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1.
基于基因工程技术 ,用PCR法扩增出编码ICA6 9的cDNA片段 ,直接克隆到pSPORT 1质粒上 ,经DNA序列测定 ,插入到GST融合蛋白表达载体 pGEX 2T ,构成重组质粒 p2T ICA6 9,得到的表达产物GST ICA6 9融合蛋白用间接ELISA法检测其免疫原性 .测序结果表明 ,所获PCR产物已正确重组到PGEX 2T表达型质粒中 .重组质粒在原核细胞中表达的融合蛋白具有免疫原性 ,并能应用于Ⅰ型糖尿病病人血清中抗ICA6 9抗体的检测 .所获得的表达产物为重组ICA6 9融合抗原 ,有助于提高Ⅰ型糖尿病的预报率和确诊率 . 相似文献
2.
通过PCR技术扩增出人铁蛋白基因,经酶切后与表达载体质粒pGEX-4T-2连接,重组质粒转化感受态大肠杆菌,利用菌落PCR、质粒双酶切、测序,证实成功地构建了人铁蛋白基因表达载体,利用IPTG对重组菌进行诱导表达,通过尿素洗涤纯化目的蛋白用于制备抗体． 相似文献
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4.
从假单胞菌（Pseudomonassp．）XZG36中克隆弹性蛋白酶基因,构建原核表达载体,实现其在大肠杆菌（Escherichiacoli）中的高效表达,并对表达产物进行酶学性质分析,为微生物发酵生产弹性蛋白酶奠定基础．以假单胞菌基因组DNA为模板,PCR扩增弹性蛋白酶基因,并将其开放阅读框（0RF）克隆至融合表达载体pET30a（＋）进一步IPTG诱导表达;表达产物经His·Bind亲和层析纯化后对弹性蛋白酶进行酶学性质分析．实验成功克隆了弹性蛋白酶基因,DNA基因片段为1672bp、编码497个氨基酸残基的多肽,与预计长度相符合;实现了其在E．coli中的高效表达,表达量约占菌体总蛋白的20％;经SDS-PAGE分析,相对分子质量为48000,与预期的一致;提纯后的表达蛋白SDS-PAGE分析可见单一条带,纯度可达92％以上．表达蛋白具有良好活性． 相似文献
5.
《天津大学学报(英文版)》2015,(1)
The Bacillus strain BH072 isolated from a honey sample showed strong antifungal activity against phytopathogen. Gene cloning test demonstrated that the strain had a tas A gene encoding an antifungal Tas A protein. Although the wild strain simultaneously produced various antifungal substances, only the physicochemical property and antifungal activity of Tas A protein were unclear due to the difficulty in extraction. In this study, tas A gene encoding the protein from Bacillus sp. BH072 was amplified by using the polymerase chain reaction(PCR) method and cloned into p ET 28a(+) vector, and then expressed in host cells Escherichia coli BL21(DE3). The expressed proteins were collected by centrifugation and ultrasonic treatment, and then purified by using nickel-nitrilotriacetic acid(Ni-NTA)metal affinity column and dialysis methods. The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) test showed that an expected protein band appeared with a size of 31 k Da. The expressed products possessed antifungal activity against the phytopathogenic indicator strain Botrytis cinerea. A genetically engineered strain tas A of E. coli was established in this study which can efficiently express Tas A protein. 相似文献
6.
目的:在大肠杆菌(BL21)中构建可溶性表达的金黄色葡萄球菌B型肠毒素(SEB)受体拮抗剂。方法:首先确定SEB受体桔抗剂的基因序列,然后用含有SEB受体桔抗剂的基因序列重组质粒表达载体PGEX-4T-1转化大肠杆菌BL21(DE3),利用IPTG诱导表达获得蛋白,产物经GST柱纯化后,利用ELISA检测其与SEB的结合能力并进行其体内外药效学实验。结果:该质粒成功转化为可溶性表达,ELISA结果显示表达产物可与SEB特异性结合。结论:本研究成功对SEB受体拮抗剂GST可溶性表达并对其活性进行初步分析。 相似文献
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To construct and identify further a recombinant of Adeno-associated virus and interferon-gamma for gene therapy, the full-length
IFN-γcDNA containing signal peptide was amplified by PCR, and then cloned into the pUC18. After screening, the fragment from
the positive clone was then subcloned into pwp19. After the correct recombinant was identified by digestion with SacI and
BamHI, it was transfected into lymphocyte cell line H9 mediated by calcium phosphate, and the expression of IFN-γ was detected
by RT-PCR and ELISA. The result showed that the IFN-γ were expressed in the H9 cells transfected with pwp/IFN-γ. The so constructed
recombinant plasmid pwp19/IFN-γ containing the full-length IFN-γ gene was expressed in mammalian cells.
Project (39570653) supported by NFSC. 相似文献
8.
根据GenBank数据库中猪圆环病毒Ⅱ型的基因组序列设计引物,采用PCR技术从病料基因组DNA中扩增出PCVⅡ河南地方株ORF4基因,全长180 bp,编码59个氨基酸.将该基因克隆至载体pGEX-4T-3中形成pGEX-4T-3-ORF4表达载体.经PCR、酶切和测序鉴定后,转化表达菌株BL21（DE3）诱导表达.SDS-PAGE结果显示：ORF4能够在大肠杆菌中表达,产物的分子量约为32 kD,且以包涵体形式存在.Western Blot检测结果显示,纯化后的ORF4蛋白能够与鼠抗6＆#215;His标签单克隆抗体发生特异性反应,为进一步研究PCVⅡORF4蛋白的特性与功能奠定了基础. 相似文献
9.
Zhong-hui ZHANG Li-juan DU Di XIANG Shun-ying ZHU Ming-yuan WU Hui-li LU Yan YU Wei HAN 《Journal of Zhejiang University. Science. B》2009,10(2)
Midkine is a heparin-binding growth factor,which plays important roles in the regulation of cell growth and differentiation.The non-tagged recombinant human midkine (rhMK) is therefore required to facilitate its functional studies of this important growth factor.In the present work,rhMK was expressed in Escherichia coli (E.coli) BL21 (DE3).The expression of midkine was efficiently induced by isopropyl-β-D-thiogalactopyranoside (IPTG).After sonication,midkine was recovered in an insoluble form,and was dissolved in guaoidine hydrochloride buffer.Renaturation of the denatured protein was carried out in the defined protein refolding buffer,and the refolded protein was purified using S-Sepharose ion-exchange chromatography.The final preparation of the rhMK was greater than 98% pure as measured by sodium dodecylsulfate-polyacrylamid gel electrophoresis (SDS-PAGE) and reverse phase high performance liquid chromatography (RP-HPLC).The purified rhMK enhanced the proliferation of NIH3T3 cells. 相似文献
10.
利用RT—PCR技术从HEK293细胞中克隆到人Rab7基因,通过双酶切将其克隆到原核表达载体pGEX-4T-2中,转化大肠杆菌E．coliDH5α感受态细胞,获得阳性克隆．24℃下IPTG诱导表达,Glutathi—one Sepharose 4B纯化后获得高纯度的Rab7蛋白．并以此为抗原免疫小白鼠,获得了Rab7的特异性抗体． 相似文献
11.
利用RT-PCR技术从日本对虾中克隆到β-actin基因的cDNA,通过双酶切将其克隆到原核表达载体pGEX-4T-2中,转化大肠杆菌E.coli BL21感受态细胞,获得阳性克隆.4℃下IPTG诱导表达,Glutathione Sepharose 4B纯化后获得高纯度的β-actin蛋白.① 相似文献
12.
1IntroductionNeural stemcells(NSCs)are a subtype of progenitorcells in the nervous systemthat can differentiate intoneurons and glia[1-3].Due to their feature of self-re-newal,NSCs have expectations for treatment of ner-vous system diseases such as Parkin… 相似文献
13.
孙晓东 《陕西教育学院学报》2010,26(2):106-107,121
为了构建人C6orf210基因原核表达载体,并在E．coliBL21中表达并纯化。采用RT—PCR扩增人C6orf210基因片段,并将其克隆到原核表达载体Pet21a中,构建重组质粒pET21a—C6ort210。经限制性内切酶EcoRI与XhoI双酶切鉴定及序列测定后,转化E．coliBL21,经IPTG诱导表达融合蛋白。结果显示获得全长为1188bp的人的CAiorf210基因片段。以构建的重组质粒pET21a—C6orf210转化E．coliBL21后,经IPTG诱导,表达出相对分子质量（Mr）约66000的融合蛋白。经SINS—PAGE、Western blot分析显示,诱导表达的蛋白为C6orf210。 相似文献
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将人细胞周期蛋白D1基因克隆入原核表达载体pET-20b中获得重组质粒pET-20b-eyeD,经酶切鉴定正确后转化大肠杆菌BL21PlaysS后获得表达菌株．该菌株经IPTG诱导后表达的目的蛋白有部分分泌到培养基上清中,将培养基上清中的蛋白沉淀后用Ni^2＋螯合柱进行纯化,最后可得到纯度达到95％以上的目的蛋白．蛋白电泳显示纯化蛋白的分子量约为33KD,Westemblot分析表明,在电泳胶的相应分子量处出现特异性条带,说明已经成功表达和纯化了重组人细胞周期蛋白D1． 相似文献
15.
利用PCR技术从肝素黄杆菌克隆到肝素酶HepI基因,通过双酶切将其克隆到原核表达载体pGEX-4T-2中,转化大肠杆菌E．coliBL21感受态细胞,获得基因工程重组菌．12℃下IPTG诱导表达12h,Glutathione Sepharose 4B纯化后获得较高纯度的HepI酶蛋白． 相似文献
16.
将斑节对虾酚氧化酶原（prophenoloxidase,proPO）基因克隆进pET28a（＋）,在大肠杆菌BL21菌株中诱导表达,SDS-PAGE和Western-blotting分析均能检测到一条分子量为79．4kDa的特异性条带,与推导的融合蛋白理论分子量82．4kDa基本相符;包涵体变性后对经Ni—NTAagarose纯化的变性液进行复性,表现出0．3851U／mg．min的PO活性;不同条件下的诱导表达结果显示,18℃时诱导培养能检测到目的蛋白的可溶性表达,经诱导9h后可溶性表达比例达到最高,约占菌体可溶性蛋白总量的s．56％;可溶性表达的目的蛋白纯化后经胰酶消化,可检测到分子量为60kDa、42．7kDa的特异性条带,并表现出0．4249U／mg．min的PO活性． 相似文献
17.
The Bacillus strain BH072 isolated from a honey sample showed strong antifungal activity against phytopathogen. Gene cloning test demonstrated that the strain had a tasA gene encoding an antifungal TasA protein. Although the wild strain simultaneously produced various antifungal substances, only the physicochemical property and antifungal activity of TasA protein were unclear due to the difficulty in extraction. In this study, tasA gene encoding the protein from Bacillus sp. BH072 was amplified by using the polymerase chain reaction (PCR) method and cloned into pET 28a (+) vector, and then expressed in host cells Escherichia coli BL21 (DE3). The expressed proteins were collected by centrifugation and ultrasonic treatment, and then purified by using nickel-nitrilotriacetic acid (Ni-NTA) metal affinity column and dialysis methods. The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) test showed that an expected protein band appeared with a size of 31 kDa. The expressed products possessed antifungal activity against the phytopathogenic indicator strain Botrytis cinerea. A genetically engineered strain tasA of E. coli was established in this study which can efficiently express Tas A protein. 相似文献
18.
Two heterologous expression systems using thioredoxin (trxA) as a gene fusion part in Escherichia coli were developed to produce recombinant pediocin PA-1. Pediocin PA-1 structural gene pedA was isolated from Pediococcus acidilactici PA003 by the method of polymerase chain reaction (PCR), then cloned into vector pET32a(+), and expressed as thioredoxin-PedA
fusion protein in the host strain E. coli BL21 (DE3). The fusion protein was in the form of inclusion body and was refolded before purification by nickel-iminodiacetic
acid (Ni-IDA) agarose resin column. Biological activity of recombinant pediocin PA-1 was analyzed after cleavage of the fusion
protein by enterokinase. Agar diffusion test revealed that 512-arbitrary unit (AU) recombinant pediocin PA-1 was obtained
from 1 ml culture medium of E. coli (pPA003PED1) using Listeria monocytogenes as the indicator strain. Thioredoxin-PedA fusion gene was further cloned into pET20b(+). Thioredoxin-PedA fusion protein
was detected in both the periplasmic and cytoplasmic spaces. The recombinant pediocin PA-1 from the soluble fraction attained
384 AU from 1 ml culture medium of E. coli (pPA003PED2). Therefore, biologically active pediocin PA-1 could be obtained by these two hybrid gene expression methods. 相似文献
19.
骨形态发生蛋白-2（bone morphogenetic protein,BMP-2）具有多种生物活性,有潜在的药用价值.为探索利用基因工程技术重组BMP-2并在大肠杆菌表达系统表达的可行性.在大肠杆菌中重组和筛选BMp-2基因,SDS-PAGE电泳分析,显示外源蛋白带在相对分子量约12kD处,以离子交换层析DEAE和分子筛纯化蛋白,缓慢复性并在C2C12细胞内检测到其蛋白活性. 相似文献
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从人直肠癌细胞株Colo320中提取总RNA,经RT-PCR扩增出SOD基因片段,经限制性内切酶（BamHI,Sinai）酶切后按正确的读码框顺序插入到pGEX-4T-2表达载体上,重组质粒转化大肠杆菌,经菌落PCR和质粒双酶切鉴定、序列测定确认,证实成功地构建了人SOD基因融合表达载体．转化菌经IPTG诱导表达,SDS-PAGE显示有与预期大小约44kD相吻合的融合蛋白带,获得了可溶性表达的目的蛋白,采用Glutathione Sepha—rose4B亲和层析对重组蛋白进行纯化,获得了纯度很高的目的蛋白． 相似文献