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1.
The gene of human thyrnosin alpha 1 (hT(1)was synthesised according to favorite eodons of Pichia pastoris by PCR. N-terminal 28 amino acid residues of 40S ribosomal protein (RP), S24Ethat is N-aeetylserine were replaced by hT( 1 for the constitution of hT(1-RP fusion gene in order to express acetyllated thyrnosin α1. And also, the Asn-Gly bond was designed to faeiliate isolation of the target protein. The fusion gene was cloned into the expression vector, pPIC/gK. The constructs were transformed into HIS4 mutant strain GS115 by eleetroporation. Both SDS-PAGE analysis and Western blot analysis indicated that the fusion protein was expressed successfully.  相似文献   

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Earthworm fibrinolytic enzyme component A(EFEa),a protein with dual fibrinolytic activity ,is one of the major therapeutically important earthworm fibrinoltic enzyme components .The cDNA fragment encoded the mature protein was cloned from earthworm (Eisenia fetida )by the RT-PCR technique,The deduced amino acid sequence of the EFE component A show high homology with some members of serine proteases trypsin family,and the amino acid residues constituting the active sites are conserved in the EFEa as compared with the other proteins of the trypsin family ,The cDNA fragment was subcloned into the expression vector pQE31 and pMAL-c2X of E.coli.The resulting expression plasmids,pQE-efea and pMAL-efea ,were used to transform the E.coli strain M15.Recombinant protein bands corresponding with calcuated molecular witht were induced .The induced His6-EFEa fusion protein with pQE-efea was accumulated into inclusion body ,while the induced MBP-EFEa fusion protein with pMAL-efea was soluble and showed fibrinoloytic activities.  相似文献   

3.
In this study, a recombinant pET28c-gBTB/POZ was constructed by cloning the sequence of the BTB/POZ domain of the zebrafish gcl (germ cell-less) into the expression vector pET28c, and pET28c-gBTB/POZ was transformed into BL21(DE3) pLysS strain to express the fusion protein for the preparation of antibody. Polyclonal-antibody against the GCL-BTB/POZ domain was prepared by immunizing rabbit with the fusion protein, and the Western Blot and immuno-histochemical analysis were performed to detect the quantity of the polyclonal-antibody. The result indicates that the polyclonal-antibodies were of good quantity and specification. Further studies will be performed to demonstrate the function and expression pattern of the GCL protein during the development process of zebrafish with the polyclonal-antibody.  相似文献   

4.
Glucagon-like peptide-1 (GLP-1) is a 30-amino acid peptide hormone, which has the regulatory function of stimulating the secretion of insulin to balance blood glucose levels. In this study, marker-free expression vector pX6 with a fusion gene of ten tandem repeated GLP-1 analog ([Ser8, Gln26 and Asp34]-GLP-1) genes was constructed and transformed into cucumbers by Agrobacterium tumefaciens-mediated transformation method. Four transgenic lines were obtained by PCR and Southern blotting analysis and two transgenic lines successfully expressed the fusion protein (named GLP-T), which was revealed by Western blotting analysis. The biological activity test results showed that the serum glucose level of diabetic rats was significantly decreased after oral administration of the fusion protein GLP-T extracted from the transgenic cucumber fruitage. These results suggest that this may provide a brand-new strategy to prevent and cure the diabetes with no pain.  相似文献   

5.
The coat protein (CP) gene of Cocksfoot mottle virus (CfMV) was amplified by RT-PCR and inserted into expression vector pGEX-4T-1, and the resulting plasmid was designated as pGEXCfMV-JANCP. The fusion protein GST-CP was expressed in BL21 (DE3) pLysS after IPTG induction. The results of SDS-PAGE and Western blot analysis showed that the CfMV-CP gene was efficiently expressed in E. coli BL21 (DE3) pLysS through IPTG induction and the 56.0 kD protein was obtained.  相似文献   

6.
The coat protein (CP) gene of Cocksfoot mottle virus (CfMV) was amplified by RT-PCR and inserted into expression vector pGEX-4T-1, and the resulting plasmid was designated as pGEXCfMV-JANCP. The fusion protein GST-CP was expressed in BL21 (DE3) pLysS after IPTG induction. The results of SDS-PAGE and Western blot analysis showed that the CfMV-CP gene was efficiently expressed in E.coli BL21 (DE3) pLysS through IPTG induction and the 56.0 kD protein was obtained.  相似文献   

7.
A recombinant plasmid pET-racd was first constructed by cloning osRACD,the development-regulating gene that controls photoperiod fertility transformation in the photoperiod sensitive genic male sterile rice Nongken 58S,into a prokaryote expression vector pET28a(+).It was then transformed into E.Coli BL-21.Cutting with thrombin of the fusion protein extracted from transformants and PAGE separation yielded pure osRACD protein,which was further concentrated using ultrafiltration and renatured using glutathione oxidation/reduction refolding system for later functional study.As demonstrated by in vitro functional assay,the osRACD protein expressed in E.Coli B-21 shows remarkable activity in binding GTP specifically and hydrolyzing it.  相似文献   

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The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtil& was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone a-factor (MFals), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-βG) was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/(h·ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.  相似文献   

11.
The fusion gene of actin (cDNA ofChlamydomonas reinhardtii) and green fluorescence protein (gfp) had been constructed into two expression vectors which could be expressed inE. coli and tobacco suspension cells BY2. The correct expression was observed inE. coli and BY2 with a fluorescence microscopy. The fusion protein, which took part in the membrane skeleton, was mainly located peripherally along the membrane, specially the fusion protein was distributed around nucleus and cell plate, while the fusion protein also forms F-actin in the cell. The fusion protein was purified from Bl21plus by ammonium sulfate fractionation, ion exchange chromatography and hydrophobic interaction chromatography. The purified production could polymerize into F-actin when the actin polymerizing buffer was added. It was demonstrated that the characteristics and function of actin inChlamydomonas was similar with those of animals and higher plants.  相似文献   

12.
The recombinant expression vector pGEMD-fhit which contains full encoding region offhit gene was constructed. The recombinant was introduced into the BL21 (DE3) strain ofE. coli and induced by 1 mmol/L IPTG to express a 29×103 polypeptide offhit fusion protein. And the 29×103 protein was sensitive and specific in reaction with anti-fhit antibody in Western blot. Foundation item: Supported by the National Natural Science Foundation of China (39770373) Biography: SUN Yan (1975−), female, Master of science, Research direction: gene engineering  相似文献   

13.
The DNA fragment encoding matureMycobacterium tuberculosis major secretory protein Ag85B was inserted into thePichia pastoris secretory expression vector pHBM905A, under the control of theAOX1 promoter. The recombinant plasmid pHBM905A-85B linearized bySal I was introduced intoPichia pastoris strain GS115 by PEG1000 transformation method. After phenotype screening and PCR identification, the resulting GS115-pHBM905A-85B strain was cultivated and induced with methanol. The recombinant Ag85B protein in secreted form was attained with molecular weight of 35×103 approximately detected by SDS-PAGE and Western blot. ELISA experiment proved that the protein had good antigen specificity. Secretory expression of recombinantM. tuberculosis Ag85B inP. pastoris will open a door to mass production of the protein in heterologous host and allow ready evaluation of its immunological function. Foundation item: Supported by the Key Scientific and Technological Project of Wuhan(301121028) Biography: LIU Yan(1971-), female, Ph. D candidate, research direction: vaccine against tuberculosis.  相似文献   

14.
An interspecies conservedPlasmodium asparagine rich antigen, designated as ARK26, was isolated by immunoscreeningP. falciparum genomic DNA expression library with mouse convalescent anti-P. yeolii serum. Partial DNA sequence analysis reveals that ARK26 contains clusters of asparagines and no randomly repeated amino acid sequence motifs are observed. A 65×103 GST fusion protein is expressed by recombinant plasmid PGEX-5X-1 (ARK26) inE. coli C strain ABLE-K. Computer programs predict that two asparagine rich regions are among the possible antigenic epitopes of p37 encoded by ARK26. Interestingly, the sequence of ARK26 displays significant similarity to yeast and several other species’ mitochondrial genes, and its possible function is discussed. Supported by a fellowship offered by International Center for Genetic Engineering & Biotechnology(ICGEB) Ma Donghui: born in 1969, Graduate student  相似文献   

15.
Based on a genetically modified radioresistant bacteria Deinococcus radiodurans, we constructed a real time whole cell biosensor to monitor radioactivity and genotoxicity in highly radioactive environment. The enhanced green fluorescence protein (eGFP) was fused to the promoter of the crucial DNA damage-inducible recA gene from D. radiodurans, and the consequent DNA fragment (PrecA-egfp) carried by plasmid was introduced into D. radiodurans R1 strain to obtain the biosensor strain DRG300. This engineered strain can express eGFP protein and generate fluorescence in induction of the recA gene promoter. Based on the correlation between fluorescence intensity and protein expression level in live D. radiodurans cells, we discovered that the fluorescence induction of strain DRG300 responds in a remarkable dose-dependent manner when treated with DNA damage sources such as gamma radiation and mitomycin C. It is encouraging to find the widely detective range and high sensitivity of this reconstructed strain comparing with other whole cell biosensors in former reports. These results suggest that the strain DRG300 is a potential whole cell biosensor to construct a detective system to monitor the biological hazards of radioactive and toxic pollutants in environment in real time.  相似文献   

16.
 采用PCR方法扩增HSV-1病毒型特异性包膜糖蛋白L(gL)基因片段并克隆至原核表达载体pGEX-5X-1获得重组质粒pGEX-5X-1-gL,将重组质粒转化E.coli BL21表达菌后经IPTG诱导表达目的蛋白.SDS-PAGE蛋白检测表明,在分子质量56 ku处有HSV-1 GST-gL融合蛋白的高效表达,通过IPTG浓度筛选和诱导前表达菌扩增培养时间的比较分析对诱导条件进行了优化,GST-gL融合蛋白表达量可达到菌体蛋白总量的48.65%.Western blot中利用HSV-1灭活病毒获得的多克隆抗体确证所表达蛋白为HSV-1病毒组分.这一表达系统的建立和优化对进一步探讨HSV-1 gL蛋白功能及其免疫原性提供了有利条件.  相似文献   

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Fruit specific promoter (2A12) from Lycopersicom esculentum and cDNA of isopentenyl-transferase (ipt) from Ti plasmid of Agrobacterium tumerfaciens C58 were cloned by PCR procedure respectively. Two plant expression vectors with 2A12/gus or 2A12/ipt were respectively constructed. These two chimeric genes were transferred into tomato by Agrobacterium mediated procedure. The results of Southern hybridization showed that the fusion genes had been integrated into tomatoes. The result of gus histochemical staining showed that 2A12 had high fruit specific expressive capability in transgenic tomato. The ipt expression resulted in accumulation of high level of cytokinins (CTKs) in fruit lead to developmental changes in fruits and seeds. The fruit of ipt transformed tomato had the hyperplastic placenta with very few seeds or even seedless. The shelf life of transgenic fruits elongated for 1–2 weeks. The ratio of fruit set, the dry weight of fruit and the crude protein content in fruit were increased, while the soluble sugar of fruits decreased.  相似文献   

19.
为使用外源表达载体表达并纯化有活性的水稻ATP酶蛋白Os AAA1,构建了p ET-32a-Os AAA1原核表达载体并进行体外IPTG诱导表达和Ni+柱亲和层析纯化.利用SDS-PAGE和Western Blot检测了目的蛋白.结果表明:将成功构建的p ET-32a-Os AAA1原核表达载体,转化到大肠杆菌Origami菌株后,在70~100 KD之间检测到可溶性目的蛋白带,并优化了诱导表达的较适温度、IPTG诱导浓度和诱导表达时间.故成功构建了p ET-32a-Os AAA1原核表达载体并获得了可溶性Os AAA1目的蛋白,为其后续研究奠定了基础.  相似文献   

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