酿酒酵母乙醇脱氢酶2(ADH2)基因的克隆表达及活性验证

为了从酿酒酵母Saccharomyces cerevisiae中克隆出乙醇脱氢酶2(Alcoholdehy drogenase2,ADH2)基因并使之在大肠杆菌中高效表达。以酿酒酵母细胞中提取的总RNA为模板,通过反转录获得酿酒酵母乙醇脱氢酶2基因,连接到表达载体pTAT上,得到重组表达质粒pTAT-ADH2,将此重组质粒转化到大肠杆菌BL21中,重组工程菌株经IPTG诱导表达得到ADH2蛋白。将该蛋白纯化后,在体外进行活性检测和小鼠体内进行毒理试验,检测ADH2的酶活性。测序结果表明克隆的基因与GenBank中所报道的adh2基因序列有90%的同源性,经SDS-PAGE电泳分析,目的蛋白得到了有效表达,蛋白条带扫描分析表明,表达量占总蛋白的50%左右,纯化得到的蛋白在小鼠体内进行毒理试验,显示出一定的活性。酿酒酵母adh2基因的克隆正确,不仅在大肠杆菌中进行了高效表达而且表现出了较好的酶活性。     

以重组P-gp为抗原建立检测MDR 1抗体间接ELISA方法的研究

目的:构建MDR 1基因原核表达质粒,表达P-gp重组蛋白,建立检测MDR1抗体的间接ELISA方法。方法:利用重组PCR技术扩增MDR 1基因的1kb片段,克隆至pET-28b（＋）中,构建原核表达质粒pETP-gp,转染感受态菌BL21（DE3）和BL21（DE3）plyss;以E.coli高效表达的P-gp基因主要抗原编码区重组蛋白为抗原,以HRP标记的兔抗人IgG为二抗,建立间接ELISA检测方法。结果:正确构建了pETP-gp原核表达质粒,并可在E.coli中高效表达,表达蛋白可用作检测MDR 1抗体ELISA抗原。结论:成功表达出重组蛋白P-gp,建立了检测MDR 1抗体的间接ELISA方法。     

人SCYL1-BP1重组蛋白的原核表达及分离纯化鉴定

前期研究结果发现,SCYL1-BP1具有细胞周期调控功能,同时具有肿瘤抑制因子的特性。目的:采用基因工程技术,构建SCYL1-BP1的大肠杆菌重组表达菌株,以获得足够量的高纯度目的蛋白,为后面进行一系列药理学检测及新药安全性测试奠定基础。方法:利用从人胎脑cDNA文库中克隆得到SCYL1-BP1基因克隆为模板,经PCR扩增,通过酶切位点克隆到新型原核表达载体pET-28b-SUMO上,转化大肠杆菌表达菌BL21(DE3)。经IPTG诱导表达,摸索优化表达条件,表达产物经Ni柱进行亲和层析纯化,后再进行SDS-PAGE和Western blot等分析鉴定。结果:成功构建了SCYL1-BP1的原核表达工程菌BL21(DE3)/pET-28b-SUMO-SCYL1BP1。SDS-PAGE和Western blot检验结果表明,诱导表达的融合蛋白His6-SUMO-SCYL1BP1的分子量约为65 kDa,主要以可溶的形式存在,且能被His标签抗体和SCYL1-BP1单克隆抗体特异性识别。结论:原核表达并纯化了人SCYL1-BP1融合蛋白,为其后续功能研究及性质实验奠定基础。     

SUMO蛋白酶活性片段的表达、纯化及活性测定

利用PCR技术人工合成编码酿酒酵母泛素样特异性蛋白酶1 (Ubiquitin-like specific protease 1,Ulp1)第403到621个氨基酸残基之间的DNA片段Ulp1p,并连接到大肠杆菌表达载体pET-3c中,构建出重组表达质粒pET-Ulp1p。将重组质粒转化至大肠杆菌BL21(DE3)中,氨苄青霉素抗性筛选转化子。经IPTG诱导4h后, SDS-PAGE结果显示,Ulp1p为可溶性表达,表达量占菌体总蛋白的50.8%。通过Ni-NTA凝胶亲和层析和G-25凝胶层析联用可以获得纯度大于95%的Ulp1p。Western-blotting分析表明,Ulp1p能与6xHis抗体产生免疫反应。以重组蛋白SUMO-hEGF(人表皮生长因子)和GST-SUMO-MT(金属硫蛋白)为底物进行酶切分析,结果显示,Ulp1p能特异性水解这两种SUMO融合蛋白,其比活为1.386 x104U/mg。     

凋亡素融合蛋白的可溶性表达及活性分析

目的:构建PET-28a-SPA原核表达载体,在大肠杆菌BL21(DE3)中实现其高效可溶性表达,测定对肿瘤细胞的凋亡效果。方法:本实验在获得凋亡蛋白融合基因的基础上,成功地构建了重组表达质粒PET-28a-SPA,将阳性重组质粒转化表达受体菌BL21(DE3)感受态细胞中,经IPTG诱导表达,表达产物经聚丙烯酰胺凝胶电泳检测和Western blot检测,并采用MTT法检测其对肿瘤细胞的增殖抑制。结果:表达产物经聚丙烯酰胺凝胶电泳检测,凋亡蛋白融合基因获得高效表达,软件分析表明表达蛋白占菌体蛋白20%左右。上清表达量约为10%。上清蛋白经纯化后,Western blot结果显示,利用凋亡蛋白单克隆抗体可以很好地和所表达的蛋白带特异性结合,并且对A549肺癌细胞及Hela细胞具有一定的凋亡作用。结论:所获凋亡蛋白以高效胞质可溶形式表达,为其研制有效的肿瘤免疫治疗靶向药物提供一定的基础。     

hGH-双精氨酸C肽人胰岛素原基因的克隆表达及纯化研究

旨在提高基因重组人胰岛素在大肠杆菌中表达的稳定性及表达包涵体蛋白的复性水平.在人胰岛素原N端前融合人生长素N端的一段序列来充当前导肽,同时将C肽设计为两个精氨酸,分10段合成长链寡核苷酸链,利用重叠延伸PCR技术(SOE PCR)扩增得到该基因片段.与表达载体PET-30a连接,转化E.coli BL21(DE3),IPTG诱导表达.表达的融合蛋白采用Ni-NTA亲和层析纯化,纯化后的蛋白经复性、冻干等步骤后用胰蛋白酶,羧肽酶B双酶切再过DEAE Sepharose Fast Flow阴离子交换柱,收集洗脱峰.对制备所得的胰岛素用SDS-PAGE,Western blot进行性质鉴定,及皮下注射小鼠测定生物活性.结果显示,目的蛋白在大肠杆菌BL21(DE3)中得到了表达,表达产物以不溶性包涵体形式纯在,约占大肠杆菌总蛋白的30%.经Ni-NTA亲和层析得到的重组蛋白纯度为85%,DEAE Sepharose Fast Flow阴离子交换纯化得到单组分胰岛素.Western Blot显示制备所得的胰岛素具有胰岛素免疫原性,皮下给药注射小鼠活性测定表明具有明显的降血糖活性.获得了一种高效生产基因重组人胰岛素的方法,为研究胰岛素类似物奠定了前期基础同时也为今后探索胰岛素的非注射给药途径提供了原料.     

The regulatory elements of araBAD operon,contrary to lac‐based expression systems,afford hypersynthesis of murine,and human interferons in Escherichia coli

The overexpression of four different interferons, i.e., murine interferon α1 and human interferons α1, α8, and α21 was challenged in Escherichia coli. Synthetic genes coding for these interferons were designed, assembled, and cloned into the vector pET9a (using the NdeI and BamHI sites), placing interferon expression under the control of phage T7 promoter. Despite an intensive screening for optimal culture conditions, no interferon synthesis was observed using overexpression systems based on the regulatory elements of lac operon (e.g., in E. coli BL21DE3). On the contrary, high levels of interferon expression were detected in E. coli BL21AI, which chromosome contains the gene coding for phage T7 RNA polymerase under the control of the araBAD promoter. To analyze the reasons of this striking difference, the molecular events associated with the lack of interferon expression in E. coli BL21DE3 were studied, and murine interferon α1 was chosen as a model system. Surprisingly, it was observed that this interferon represses the synthesis of T7 RNA polymerase in E. coli BL21DE3 and, in particular, the expression of lac operon. In fact, by determining β‐galactosidase activity in E. coli BL21AI, a significantly lower LacZ activity was observed in cells induced to interferon synthesis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Production of recombinant salmon calcitonin by amidation of precursor peptide using enzymatic transacylation and photolysis in vitro

The C terminal amidation is required for full biological activity of salmon calcitonin (sCT). We constructed BL21(DE3)/pGEX-sCT-Ala, an engineering Escherichia coli strain. The soluble fusion protein of GST-sCT-Ala expressed from BL21(DE3)/pGEX-sCT-Ala was purified by affinity chromatography after high density, high expression culture and sonication of bacteria. Following S-sulfonation of the fusion protein, the 33 alanine-extended peptides were released from the fusion protein by cyanogen bromide. The S-sulfonated precursor peptide was transacylated by CPD-Y, o-PNGA as a nucleophile, to produce photosensitive SO(-)(3)-sCT-o-PNGA. After photolysis and folding, the biological activity of sCT was assayed as standard.     

Optimization of expression and purification of two biologically active chimeric fusion proteins that consist of human interleukin-13 and Pseudomonas exotoxin in Escherichia coli

We have previously reported that a variety of solid human tumor cell lines express a large number of receptors for interleukin-13 (IL-13). These receptors could be targeted with a chimeric fusion protein consisting of human IL-13 and a truncated form of Pseudomonas exotoxin (PE). We describe here optimization of critical steps involved in high yield expression of two recombinant chimeric fusion proteins for obtaining highly purified and biologically active cytotoxins in Escherichia coli. The chimeric constructs of human IL-13 and two 38 kDa truncated PEs: (i) PE38 and (ii) PE38QQR, (three lysine residues in PE38 at 590, 606, and 613 substituted with two glutamine and one arginine) were used for protein expression in pET prokaryotic expression vector system with kanamycin as a selection antibiotic. Our results suggest that fresh transformation of E. coli and induction by isopropyl-beta-D-thiogalactopyranoside (IPTG) for 6 h resulted in maximum protein expression. To further improve the yield, we used a genetically modified E. coli strain, BL21(DE3)pLysS, which carries a plasmid for lysozyme with a weak promoter that inhibits T7 RNA polymerase and minimizes protein production in the absence of IPTG. Use of this strain eliminated the need for lysozyme digestion of the induced bacteria to release inclusion bodies, which resulted in expression of purer protein as compared to the conventional BL21(DE3) strain. Additional protocol optimizations included 16 h solubilization of inclusion bodies, constitution of refolding buffer, and timing of dialysis. These proteins were finally purified by Q-Sepharose, mono-Q, and gel filtration chromatography. Between 14-22 and 21-28 mg highly purified and biologically active protein was obtained from 1L of BL21 (DE3) and BL21 (DE3) pLysS bacteria culture, respectively. As IL-13R targeting for brain tumor therapy offers an exciting treatment option, optimization of production of IL-13PE will enhance production of clinical grade material for Phase III clinical trials.     

HIV-1 Vpu蛋白的原核表达、鉴定和纯化

目的：克隆、表达、纯化人免疫缺陷病毒Ⅰ型（HIV-1）Vpu蛋白,为其功能及免疫学研究奠定基础。方法：PCR扩增Vpu基因,纯化、酶切后克隆到原核表达载体pET32a中,转化大肠杆菌BL21（DE3）菌株获得表达工程菌株,IPTG诱导蛋白表达,免疫印迹鉴定目的蛋白,亲和层析纯化蛋白。结果：构建了HIV-1Vpu蛋白的原核表达载体Vpu-pET32a,并在大肠杆菌中高效表达,目的蛋白呈可溶性形式存在,免疫印迹检测显示为目的蛋白,经Ni—NTAAgarose纯化获得了高纯度的目的蛋白。结论：在原核表达系统中表达了可溶性HIV-1Vpu蛋白,为进一步进行HIV-1Vpu蛋白的免疫原性和功能研究奠定了基础。     

人B淋巴细胞刺激因子C端肽的免疫增强作用

用套式PCR从人胎脑cDNA文库中克隆了B淋巴细胞刺激因子C端肽 (C terminalpeptideofBlymphocytestimulator,C BLyS)的cDNA。在大肠杆菌BL2 1CodonPlus (DE3)RIL中以包含体形式表达了C BLyS。对包含体的复性条件进行了摸索 ,建立了C BLyS的透析复性与纯化方法。经复性和纯化的C BLyS可结合其受体B细胞成熟抗原 (Bcellmaturationantigen ,BCMA) 人IgG1Fc融合蛋白 ,刺激体外培养的小鼠脾脏细胞增殖 ,并且可明显增强小鼠对溶菌酶的免疫应答水平。     

High-level expression of functional tumor suppressor LKB1 in Escherichia coli

The human LKB 1 tumor suppressor has been implicated as an important regulator of many cellular processes and signaling pathways, indicating that it could be a good candidate for anticancer drugs. The failure of its obtain high-level expression has been a major obstacle to study its protein structure and function in vitro. Here, we describe the high-level expression of human LKB 1 in Escherichia coli and show its kinase activity and anticancer effects on a tumor cell line. The gene encoding LKB 1 was optimized by replacing rare codons with codons frequently used in E. coli and synthesized with overlapping primers. The recombinant His-LKB 1 was expressed in hosts BL21（DE3） （BL） and Rosetta-gami（DE3）pLysS （RG）. His- LKB 1 from BL was present mainly as inclusion body. The soluble His-LKB 1 from RG accounted for 34. 1% of total proteins and the yield of purified His-LKB 1 was approximately 92 μg/ml. Purified His-LKB 1 protein from both hosts was functionally active, as shown by reversible autophosphorylation and kinase activity in the absence of any other associated kinase. The growth inhibitory ratio of the purified BL-derived and RG-derived His-LKB 1 on hepatic carcinoma SMMC-7721 cells was 24.97% and 45.68%, respectively, and both could produce significant cell-cycle arrest.     

HCV包膜蛋白E2的克隆、表达与检测

构建丙型肝炎HCV包膜蛋白糖蛋白的E2基因原核表达载体,获得大量重组HCVE2蛋白,进行E2蛋白的抗原性及潜在保护作用研究。通过RT-PCR从HCVRNA阳性血清标本中扩增出975bp(383～708)E2基因片段,PCR产物经EcoR I和Sall I双酶切后连接到经同样酶切的PET-41a原核表达载体上,转化到大肠杆菌BL21(DE3)菌株,经Amp筛选,得到阳性重组质粒PET41a-HCVE2菌株,并以IPTG诱导蛋白表达,SDS-PAGE鉴定,表达产物经固定化金属配体亲和层析纯化,用ELLSA方法检测生物学活性。结果表明,构建的HCVE2包膜蛋白基因片段原核表达质粒所表达产物主要以包涵体形式存在,表达的融合蛋白与HCV阳性血清具有较好的反应原性。以HCVE2融合蛋白检测患者阳性血清具有良好的抗原性,有望能提高HCV抗体检测试剂盒的检出率。     

KDR酪氨酸激酶的克隆表达及纯化

VEGF作为一种血管内皮细胞有丝分裂原,通过与内皮细胞表面特定受体结合,从而导致新生血管的形成,其中VEGFR-2(KDR/FIk-1)在肿瘤血管形成中起重要作用,因此其抑制剂有可能发展成为治疗肿瘤的新药.采用大肠杆菌成功表达了具有酪氨酸激酶活性的KDR酪氨酸激酶催化域(KDR-CD).采用RT-PCR从人脐静脉内皮细胞中提取总RNA,获得KDR-CD的编码基因,将其克隆至pET-30a载体,在E. coli BL21(DE3)中进行了成功表达,采用Ni-NTA亲和层析对其进行了纯化,Western blotting结果显示表迭的KDR-CD蛋白自身磷酸化,重组KDR-CD蛋白具有利用ATP催化底物发生磷酸化反应的激酶活性.     

Angioarrestin(hARPl)C端FD区的克隆、表达及其免疫活性鉴定

 Angioarrestin是一种具有潜在应用价值的肿瘤血管形成抑制因子.利用DNA重组法构建了angioarrestin C端 hFD cDNA 和麦芽糖结合蛋白(MBP)重组原核表达质粒 pMAL-C2-hFD.将重组质粒转入大肠杆菌E.coli BL21（DE3）,经0.3 mmol/LIPTG 在37℃条件下诱导表达4h,SDS-PAGE 检测,融合蛋白表达量约占细菌总蛋白的20%.Western印迹证实,目的蛋白N端带有MBP标签.取表达上清纯化、透析、浓缩并冻干,以此为抗原免疫Balb/c小鼠制备多克隆抗体.此多抗可以与pET 22b（+）表达系统获得的 hFD重组蛋白发生良好的抗原抗体反应,ELISA检测多抗效价达1∶10240.实验证明：通过基因重组可获得angioarrestin C端hFD在大肠杆菌中的高效表达蛋白,且该蛋白具有较高的免疫活性.以此为抗原制备的抗angioarrestin多克隆抗体为深入研究angioarrestin提供了材料.     

重组人MBD4蛋白在大肠杆菌中的表达、纯化及活性分析

为获得重组人MBD4蛋白,将编码MBD4的开放式阅读框（ORF）插入原核表达载体pGEX6P1 GST基因下游的多克隆位点（MCS）.将获得的表达质粒转化入大肠杆菌BL21(DE3) 菌株扩大培养并用IPTG诱导融合蛋白的表达.用谷胱甘肽琼脂糖凝胶 4B亲和介质从菌体裂解液中纯化了GST-MBD4融合蛋白.经过Prescision protease专一性裂解成功去除了融合蛋白上的GST标签.通过Mono Q阴离子交换层析获得了纯度达94%以上的MBD4蛋白,该蛋白具有甲基化DNA结合和糖苷酶生物活性.     

应用分子伴侣共表达系统表达pfu基因及酶活性测定

将通过In-fution方法构建的pET32a-pfu质粒与可以促进可溶性表达的HG-PGR07质粒一起转入大肠杆菌B121（DE3）共表达,以pET32a-pfu单独在B121（DE3）中表达作为对照。用热变性和（NH4）2SO4沉淀去除部分杂蛋白,再经Ni—NAT亲和层析柱纯化分离尼血蛋白,SDS-PAGE检测结果表明目的蛋白大小约为90kD,与预计的分子量大小一致。最后对其酶活性测定结果表明分子伴侣能够促进pfu基因表达,并能够提高酶活性。     

人角质化细胞生长因子-2基因的克隆、表达及产物的纯化、鉴定

用高表达菌株BL21codon plus compentent cells表达重组人角质化细胞生长因子(Hkgf-2)蛋白并初步纯化和检测其活性。通过RTPCR从流产胎儿肺组织中钓取hKGF-2cDNA,将其克隆入pBV220载体质粒。在大肠杆菌BL-21codon plus compent cells中表达hKGF-2蛋白。采用亲和层析和离子交换层析分离纯化,以细胞增殖实验测定表达蛋白的生物活性。结果显示,hKGF-2蛋白在BL21中得到高效表达;hKGF-2蛋白能刺激NIH3T3细胞的增殖,具有显著的促有丝分裂活性。     

植原体免疫主导膜蛋白Imp基因原核表达载体构建及表达

旨在构建植原体免疫主导膜蛋白Imp基因原核表达载体,并进行初步表达。以重组克隆质粒pMD18-T-Imp为模板,PCR扩增Imp基因片段。构建表达载体pET-28a(+)-Imp,转化宿主菌E.coliBL21(DE3)。筛选阳性克隆,提取重组质粒作PCR鉴定、酶切鉴定及IPTG诱导表达鉴定。PCR及双酶切结果显示,重组质粒pET-28a(+)-Imp构建成功。经IPTG诱导BL21(pET-28a(+)-Imp)表达约20 kD的蛋白,与预期的携带6×His-Tag的目的蛋白(19.5 kD)大小相符,主要以包涵体形式存在。结果显示,构建的表达载体pET-28a(+)-Imp在E.coliBL21(DE3)中能够达一定量表达,为进一步纯化Imp蛋白奠定基础。     

重组人β防御素3在大肠杆菌中的表达和活性分析

防御素是生物界广泛分布的一类低分子短肽,具有广谱高效的杀菌、抗肿瘤作用,并且不易使微生物产生抗药性,具有很高的应用价值,其中最引人注目的是β防御素[1,2].人β防御素3(humanβ-defensin3,hBD3)是最近发现的第3种人源性β防御素,与其它人防御素相比,在抗菌活性等方面具有明显优势,是所有防御素中抗菌能力最强的之一[3~7],具有独特的研究和开发价值.为了得到高效表达hBD3的工程菌株,本实验按照细菌对密码子的偏爱,人工合成了hBD3的寡核苷酸片段,构建了其表达载体.经IPTG诱导、分离纯化和肠激酶切割,得到了与天然hBD3活性基本相同的…