here describe for the first time the production of a recombinant fusion of a protein analyte with horseradish peroxidase in Escherichia coli, employing refolding of inclusion bodies and reconstitution with heme. The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and defined structure. As a protein analyte, the human heart fatty acid binding protein (H-FABP) was chosen, which is a new and sensitive marker for acute myocardial infarction. The recombinant conjugate was fully active [650 U/mg with 2,2-azino-bis(3-ethyl-thiazoline-6-sulfonate) as substrate] and obtained in a yield of 12 mg/L of E. coli culture, which is better than that for recombinant peroxidase alone. The competitive immunoassay that was developed with the recombinant conjugate requires fewer incubation steps than the traditional sandwich ELISA format. It permitted the detection of H-FABP directly in plasma in the range of 10-1500 ng/mL which is the relevant range for clinical decision-making.     

利用自剪切融合蛋白在大肠杆菌中表达并纯化兔防御素

将源自兔嗜中性细胞的防御素基因NP-1插入原核表达载体pTYB2,并将重组质粒pTYB2-NP1转入大肠杆菌BL21中,用IPTG诱导使防御素以融合蛋白的形式表达。然后,通过亲和层析利用自剪切融合蛋白分离提纯目的蛋白。蛋白电泳检测到以二聚体形式存在的防御素,但此蛋白没有对大肠杆菌的抑菌活性。同时,对该表达纯化系统的特点和用原核生物大肠杆菌表达真核生物防御素的问题进行了分析和讨论。     

海洋哈维氏弧菌溶血素蛋白在酵母菌细胞表面上的展示及其活性的测定

为制备以酒精酵母为载体的基因工程疫苗,参照其Genbank中基因序列设计一对引物,PCR扩增得到预期长度的产物,双酶切插入用于酒精酵母表面展示的穿梭质粒载体pYD1,转化大肠杆菌TOP10,提取阳性质粒转化酒精酵母菌株EBY100,诱导表达后,用免疫荧光和流式细胞仪检测外源蛋白的表达,结果测得最佳诱导时间为36～48h,诱导培养后约30.0%左右的酵母细胞表达外源蛋白;同时以EBY100和转空质粒的酵母菌株为对照,测得诱导培养后的阳性酵母转化株的细胞对牙鲆鱼血细胞具有溶血活性,以此酵母活细胞分设高中低三个浓度组,腹腔注射养殖牙鲆幼鱼,检测其毒性,结果表明此重组酵母对牙鲆是安全的.该研究为下一步鱼用活载体疫苗免疫效果的研究奠定了基础.