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1.
人铜锌超氧化物歧化酶基因的克隆和乳酸乳球菌中的表达   总被引:5,自引:0,他引:5  
采用RT-PCR技术从人肝总RNA中分离扩增了0.45kb的人铜锌超氧化物歧化酶(Cu/ZnSOD)基因的cDNA序列,首先克隆至大肠杆菌表达质粒pET23b,进行了序列测定和超高表达,将Cu/Zn,SODcDNA亚克隆至乳酸乳球菌表达载体pMG36e,用电穿孔法将重组质粒pMG36esod转化到乳酸乳球菌,获得Cu/Zn SOD的组成型表达,其表达量约占乳酸乳球菌可溶性蛋白的5%以上,活性染色表  相似文献   

2.
人Mn—SOD cDNA的克隆及高效表达   总被引:13,自引:0,他引:13  
用逆转录-聚合酶链反应(RT-PCR),以人肝细胞株(L02)总RNA为模板,扩增了人锰超氧化物歧化酶(hMn-SOD)的cDNA。重组到T7启动子控制下的表达载体pET-24a(+)中,构建表爱质粒pET-MnSOD,并转化大肠杆菌BL21(DE3)。SDS-PAGE及蛋白质印迹分析表明,经1mmol/L异丙基硫代-β-D-半乳糖苷(IPTG)诱导后,可高效表达一分子量为22kD的蛋白质,与抗人  相似文献   

3.
人GM—CSF cDNA的克隆和在大肠杆菌中的表达   总被引:3,自引:0,他引:3  
从诱导的人胚肺细胞HFL株中提取总RNA.经RT-PCR反应获取了人GM-CSFcDNA,DNA序列测定表明其顺序与文献报道完全一致。为了获得高效表达,应用PCR改造了人GM-CSF的cDNA5’端核苷酸序列,并将改造的人GM-CSF基因插入含T7启动子的质粒pET-11d构建成表达质粒pETC-5,将此质粒转化大肠杆菌株BL21(DE3)得到表达菌株BLEC4。表达菌株用0.5mol/LIPTG诱导2小时后,产生大量重组蛋白并形成包涵体。SDS—PAGE电泳图谱扫描结果表明,rhGM-CSF产量占菌体总蛋白量的16%。ELISA和TF-1细胞培养测定表明,初步纯化和复性的rhGM-CSF具有天然的hGM-CSF生物活性。  相似文献   

4.
将Mn-SOD与抗癌胚抗原(CEA)单链抗体基因(Sc-Fv gene)融合,重组到含T7启动子的表达载体pET-22b(+)中,构建表达质粒pETMn-SOD-ScFv,并转化大肠杆菌BL21(DE3),进行高效表达,表达物占菌体可溶性总蛋白的24%。SDS-PAGE和蛋白质和迹图谱显示表达物分子量为45kD与融合基因编码蛋白质的理论值相符。该蛋白质在大肠杆菌中为泌型表达有利于纯化。RIA测定表  相似文献   

5.
江浙蝮蛇神经生长因子在大肠杆菌中的表达及纯化   总被引:2,自引:0,他引:2  
将江浙蝮蛇(AgkistrodonhalysPalas,A.h.P.)神经生长因子(Nervegrowthfactor,NGF)基因克隆于分泌型原核表达载体pET22b+,以C端融合了6个组氨酸的形式在大肠杆菌BL21(DE3)中进行了IPTG诱导表达。SDSPAGE分析确定有一比理论值略大的诱导表达条带,其表达量占全菌蛋白质的20%左右,且表达蛋白质主要是以包涵体的形式存在。用6mol/L的盐酸胍溶解包涵体后,利用固定化金属离子(Ni2+)配体亲和层析一步纯化目的蛋白质,纯度可达80%左右。对该重组肽进行变复性研究。利用PC12细胞进行生物活力测定,证明表达产物具有NGF生物活力。  相似文献   

6.
利用PCR扩增得到粒细胞-巨噬细胞集落刺激因子(GM-CSF)、白细胞介素-3(IL-3)完整基因片段,将其分别克隆pGEM-T构建成GM-CSF/IL-3融合蛋白基因,DNA序列与设计预期一致。将得到的融合蛋白基因克隆对72RNA聚合酶表达载体pT7zz,得到表达质粒pFu,经转化至表达宿主E.coli BL21(DE3),在IPTG诱导下获得融合蛋白目的产物的直接表达。经SDS-PAGE电泳鉴  相似文献   

7.
促黄体激素/人绒毛膜促性腺激素受体(LH/hCF receptor)是一种与蛋白偶联的糖蛋白。本文报道了从大鼠卵巢cDNA库中筛选LH/hCG受体cDNA及其在昆虫细胞中的高效表达。LH/hCG受体cDNA全长2403bp,编码受体信号肽和成熟受体674个氨基酸。用多角体病毒表达载体pVL1393,LH/hCG受体cDNA在昆虫细胞中得到高效表达。在非还原和还原条件下的SDS-PAGE分析显示,用  相似文献   

8.
利用COS7细胞暂时表达系统,研究转译起始序列对EPO-cDNA表达的影响。通过DNA重组技术,构建了原EPO-cDNA表达载体pCSV-EPO(1),其转译起始序列为5'AATTCATGG3'。同时通过定点突变技术,将起始序列改变成5'CCACCATGG3',而构建了另一表达载体PCSV-EPO(2)。后经序列分析证明无误后和前均通过DEAE-dextran法转染COS7细胞上清,测定结果为  相似文献   

9.
化学合成的人尿激酶原cDNA克隆在表达质粒pET11d中,在T7启动子的作用下,经0.1mmol/LIPTG诱导,在大肠杆菌BL21(DE3)pLysS中获得表达。其表达量占菌体总蛋白的15%,表达产物以无活性的包涵体形式存在。经体外变复性,Zn2+选择性沉淀,抗体亲和柱层析,及Benzamidine亲和吸附,所表达的人尿激酶原被纯化为单一条带,其比活约110000IU/mg。  相似文献   

10.
霍乱弧菌zot基因的克隆及其在大肠杆菌中的表达   总被引:2,自引:0,他引:2  
从霍乱疫苗菌中抽提基因组DNA,用PCR的方法扩增zot基因。序列分析表明,zot基因编码399个氨基酸,其中4个氨基酸与文献报道有差异。将zot基因插入含T7启动子的质粒pET-28(a+)构建表达质粒pET-ZOT,转化大肠檑菌BL21(DE3)筛有达菌株BLZOT。表达菌株经1mmol/LT IPTG诱导表达3-5h后,表达大量ZOT蛋白,并形成包涵体,经SDS-PAGE分析重组ZOT蛋白分  相似文献   

11.
陆地棉叶绿体铜锌超氧化物歧化酶基因的克隆与表达   总被引:1,自引:0,他引:1  
以陆地棉‘CRI36'的叶片为材料,使用RACE技术克隆到了棉花叶绿体Cu/Zn-SOD酶基因。基因序列全长共1 043 bp,含有完整的开放阅读框。推导的氨基酸序列分析显示含有叶绿体信号肽,和已知植物的叶绿体Cu/Zn-SOD酶蛋白的氨基酸残基的同源性在66%~74%之间。基因的表达谱分析显示:棉花叶绿体Cu/Zn-SOD酶基因主要在叶片、茎中表达,根、花和下胚轴中没有检测到信号,即基因的表达主要在棉花的绿色组织。不同生育期的表达谱结果证实:该基因主要在苗期表达,以后表达逐渐减少。用pET-21a(+)构建了原核表达载体,在大肠杆菌BL21(DE3)的表达结果显示:表达后得到一个29.0 kD的新蛋白,其分子量与预期目标一致。对SOD酶活性的分析证实,重组菌的酶活性显著增加,证明克隆的基因具有活性。  相似文献   

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13.
Because of its capacity to rapidly convert superoxide to hydrogen peroxide, superoxide dismutase (SOD) is crucial in both intracellular signalling and regulation of oxidative stress. In this paper we report the cloning of a Cu/Zn SOD (designated as pfSOD) from the pearl oyster (Pinctada fucata) using rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of this Cu/Zn SOD contains an open reading frame (ORF) of 471 bp coding for 156 amino acids. No signal peptide was identified at the N-terminal amino acid sequence of Cu/Zn SOD indicating that this pfSOD encodes a cytoplasmic Cu/Zn SOD. This is supported by the presence of conserved amino acids required for binding copper and zinc. Semi-quantitative analysis in adult tissues showed that the pfSOD mRNA was abundantly expressed in haemocytes and gill and scarcely expressed in other tissues tested. After challenge with lipopolysaccharide (LPS), expression of pfSOD mRNA in haemocytes was increased, reaching the highest level at 8 h, then dropping to basal levels at 36 h. These results suggest that Cu/Zn SOD might be used as a bioindicator of the aquatic environmental pollution and cellular stress in pearl oyster.  相似文献   

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15.
人铜锌超氧化物歧化酶基因在乳酸乳球菌中的食品级表达   总被引:5,自引:1,他引:4  
以lacF基因为食品级选择标记,构建了乳酸乳球菌食品级基因表达系统,并进而实现了人铜锌超氧化物歧化酶基因在乳酸乳球菌中的食品级表达。首先构建了含有lacF基因两侧同源DNA序列(0.5kb)的整合型质粒pUCEmDE,通过pUCEmDE与乳酸乳球菌MG5267染色体上单拷贝的乳糖操纵子之间的同源双交换,构建了lacF基因缺失突变的食品级受体菌WZ103 (Lac-),并经PCR及Lac表型检测所验证。然后构建了互补质粒pMG36eF,其lacF基因的表达受组成型的强启动子P32的控制。将其电转化导入WZ103后,Lac+表型得到恢复,表明WZ103中lacF基因的功能可被互补质粒pMG36eF上的lacF基因互补。随后,以互补质粒pMG36eF为基础,构建了不含任何抗生素抗性选择标记的人铜锌超氧化物歧化酶基因的食品级表达质粒pWZ104。通过非变性聚丙烯酰胺凝胶电泳和SOD活性凝胶染色分析,检测到WZ103(pWZ104)中Cu/Zn SOD的表达,并且具有生物活性。  相似文献   

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Abstract A polyclonal antibody against purified Cu,Zn superoxide dismutase (SOD) from the pathogen Aspergillus fumigatus was raised in a sheep. This antibody recognised purified A. fumigatus SOD, together with a single band of 19 kDa in A. fumigatus cytoplasmic antigen, by immunodevelopment of Western blots. The polyclonal serum did not recognise either the manganese or iron containing forms of the enzyme; however, it was reactive against putative Cu,Zn SODs in other members of the genus Aspergillus . Immunofluorescent staining of A. fumigatus cultures demonstrated expression of the Cu,Zn SOD in conidia and hyphae, with the cell wall staining particularly intensely. Conidiophores were stained in an uniformly intense pattern. Immunoelectron microscopy confirmed that the SOD was present within the hyphal cell wall, although there was also labelling in the cytoplasm. SOD may protect Aspergillus against oxidants produced by immune effector cells and these observations demonstrate that the enzyme is available to perform its antioxidant function within the cell wall.  相似文献   

18.
As a special species of avian, Peking duck is often used as a model for exploring effective factors against cardio-cerebrovascular diseases, and therefore investigations of antioxidant enzymes including superoxide dismutase are intriguing. By using 3(')-RACE with a gene-specific primer, a cDNA encoding duck Cu,Zn SOD was amplified from the total RNA extracted from Peking duck liver. Three free cysteine residues are found in the deduced amino acid sequence of duck SOD, among which Cys153 at the carbonyl-terminal is a distinctive feature. Production with a high yield of recombinant duck Cu,Zn SOD was achieved in Escherichia coli after the reconstituted expression vector pET-3a-dSOD was transformed into the bacterial strain BL21(DE3)pLysS. After two steps of anion exchange chromatography, a great quantity of the purified enzyme (100mg/L fermented culture) with an enzymatic activity comparable to that of native duck and bovine SOD was finally obtained. Duck SOD is a homodimer with 153 residues for each subunit. The molecular mass of the recombinant enzyme is 15,540.0Da measured by mass spectrum, which well coincides with the estimated size of the sequence but significantly differs from that of the native counterpart. Five charge isomers were observed on isoelectricfocusing (IEF). The most interesting observation is that the thermal stability of duck SOD is much lower than that of the bovine enzyme as revealed by irreversible heat inactivation at 70 degrees C. These properties are discussed in relation to the distinctive free Cys residues in duck Cu,Zn SOD.  相似文献   

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