膜蛋白KCNN1在昆虫细胞Sf9中的表达与纯化

目的:利用昆虫细胞表达系统真核表达并纯化小电导钙激活钾离子通道蛋白1(KCNN1)。方法:以基因重组方法构建杆状病毒穿梭质粒reBacmid-KCNN1,将其转染至杆状病毒/Sf9细胞表达系统表达目的蛋白,并用Western印迹鉴定KCNN1的表达水平;用Ni-IDA-Sepharose CL-6B亲和层析柱纯化裂解细胞上清中的KCNN1,并用Western印迹鉴定纯化结果。结果:KCNN1在Sf9细胞中高效表达,通过亲和层析获得了纯化的KCNN1。结论:膜蛋白KCCN1在昆虫细胞Sf9中的表达与纯化,为深入研究其分子生物学功能提供了材料,也为全长膜蛋白的体外表达提供了一套可借鉴的实验方法。     

Expression, Purification, and Bioassay of Human Stanniocalcin from Baculovirus-Infected Insect Cells and Recombinant CHO Cells

Stanniocalcin is a calcium- and phosphate-regulating glycoprotein hormone that was first described in fish where it functions in preventing hypercalcemia. Human cDNA clones encoding the homolog of stanniocalcin have been recently isolated. In this study, the full-length cDNA coding for human stanniocalcin (hSTC) was cloned into both baculovirus and CHO expression vectors. Recombinant hSTC was then produced efficiently from both baculovirus-infected insect cells and CHO cells in large-scale bioreactors. Purification protocols were developed and used to purify recombinant hSTC from both sources in four chromatography steps. The hSTCs from both expression systems were secreted as glycosylated proteins and as disulfide-linked homodimers. The results from glycosylation studies indicated that stanniocalcin from both sources contained N-linked oligosaccharides but no O-linked sugars. In anin vivobioassay based on the inhibition of gill calcium transport in fishes, the baculovirus and CHO-expressed protein showed biological activity which is dose dependent. The inhibitory effects of hSTC produced from both systems were essentially equipotent in fishes, despite the differences in glycosylation. Consequently, the precise role of the carbohydrate moiety in recombinant hSTC remains to be determined.     

增强型绿色荧光蛋白的真核表达和纯化

根据编码增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的开放读码框(open reading frame,ORF)设计引物,PCR方法扩增出5'端带His标签的EGF PORF,利用杆状病毒表达系统构建表达EGFP基因的重组杆状病毒DNA分子,转染sf9细胞.取细胞...     

庚型肝炎病毒NS3蛋白在昆虫细胞中的表达

庚型肝炎病毒(HGV)/GB病毒C(GBV-C)疑似引起人类庚型肝炎[1～3].HGV和GBV-C为同一病毒的两个不同分离株,本文将其称为GBV-C/HGV.GBV-C/HGV属黄病毒科,为单股正链RNA病毒,全长约9.4kb.基因组中仅含有一个单一开放阅读框,编码E1、E2结构蛋白和NS2、NS3、NS4及NS5非结构蛋白.GBV-C/HGV的NS3蛋白具备丝氨酸蛋白酶活性和解旋酶活性[3],在NS3蛋白中还存在线性抗原表位[4],因此,NS3蛋白是GBV-C/HGV的重要功能蛋白.     

弗氏志贺菌5a 型 M90T 株 ClpB 蛋白在大肠杆菌中的融合表达和纯化

目的：构建弗氏志贺菌cZp8基因的原核表达质粒,在大肠杆菌中表达后,纯化带T7标签的ClpB蛋白。方法与结果：PCR扩增得到线性化表达载体pET24a与2574bp的c加曰基因片段,利用不依赖连接反应的克隆法（LIC）进行克隆,得到重组质粒pET-ClpB,转入大肠杆菌BL21（DE3）中进行诱导表达,通过一系列条件优化,确定可溶性表达条件为在30℃下、用1mmol／LIPTG诱导2h;利用抗T7单克隆抗体琼脂糖珠进行亲和纯化,得到了纯度很高的相对分子质量为95×10^3的ClpB-T7融合蛋白。结论：实现了ClpB-T7在大肠杆菌中的可溶性表达,并纯化获得了高纯度的融合蛋白。     

Rapid cloning and purification of proteins: gateway vectors for protein purification by self-cleaving tags

We have combined Invitrogen's Gateway cloning technology with self-cleaving purification tags to generate a new system for rapid production of recombinant protein products. To accomplish this, we engineered our previously reported DeltaI-CM cleaving intein to include a Gateway cloning recognition sequence, and demonstrated that the resulting Gateway-competent intein is unaffected. This intein can therefore be used in several previously reported purification methods, while at the same time being compatible with Gateway cloning. We have incorporated this intein into a set of Gateway vectors, which include self-cleaving elastin-like polypeptide (ELP), chitin binding domain (CBD), phasin (polyhydroxybutyrate-binding), or maltose binding domain (MBD) tags. These vectors were verified by Gateway cloning of TEM-1 beta-lactamase and Escherichia coli catalase genes, and the expressed target proteins were purified using the four methods encoded on the vectors. The purification methods were unaffected by replacing the DeltaI-CM intein with the Gateway intein. It was observed that some purification methods were more appropriate for each target than others, suggesting utility of this technology for rapid process identification and optimization. The modular design of the Gateway system and intein purification method suggests that any tag and promoter can be trivially added to this system for the development of additional expression vectors. This technology could greatly facilitate process optimization, allowing several targets and methods to be tested in a high-throughput manner.     

伪狂犬病毒gE基因在昆虫细胞中的高效表达

In order to develop a simple and safe test for the detection of vaccinated as well as wild type Pseudorabies virus (PRV) infected pigs, the modified gE gene of PRV Ea strain, obtained by cutting the 5' UTR using PCR and DNA recombinant technique, was inserted into baculovirus expression vector pFastBac 1, resulting the trans-position plamid pFE1.75. After homologous recombination, recombinant baculovirus rvBacE1.75 was gained and high level expression of glycoprotein E (gE) was observed after the infection of rvBacE1.75 to Tn-5B1-4 cells. The expression product was 80-88 kD and was specific to antisera against PRV Ea strain by Western-blotting. Purified recombinant proteins were used as an antigen in Latex Agglutination Test(gE-LAT) and the test was specific, sensitive, safe and simple.     

Expression of Rice Gall Dwarf Virus Outer Coat Protein Gene (S8) in Insect Cells

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected b...     

Memo蛋白的融合表达和纯化

目的:一些临床预后较差的恶性肿瘤一般具有很高的转移性和侵袭性,近来的研究表明,Memo蛋白与恶性肿瘤的这些特性有十分密切的关系。本研究拟克隆、融合表达和纯化Memo蛋白。方法:用PCR方法从乳腺文库中扩增Memo蛋白编码序列,并将其以正确相位与pGST载体中的GST编码序列融合,构建成重组质粒pGST-Memo。将此重组质粒转化大肠杆菌DH5α后,用谷胱甘肽-Sepharose4B纯化融合蛋白,并用Western印迹检测融合蛋白的表达。结果:构建了Memo蛋白的融合表达载体,并在大肠杆菌DH5α中得到表达,经谷胱甘肽-Sepharose4B亲和层析获得了纯化的GST-Memo融合蛋白。Western印迹检测表明,此蛋白可与GST抗体反应。结论:Memo蛋白的克隆、融合表达和纯化,为进一步研究恶性肿瘤的转移性、侵袭性及其相关机理提供了有用的材料。     

大鼠肝脏F抗原基因的克隆、表达及产物纯化

肝脏F抗原是一种新型的能直接反映肝损伤程度的血清学标志,它是1968年由美国学者Fravi从小鼠肝中分离出来的[1].F抗原是存在于哺乳动物肝细胞质中的一种水溶性不稳定的单链蛋白质,相对分子量为43kD[2],正常肝组织中的F抗原含量是血清中的1370倍,只有肝细胞被破坏时,F抗原才释放     

人vasorin蛋白的基因克隆、表达及纯化简

目的：克隆、表达人vasorin（VASN）蛋白。方法：利用PCR方法从HepG2细胞的cDNA中扩增获得目的基因,并插入带有6xHis标签的原核高效可溶性表达载体pET28a中,构建重组表达质粒pET28a-VASN,将重组表达质粒转化大肠杆菌BL21（DE3）,经IPTG诱导后目的基因获得表达,对融合目的蛋白进行Ni^2＋金属螯合柱纯化。结果：内切酶鉴定及基因序列测定证实重组表达质粒构建成功;对目的蛋白进行了原核表达,SDS-PAGE显示相对分子质量为61x10^3的特异表达条带;Western印迹证实目的蛋白为VASN,且主要以包涵体形式存在;对经尿素变性的表达产物进行了亲和层析纯化,有利于以后的变性、复性过程。结论：获得了人VASN融合蛋白,为其进一步的生物学功能研究奠定了基础。     

大连蛇岛蝮蛇类凝血酶基因在毕赤酵母中的克隆表达及分离纯化

根据同源性分析设计引物,通过RT-PCR方法从大连蛇岛蝮蛇毒腺总RNA中合成扩增出类凝血酶基因,之后将该基因克隆到表达载体pPIC9K中,经电激转化后整合至毕赤酵母细胞基因组中.经筛选得到甲醇快速生长型转化子His+Mut+在500 ml摇瓶中培养,甲醇诱导分泌表达.上清液中重组类凝血酶是通过两步柱层析得到:Q Sepharose FF和Benzamidine-Sepharose 4BCL.与天然蛇毒类凝血酶一致,分泌表达的重组类凝血酶具有较强的酯酶活性,但精氨酸甲酯如TAME的水解活性较弱.此重组类凝血酶在37℃中性溶液中保存过夜将分解成小肽,但在0℃下很稳定.该酶的最适pH为8.0.     

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。     

大连蛇岛蝮蛇类凝血酶基因在毕赤酵母中的克隆表达及分离纯化(英)

根据同源性分析设计引物,通过RT-PCR方法从大连蛇岛蝮蛇毒腺总RNA中合成扩增出类凝血酶基因,之后将该基因克隆到表达载体pPIC9K中,经电激转化后整合至毕赤酵母细胞基因组中.经筛选得到甲醇快速生长型转化子His⁺Mut⁺在500 ml摇瓶中培养,甲醇诱导分泌表达.上清液中重组类凝血酶是通过两步柱层析得到：Q Sepharose FF和Benzamidine-Sepharose 4BCL.与天然蛇毒类凝血酶一致,分泌表达的重组类凝血酶具有较强的酯酶活性,但精氨酸甲酯如TAME的水解活性较弱.此重组类凝血酶在37℃中性溶液中保存过夜将分解成小肽,但在0℃下很稳定.该酶的最适pH为8.0.     

成熟志贺毒素全长与截短A亚单位在E.coli中的表达及纯化

应用PCR技术从Ⅰ型痢疾志贺菌(Shigella dysenteriaetype I)中,扩增出约895 bp的成熟ShT-A基因片段,克隆至pGEM-T载体中,经蓝白斑筛选、PCR和双酶切鉴定正确后,命名为pGEM-TA2。测序结果表明,ShT-A与GenBank中ShT-A序列完全一致。用BamHⅠ和KpnⅠ双酶切克隆质粒,得到为895 bp成熟ShT-A与pQE30原核重组表达载体连接,构建原核重组表达质粒。经IPTG诱导,SDS-PAGE电泳观察,没有目的蛋白表达。DNAsis软件分析ShT-A基因序列,发现513 bp处有HindⅢ位点,故以ShT-A上游引物的BamHⅠ和HindⅢ从pGEM-TA2切出一个513 bp的截短片段,重新插入pQE30表达载体,得到pQE30-A513重组表达质粒,转化E.coliM15,经IPTG诱导,SDS-PAGE电泳观察,在20 ku处出现1条特异性的表达蛋白带,与截短ShT-A513分子量相符,表达量约占菌体总蛋白的37.4%,表达形式为包涵体。为抗体的制备提供了必要的物质基础。     

CHO/dhfr-细胞定点整合高效表达系统的建立

为了克服随机整合建立高表达细胞株时“位置效应”所带来的不可预知的后果,我们尝试建立基于定点整合的CHO高效表达系统。首先设计一个新的高效筛选载体pMCEscan。该载体含有报告基因(k₂tPA)、扩增基因（dhfr）、重组酶识别序列(FRT)及筛选基因（neo）,且neo基因的表达经过系统的弱化,确保能够对基因组中的整合位点进行大规模的高效筛选。然后利用该载体转染CHO/dhfr^－细胞并进行大规模筛选以获得足够多的阳性克隆,并对阳性克隆进行系统分析,筛选出报告基因表达水平高、单拷贝且扩增效果好的克隆,此克隆被认为筛选载体整合入CHO细胞基因组中转录热点（Hot spot）区域,从而获得了能够实现外源基因在基因组中定点整合和有效表达的CHO/dhfr-细胞系。随后利用位点特异性重组系统（FLP-FRT）将外源基因定点整合到Hot spot区域,以实现外源基因在CHO细胞基因组中的定点整合及高效表达。并利用该细胞系实现了k₂tPA的高表达,表达量达到17.1μg/10⁶cell·24h。该研究致力于CHO细胞基因组中高表达位点的寻找和确认,建立基于定点整合的哺乳动物细胞高效表达系统。     

CHO/dhfr-细胞定点整合高效表达系统的建立

为了克服随机整合建立高表达细胞株时“位置效应”所带来的不可预知的后果,我们尝试建立基于定点整合的CHO高效表达系统。首先设计一个新的高效筛选载体pMCEscan。该载体含有报告基因(k2tPA)、扩增基因(dhfr)、重组酶识别序列(FRT)及筛选基因(neo),且neo基因的表达经过系统的弱化,确保能够对基因组中的整合位点进行大规模的高效筛选。然后利用该载体转染CHO/dhfr-细胞并进行大规模筛选以获得足够多的阳性克隆,并对阳性克隆进行系统分析,筛选出报告基因表达水平高、单拷贝且扩增效果好的克隆,此克隆被认为筛选载体整合入CHO细胞基因组中转录热点(Hotspot)区域,从而获得了能够实现外源基因在基因组中定点整合和有效表达的CHO/dhfr-细胞系。随后利用位点特异性重组系统(FLP-FRT)将外源基因定点整合到Hotspot区域,以实现外源基因在CHO细胞基因组中的定点整合及高效表达。并利用该细胞系实现了k2tPA的高表达,表达量达到17.1μg/106cell.24h。该研究致力于CHO细胞基因组中高表达位点的寻找和确认,建立基于定点整合的哺乳动物细胞高效表达系统。     

一个新的人乙酰化酶MAK3的克隆、表达和纯化

在对乙酰GNAT家族进行研究分析的过程中,找到一个人GNAT家族的新成员MAK3。MAK3含有一个保守的乙酰化酶结构域,并且不同物种中的MAK3的蛋白质序列高度保守。通过半定量和定量RT—PCR检测了在不同组织中MAK3的mRNA水平。并且把MAK3分别克隆到了真核和原核表达质粒中,通过免疫印迹技术证实了MAK3的表达。还进一步用亲和层析的方法成功纯化了细菌中表达的MAK3,并对纯化后的MAK3的酶活性进行了测定。     

人源CLP的表达、纯化及生化特性研究

目的：为进一步研究CLP（coactosin-likeprotein）与5’-脂氧合酶、肌动蛋白的相互作用机制及功能,开展CLP克隆表达、分离纯化研究,以得到高纯度的CLP,并对其生物化学特性进行分析测定。方法：从人的胎肝cDNA文库中经PCR扩增得到CLP基因,克隆到原核表达载体pGEX-6p-1中并获得高效表达,经过Glutathione Sepharose 4B亲和层析和Su-perdex 75分子筛纯化,得到高纯度的CLP;在此基础上进行SDS-PAGE、动态光散射和分析型超速离心等实验,并进一步分析实验结果。结果：CLP在溶液中主要以单体形式呈现;CLP的摩擦率为1．909,证实该蛋白质具有线性化趋势存在的可能性,且线性化程度较高。结论：实验结果揭示了CLP作为线性化蛋白质的可能性,为进一步搭建CLP和丝状肌动蛋白的作用模型奠定了一定的数据基础。