汉滩病毒M,S基因不同拼接方式原核表达效果比较研究

为了比较汉滩病毒囊膜糖蛋白G1与核蛋白(NP)主要抗原位点片段不同拼接方式的原核表达效果，将汉滩病毒76—118株M基因编码G1的片段与S基因编码区5’端约O．7kb的片段连接，克隆入pGEX一4T2，构建嵌合基因原核表达栽体pGEX-4T2-G1S0．7，pGEX-4T2-S0．7G1，在大肠杆菌XLl一Blue中诱导表达GST—G1S0．7或GST—S0．7G1融合蛋白。经IPTG诱导后，ELISA活性测定结果表明，两种融合蛋白均可与抗汉坦病毒NP的mAb特异性结合，融合蛋白GST—G1S0．7还可与抗汉滩病毒糖蛋白的mAb特异性结合。Western blot结果显示，诱导出G1S0．7或S0．7G1与GST的融合蛋白，其中G1S0．7嵌合基因的表达产物降解较少。研究证明：两种拼接方式的嵌合基因均可在大肠杆菌中表达出有生物学活性的融合蛋白，但表达效果不同，为汉滩病毒基因工程疫苗的研究奠定了基础。     

鳗鲡病原菌二联外膜蛋白基因工程表达载体的构建

根据鳗鲡病原性嗜水气单胞菌Ⅱ型孔蛋白(porinⅡ)和迟钝爱德华氏菌外膜蛋白S(ompS2)的基因全长序列,分别选取这两个全长序列中表达其蛋白质膜外部分且理论免疫原性较好的两个基因片段,通过融合PCR技术连接这两个外膜蛋白基因片段.根据表达载体(pGEX-2T-His)的限制性酶切位点在连接序列片段的两端引入限制性酶切位点BamHⅠ和EcoRⅠ,成功构建了双外膜蛋白基因片段重组表达载体(pGEX-2T-His-porinⅡ-ompS2).表达载体理论表达产物的蛋白质结构预测表明表达产物无信号肽和跨膜区,不存在三级结构;亲水性和免疫原性预测分析表明该表达蛋白理论上为可溶性蛋白并具有丰富的抗原决定簇,本研究为该表达载体的蛋白表达、纯化以及表达产物的免疫原性研究奠定了基础.     

大肠杆菌中融合表达汉滩病毒S,M基因片段的研究

为在大肠杆菌中融合表达汉滩病毒囊膜糖蛋白G1与NP含主要抗原位点的片段 .将汉滩病毒 76 118株S基因编码区 5′端约 0 .7kb的片段与M基因编码G1的片段连接 ,克隆入 pGEX 4T2 ,构建嵌合基因原核表达载体 pGEX 4T2 S 0 .7G1,在大肠杆菌XL1 Blue中诱导GST S0 .7G1融合蛋白的表达 .表达产物用ELISA和Westernblot进行鉴定 .限制性内切酶酶切鉴定证明 ,成功构建了嵌合原核表达载体 pGEX 4T2 S0 .7G1.经IPTG诱导后 ,ELISA活性测定结果表明 ,该融合蛋白可与抗汉坦病毒NPmAb特异性结合 .Westernblot结果显示 ,诱导出相对分子质量 (Mr)大于 1× 10 5的S0 .7G1与GST的融合蛋白 ,并有一系列大小不等的可与抗汉坦病毒NPmAb特异性结合的蛋白带 .获得具有特异性结合活性的融合蛋白GST S0 .7G1,为汉滩病毒基因工程疫苗的研制奠定了基础     

牛肠激酶催化亚基cDNA的克隆及融合表达载体的构建

肠激酶(Enterokinase,EK)是目前生物制药领域纯化重组蛋白产品时用于切割融合蛋白的首选工具酶之一．为克隆表达牛肠激酶催化亚基(EKL)编码的基因,以期应用于融合蛋白的切割与纯化,从市售肉牛十二指肠组织中提取总RNA,以RT-PCR方法扩增其cDNA片段,将此片段克隆于pUCm-T载体中进行全序列分析．结果表明克隆的cDNA与GenBank上的序列相比完全一致．随后,将目的基因片段插入pET32a融合型表达载体中．经测序证实其重组DNA5’端多克隆位点与重组片段的接口处核苷酸顺序准确无误,所表达重组蛋白经SDS-PAGE分析,相对分子质量为50kD,表达量达38％,为进一步进行Enterokinase催化亚基蛋白表达及活性研究奠定了基础．     

甘蓝型油菜乙醇酸氧化酶(GO)基因片段的克隆及hpRNA表达载体构建

通过PCR从甘蓝型油菜(Brassica napus)华双4号基因组DNA中扩增出乙醇酸氧化酶(GO)基因片段,DNA序列分析表明扩增GO基因片段的外显子部分与报道序列相同.以扩增出的GO基因片段作模板从一端设计引物扩增出一个相应的小片段.将GO基因大小两个片段反向连接,插入到植物表达载体2300-nap的napin启动子和nos终止子之间,植物表达载体2300-35S的35S启动子和nos终止子之间,分别构建成可转录表达出发夹RNA(hairpin RNA,hpRNA)结构的种子特异型和组成型油菜hpRNA干扰载体.     

枯草杆菌纳豆激酶基因的克隆及其在E.coli BL21(DE3)plysS中的表达

用CTAB法从枯草杆菌(纳豆株)中提取基因组DNA，聚合酶链式反应(PCR)扩增获得837bp的DNA片段，将该基因片断克隆到pMD18-T载体上，筛选重组子，通过限制性内切酶、PCR技术和测序分析确定该重组子所含插入片断为纳豆激酶基因．利用基因重组技术构建了纳豆激酶基因的融合表达载体，转化E．coli BL21(DE3)plysS后经IPTG诱导在大肠杆菌中高效表达，经SDS—PAGE电泳及bandscan软件分析融合蛋白分子量约为33．4kD，约占菌体总蛋白29．4％．     

SARS-CoV棘突蛋白片段在E.coli中的表达及免疫活性分析

SARS-冠状病毒(SARS-CoV)棘突蛋白(Spike,S)是构成病毒包膜突起的主要成分,属于型膜糖蛋白,全长1255aa,在病毒入侵宿主细胞中起重要作用.本文利用生物信息学技术,界定冠状病毒SARS-CoVS蛋白的抗原决定区域,通过PCR扩增相应片段,克隆至原核表达载体pET-H,得到四个重组质粒pET-HS1、pET-HS3、pET-HS4、pET-HS5.经序列测定证实后,转化大肠杆菌BL21(DE3),IPTG诱导获得表达,产物经紫外薄层扫描显示目的蛋白占菌体细胞总蛋白量的20%~30%.经Westernblot检测,表达蛋白与恢复期SARS病人血清呈高反应原性.纯化相应蛋白免疫小鼠,所获抗血清效价达1∶3×103.与SARS诊断试剂反应呈阳性,这一工作将为进一步研究S蛋白的定位、功能及SARS诊断提供帮助.     

亮氨酸拉链结构蛋白基因表达载体选择

以ZG1( )、ZG1(-)、ZG2( )、ZG2(-)、ZG3( )、ZG3(-)寡聚核苷酸片段为材料，合成了含有GCN4亮氨酸拉链蛋白基区结合DNA必需的35个氨基酸的折叠片段，将此片段分别克隆到pET3b质粒和pBLZ质粒中．酶切后用2％琼脂糖电泳检测证明两者克隆成功．将这2种重组质粒分别转化到E.coli DH5a中．发现pET3b重组体在E．coli DH5a中表达成功．而pBLZ质粒重组体在E．coli DH5a中不能成功表达；从转化子中分离得到重组质粒pET3b,酶切和序到分析都证明插入序列为合成的亮氨酸拉链蛋白基因．     

汉滩病毒S基因0.7kb片段与M基因G2片段不同拼接方式嵌合基因原核表达产物的初步比较

比较汉滩病毒S、M基因部分片段的嵌合基因不同拼接方式表达产物的活性。构建了嵌合基因原核表达载体pGEX-4T-1-S0.7G2,并与已构建的pGEX-4T-1-G2S0.7的诱导表达产物进行了比较。结果表明,融合蛋白同时保持亲本蛋白的结合活性,且前者表达产物的结合活性始终比后者低。研究表明,嵌合基因的不同拼接方式对融合蛋白的活性可能有一定的影响。     

人类扭转蛋白A的基因克隆、表达与蛋白质纯化(Ⅰ)——DYT1基因克隆与原核表达

通过基因工程方法，用大肠杆菌原核表达系统表达人扭转蛋白A．从该蛋白编码序列中设计引物，以人肝cDNA文库作模板扩增到编码该蛋白的基因片段．将所得片段与pMDl8-T载体连接，转化到JM109大肠杆菌中，从转化平板上挑出菌落，用碱裂解法提取质粒，通过PCR和酶切分析，筛选到阳性克隆，测序结果与文献报道结果一致．提取质粒，用BamHI和XhoI酶切，回收目的片段，分别克隆到原核表达载体pET28a（4-）和pGEX-6P-1中，转化JM109受体菌，从JM109受体菌中提出质粒，再转化到BL21（DE3）菌中，筛选出阳性克隆，构建了人扭转蛋白A原核表达载体．IPTG诱导该工程菌，培养并离心收集．SDS-PAGE结果表明该蛋白在两种载体中均得到了高效表达．     

Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus

Spike (S) proteins of coronaviruses, including the coronavirus that causes severe acute respiratory syndrome (SARS), associate with cellular receptors to mediate infection of their target cells. Here we identify a metallopeptidase, angiotensin-converting enzyme 2 (ACE2), isolated from SARS coronavirus (SARS-CoV)-permissive Vero E6 cells, that efficiently binds the S1 domain of the SARS-CoV S protein. We found that a soluble form of ACE2, but not of the related enzyme ACE1, blocked association of the S1 domain with Vero E6 cells. 293T cells transfected with ACE2, but not those transfected with human immunodeficiency virus-1 receptors, formed multinucleated syncytia with cells expressing S protein. Furthermore, SARS-CoV replicated efficiently on ACE2-transfected but not mock-transfected 293T cells. Finally, anti-ACE2 but not anti-ACE1 antibody blocked viral replication on Vero E6 cells. Together our data indicate that ACE2 is a functional receptor for SARS-CoV.     

SARS相关冠状病毒刺突糖蛋白的研究(综述)

概述了SARS—CoV的S蛋白的结构及功能相关研究。严重急性呼吸综合症相关的冠状病毒(SARS-CoV)引起2003年我国南方非典型肺炎爆发流行，波及多个国家和地区。目前全球许多学对SARS-CoV进行了广泛的研究，发现S蛋白是病毒表面的主要蛋白，它构成冠状病毒科特征性的冠状样结构，在严重急性呼吸综合症的发病机制起着关键性作用，可介导表达相关受体的宿主细胞感染。现已鉴定出SARS-CoV的S蛋白相关受体，同时它在抗病毒感染中是一个关键靶蛋白。     

Analysis of proteins that interact with nucleocapsid protein of SARS-CoV using 1 5-mer phage-displayed library

Analysis of proteins that interact with N protein of SARS-CoV using 15-mer phage-displayed library will help to explore the virus pathogenesis and to develop new drugs and vaccines against SARS. In this study, we cloned, expressed and purified N protein of SARS-CoV. This 46-kD N protein was verified by SDS-PAGE and Western-blot. Then, the peptides binding-specific to N protein were identified using 15-mer phage-displayed library. Surprisingly, all of the 89 clones from monoclonal ELISA were positive （S/N〉2.1） and the result was further confirmed experimentally once again. Six N protein-binding peptides, designated separately as SNA1, SNA2, SNA4, SNA5, SNA9 and SNG11, were selected for sequencing. Sequence analysis suggested that SNA5 shared approximatively 100% sequence identity to SNA4, SNA2, SNA9 and SNA1. In addition, the binding specificity of the 15-mer peptides with the SARS-CoV N protein was further demonstrated by blocking ELISA using the synthetical 15-mer peptide according to the deduced amino acid sequence of SNA5. Also, the deduced amino sequence of SNA5 was compared with proteins in translated database using the tblastx program, and the results showed that the proteins with the highest homology were Ubiquinol-cytochrome c reductase iron-sulfur subunits （UCRI or UQCR）, otherwise known as the Rieske iron-sulfur proteins （RISP）. Notablely, in the [2Fe-2S] redox centre of UCRI, there were 6 residues [GGW（Y）F（Y）CP] compatible to the residues （position 2→7, GGWFCP7） of the NH2-terminal of the 15-mer peptide, which indicated higher binding specificity between the N protein of SARS-CoV and the redox centre of UCRI to some extent. Here, the possible molecular mechanisms of SARS-CoV N protein in the pathogenesis of SARS are discussed.     

A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice

Public health measures have successfully identified and contained outbreaks of the severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), but concerns remain over the possibility of future recurrences. Finding a vaccine for this virus therefore remains a high priority. Here, we show that a DNA vaccine encoding the spike (S) glycoprotein of the SARS-CoV induces T cell and neutralizing antibody responses, as well as protective immunity, in a mouse model. Alternative forms of S were analysed by DNA immunization. These expression vectors induced robust immune responses mediated by CD4 and CD8 cells, as well as significant antibody titres, measured by enzyme-linked immunosorbent assay. Moreover, antibody responses in mice vaccinated with an expression vector encoding a form of S that includes its transmembrane domain elicited neutralizing antibodies. Viral replication was reduced by more than six orders of magnitude in the lungs of mice vaccinated with these S plasmid DNA expression vectors, and protection was mediated by a humoral but not a T-cell-dependent immune mechanism. Gene-based vaccination for the SARS-CoV elicits effective immune responses that generate protective immunity in an animal model.     

Bioinformatical study on the proteomics and evolution of SARS-CoV

A novel coronavirus has been identified as the causative agent of the severe acute respiratory syndrome (SARS). For all the SARS-CoV associated proteins derivated from the SARS-CoV genome, the physiochemical properties such as the molecular weight, isoelectric point and extinction coefficient of each protein were calculated. The transmembrane segments and subeellular localization (SubLoeation) prediction and conserved protein motifs search against database were employed to analyze the function of SARS-CoV proteins. Also, the homology protein sequence alignment and evolutionary distance matrix calculation between SARS-CoV associated proteins and the corresponding proteins of other coronaviruses were employed to identify the classification and phylogenetic relationship between SARS-CoV and other coronaviruses. The results showed that SARS-CoV is a novel coronavirus which is different from any of the three previously known groups of coronviruses, but it is closer to BoCoV and MHV than to other coronaviruses. This study is in aid of experimental determination of SARS-CoV proteomics and the development of antiviral vaccine.     

SARS冠状病毒核衣壳蛋白基因的表达及其在SARS诊断中的应用

目的为SARS感染后建立一种特异性免疫学诊断方法,为基因疫苗的研制提供备选实验材料.方法利用基因重组的方法,在大肠杆菌中表达了SARS冠状病毒(SARS-CoV)N蛋白全长基因,经包涵体的初步纯化,金属螯合层析进一步纯化N蛋白,SDS-PAGE电泳和ELISA方法进行结果检测.结果N蛋白抗原与30名SARS感染患者血清结合全部为阳性,对照组30名正常人血清为阴性,30名发热但非SARS患者血清为阴性.结论利用基因重组的方法对SARS-CoV N蛋白进行了表达和纯化,证明该重组N蛋白抗原具有良好的反应特异性,是良好的应用于病毒感染早期诊断的抗原.完全可用于组构检测核心抗体的诊断试剂.并建立了免疫学的检测方法,为抗体检测提供了安全、方便的手段,并为基因工程疫苗研究奠定了基础.     

SARS的研究进展

2003年3月,发现SARS病原是一种从未在人类或动物中发现过的新的冠状病毒.其基因组结构与其他的冠状病毒相似,由29727核苷酸组成,有11个开放阅读框.通过多基因分析序列比较说明,SARS冠状病毒与已知的冠状病毒无关.针对SARS冠状病毒的各种特征及研究进展进行了综述.     

SARS冠状病毒的诊断性实验研究

严重急性呼吸综合征(SARS)是一种新的人类感染性疾病，其致病菌为SARS冠状病毒(SARS-CoV)。本文就SARS-CoV发现过程、病毒特点及检测方法的研究进展和应用情况加以概述。     

Phylogeny of SARS-CoV as inferred from complete genome comparison

SARS-CoV, as the pathogeny of severe acute respi-ratory syndrome (SARS), seems to be the first coronavirus that is lethal to humans. Coronavirus (family Coronaviri-dae, genus Coronavirus) is an enveloped, single-stranded plus sense RNA virus whose genome has approximately 30 kb size. Whereas coronaviruses may cause severe dis-ease in animals, coronaviruses human strains only cause mild diseases until SARS-CoV was discovered. To date, SARS-CoV genomes from 12 isolates have been comp…     

传染性非典型肺炎病毒核蛋白的表达与活性检测

用PCR方法，人工合成传染性非典型肺炎病毒(SARS-CoV)核蛋白(N)全编码基因，并构建原核表达载体．在大肠杆菌中进行表达．结果重组N蛋白表达产量占菌体总蛋白的40％以上，主要以可溶形式存在．用SARS患者急性期血清进行蛋白印迹检测，表明可溶形式和包含体形式均有明显活性．包含体形式的重组蛋白经纯化后纯度可达90％以上，活性与纯化前相当，可作为SARS抗体诊断试剂盒的抗原原料．