SARS病毒S蛋白的原核表达与DNA疫苗的构建

目的 探讨SARS冠状病毒的刺突(spike,S)蛋白的免疫学特性,以及S蛋白作为SARS-CoV病毒疫苗组分的可行性。方法 将S蛋白基因分段克隆入原核表达载体pET-15b,并在大肠杆菌中表达,经过亲和层析得到纯化的重组蛋白rS_a和rS_b;将全长S基因克隆入真核分泌表达载体pSecTagB,得到重组DNA疫苗pSecS,免疫小鼠,得到SAS-CoVS蛋白抗血清。然后用纯化的重组蛋白rS_a和rS_b建立的SARS-CoVS抗体ELISA检测技术研究所构建的S-DNA疫苗的免疫效果。结果 分段的重组蛋白rS_a和rS_b在大肠杆菌中均以可溶性形式得到高效表达,并能与SARS确诊病人血清以及pSecS免疫鼠血清发生特异性抗原抗体反应,原核表达的重组分段S蛋白具有SARS-CoVS蛋白相似的抗原性。结论 原核表达的两段重组S蛋白有可能作为抗原组分用于临床SARS-CoV检测中;所构建的SARS-CoV的S基因核酸疫苗能在小鼠体内产生特异性抗体,这为进一步SARSDNA疫苗的研制提供一定的借鉴作用。     

SARS病毒E蛋白基因与EGFP基因在BHK-21细胞中的融合表达

目的：构建SARS病毒E蛋白基因的真核表达载体，观察E蛋白基因在BHK-21细胞中的正常表达及定位。方法：用PCR方法扩增E蛋白基因，上、下游引物两端分别设计EcoRⅠ和BamHⅠ酶切位点，产物双酶切后定向克隆于pEGFP-N1载体，构建真核表达载体pEGFP-E，脂质体法转染BHK-21细胞，荧光显微镜观察E蛋白基因的表达。结果：经酶切和DNA序列测定鉴定，E蛋白基因正确插入到增强绿荧光蛋白EGFP基因上游，荧光显微镜显示E蛋白表达于细胞浆内，包膜上也有分布。结论：在BHK-21细胞中成功表达了SARSCovE-EGFP融合蛋白，蛋白定位于细胞浆内。     

重组Trx-ApoO融合蛋白的构建与表达

目的:构建人载脂蛋白O (apolipoprotein O, ApoO)表达质粒,用pET原核表达系统制备重组抗原Trx-ApoO融合蛋白.方法:以人类肝脏cDNA文库为模板,聚合酶链反应(PCR) 法扩增ApoO DNA,将测序正确的目的基因片段插入质粒pET-32a (+)相应位点构建重组质粒并转化E.coli B...     

Construction of Prokaryotic Expression Plasmid of Fusion Protein Including Porin A and Porin B of Neisseria Gonorrhoeae and Its Expression in E. coli

In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a ( ) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E. coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a ( ) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E. coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0. 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.     

布鲁氏菌猪种标准株外膜蛋白OMP25的原核表达

目的克隆布鲁氏菌外膜蛋白Omp25基因并构建基因的原核表达系统。方法用聚合酶链反应技术扩增得到布鲁氏菌基因组，得Omp25基因片段，T—A克隆后测定核苷酸序列，将目的基因定向插入原核表达载体pGEX-4T-1，经双酶切和DNA测序测定，将构建的重组质粒转化大肠杆菌（E．coli HB101，TOP10），经IPTG诱导后，用SDS—PAGE检测重组蛋白rOrap25表达情况。结果经DNA测序显示Omp25DNA序列和插入位点正确，成功构建了重组质粒DGEX-4T-1-Omp25，经IPTG诱导，特异性表达出以包涵体形式存在48kDa的Omp25融合蛋白。结论制备、克隆了布鲁氏菌外膜蛋白Omp25基因片段，并在大肠杆菌中表达出Omp25融合蛋白。     

弓形虫ROP2基因的体外扩增及真核表达重组质粒的构建

目的　构建弓形虫棒状体蛋白2（POP2）基因真核表达重组质粒。方法　根据ROP2基因已知序列,设计合成一对引物,上、下游引物分别引入EcoRI、SalI酶切位点,用PCR方法从弓形虫RH株基因组DNA中扩增编码ROP2的基因片段,插入pGEX-4T-1质粒,构建原核表达重组质粒pGEX-4T-1-ROP2,而后经EcoR　I、Not　I双酶切出ROP2基因片段,再亚克隆到载体pcDNA3中构建真核表达重组质粒pcDNA3-ROP2。结果　ROP2基因体外扩增产物大小约1043bp,重组质粒经酶切及PCR鉴定表明获得正确重组子,克隆基因测序结果与已知序列基本吻合。结论　在国内首次克隆了弓形虫ROP2基因并构建了真核表达质粒pcDNA3-ROP2,为下一步弓形虫DNA疫苗研究打下了基础。     

A型流感病毒M基因DNA疫苗载体构建及免疫性研究

目的构建A型流感病毒M基因DNA疫苗质粒载体并对免疫后小鼠体内免疫应答反应进行分析和评价。方法将H3N2亚型流感病毒M基因通过RT-PCR扩增后克隆构建DNA疫苗载体pBudCE4．1-M,并将pBudCE4．1-M质粒体外瞬时转染COS7细胞并通过Western blot鉴定M1蛋白的表达,然后将pBudCE4．1-M质粒免疫小鼠。通过EUSA对小鼠体内生成的M1蛋白IgG抗体进行鉴定。结果M基因被成功克隆到质粒载体pBudCE4．1上,并在体外COS7细胞中表达出28kD的M1蛋白。疫苗质粒免疫小鼠后4周,在小鼠血清中检测到M1蛋白kG抗体的吸光度有明显升高。结论成功构建了一种A型流感病毒M基因DNA疫苗载体,将其免疫小鼠后成功诱导小鼠体内产生了特异性的IgG抗体。     

Construction of Prokaryotic Expression Plasmid of Fusion Protein Including Porin A and Porin B of Neisseria Gonorrhoeae and Its Expression in E.coli

Neisseriagonorrhoeaeisacommonpathogenicmicroorganismwhichcausessextransmitteddisease .Therewereanestimated 6 0millionnew gonococcalinfectionsworld widein 1999.Theporins ,thepre dominantproteinsonthesurfaceofpathogenicNeis seria ,formafamilyofstructurallyr…     

SARS相关冠状病毒S1融合蛋白的原核表达与纯化

目的：克隆严重急性呼吸综合征相关冠状病毒(SARS-CoV)S1蛋白编码DNA，构建原核表达质粒pGEX-5T／S1，并诱导表达谷胱甘肽巯基转移酶融合蛋白。方法：采用PCR方法扩增合成S1片段，并克隆入载体中，经双酶切鉴定和测序后把S1序列定向插入原核表达载体pGEX-5T的多克隆位点，转化大肠杆菌K802，将纯化后的融合蛋白用于SARS—CoV抗体阳性血清的检测。结果：GST—S1融合蛋白以可溶形式表达，纯化后的蛋白用ELISA法检测，结果与对照相符。结论：成功诱导原核表达并纬化出融合蛋白S1，为将其应用于SARS—CoV的特异性检测和亚单位疫苗研究奠定了基础。     

SARS冠状病毒S1基因的克隆及其植物表达载体的构建

目的:为获得抗SARS的转基因植物疫苗,进行了SARS S1基因的克隆,植物表达载体构建的研究。方法:根据SARS S蛋白受体结合结构域318-510氨基酸区域,设计合成长579 bp的序列片断(S1基因)。并以之为模板,进行PCR扩增,扩增产物被克隆至pGEM-Teasy载体进行序列测定。然后将S1基因插入植物表达载体pCAMB IA2301的35S启动子和NOS终止子之间,构建植物表达载体pCS1。将重组体转化大肠杆菌E.coliXL1,并对重组体进行了鉴定。结果:酶切鉴定和序列分析显示,克隆的目的基因与设计的片断序列一致;双酶切表明,植物表达载体的构建完全正确。结论:成功地克隆了S1基因和构建了含有S1基因的植物表达载体,为进一步获得抗SARS的转基因植物疫苗打下了基础。     

人载脂蛋白M的表达与纯化

目的:表达人细胞外全长结构域重组人载脂蛋白M(apolipoprotein M,ApoM)并将其纯化.方法:利用人类肝脏cDNA文库做模板,聚合酶链反应(PCR)法扩增ApoM DNA,将测序正确的目的基因片段插入质粒pGEXT相应住点,插入E.coli JM109,转化E.coli DL21(DE3),IPTG诱导蛋白表达.结果:PCR扩增产物经琼脂糖凝胶电泳证实在SDS-PAGE上出现一条560bp的基因片段.测序结果与GenBank公布的人ApoM基因序列完全一致.ApoM cDNA基因片段经IPTG诱导表达重组蛋白,SDS-PAGE电泳分析表明在相对分子量24 kD左右出现新的蛋白条带.结论:成功克隆出人ApoM基因,重组表达出ApoM蛋白.     

Construtcion of Neisseria Gonorrhoeae Porin B Plasmid Recombinant and Its Expression in E. coli

A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E. coli DE3 was constructed in order to provide a basis of research in detection, prophylactic and therapeutic vaccine against the pathogen infection. The gene encoding PIB was amplified by PCR from Neisseria gonorrhoeae and cloned into prokaryotic expression plasmid pET-28a( ) to construct a pET-PIB recombinant, which was verified by restriction endonuclease and DNA sequencing. Protein PIB was expressed in E. coli DE3 induced with IPTG. The antigenicity of the expressed protein was evaluated by indirect ELISA. Rabbits were immunized with the protein and serum was collected after immunization. To assess the immunogenicity of the protein, the titer of serum to protein PIB was determined by ELISA. DNA sequence analysis showed that the nucleic acid sequence of PIB gene was 99.28 % of homology compared with that (NGPIB18) published in GenBank. A 41 kD fused protein was detected by SDS-PAGE and was proven to have reactivity with anti-PIB polyclonal antibody from mouse. A polyclonal antibody to PIB of 1：4000 titer determined by indirect ELISA was obtained from rabbit immunized with the purified product. Recombinant plasmid encoding PIB of Neisseria gonorrhoeae was constructed. Protein PIB with antigenicity and immunogenicity was successfully expressed.     

Construction of Prokaryotic Expression Plasmid of mtrC Protein of Neisseria gonorrhoeae and Its Expression in E. Coli

Summary： In order to provide a rational research basis for detection of resistance of Neisseria gonorrhoeae to antimierobial hydrophobie agents and study on the resistant mechanism of multiple transferable resistance （mtr） efflux system, plasmid pET-28a（＋） encoding mtrC gene was constructed and the related target protein was expressed in Escherichia colt （E. cold DE3. The fragments of mtrC gene of Neisseria gonorrhoeae from the standard strains were amplified and cloned into prokaryotic expression plasmid pET-28a（＋） with restriction endonuelease to construct recombinant pET-mtrC which was verified by restriction endonuelease and DNA sequencing. The recom- binant was transformed into E. coli DE3 to express the protein mtrC induced by IPTG. The results showed mtrC DNA fragment was proved correct through restriction endonuelease and DNA sequencing. hs sequence was 99.5 % homologus to that published on GeneBank （U14993）. A 48.5 kD fusion protein which was induced by IPTG was detected by SDS-PAGE. It was concluded that the construction of prokaryotic expression plasmid of mtrC protein of Neisseria gonorrhoeae was correct and the fusion protein was successively expressed in E. coli.     

Construction, Expression and Identification of a Recombinant BCG Vaccine Encoding Human Mycobacterium Tuberculosis Heat Shock Protein 65

Heat shock protein 65 (HSP65) is one of the most important protective immunogens against the tuberculosis infection. The signal sequence of antigen 85B and the whole HSP65 DNA se-quence of human Mycobacterium tuberculosis (M. tuberculosis) were amplified from BCG genome and plasmid pCMV-MTHSP65 respectively by polymerase chain reactions (PCR). These two se-quences were cloned into the plasmid pBCG-2100 under the control of the promoter of heat shock protein 70 (HSP70) from human M. tuberculosis, yielding the prokaryotic shuttle expression plas-mid pBCG-SP-HSP65. Results of restriction endonuclease analysis, PCR detection and DNA se-quencing analysis showed that the two cloned DNA sequences were consistent with those previously reported, and the direction of their inserting into the recombinant was correct and the reading frame had been maintained. The recombinants were electroporated into BCG to construct the recombinant BCG vaccine and induced by heating. The induced expression detected by SDS-PAGE showed that the content of 65 kD protein expressed in recombinant BCG was 35.69% in total bacterial protein and 74.09% in the cell lysate supernatants, suggesting that the recombinant HSP65 gene could express in BCG with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive recombinant proteins could specifically combine with antibody against M.tuberculosis HSP65, indicating that the recombinant proteins possess the biological activity of HSP65.     

SARS冠状病毒M蛋白B细胞抗原表位的原核表达与抗原活性检测

目的从SARS冠状病毒M蛋白的线性重叠肽链文库中筛选出5个B细胞抗原表位,通过构建原核表达载体,表达抗原表位融合蛋白,并检测其抗原活性.方法应用大肠杆菌高频密码子设计引物,通过PCR方法合成编码SARS冠状病毒M蛋白5个抗原表位(MKY1、MKY2、MKY3、MKY4和MKY5)的DNA片段,经克隆和测序分析,亚克隆至表达载体pET-CKS,转化大肠杆菌BL21;阳性菌株经IPTG诱导,SDS-PAGE分析;大量诱导表达抗原表位融合蛋白,亲和层析予以纯化;Western检测SARS病人阳性血清对融合蛋白的识别.结果成功构建SARS冠状病毒M蛋白抗原表位的表达载体,在大肠杆菌BL21中表达,融合蛋白表达量达到细菌总蛋白30%,经亲和层析纯化,融合蛋白可被SARS病人抗血清识别.结论原核表达的抗原表位融合蛋白具有良好的抗原活性,为下一步进行SARS冠状病毒诊断试剂盒的开发研究奠定基础.     

汉坦病毒H8205株G1-人源IL-2融合基因的克隆与表达

目的 构建融合基因pcDNA3．1／His-B-IL-2-G1，为进一步研究汉坦病毒新型裸DNA疫苗奠定基础。方法 采用PCR从含有人源IL-2的质粒中扩增出IL-2片段，将人源IL-2插入真核表达载体pcDNA3．1／His—B中，构建的质粒命名为pcDNA3．1／His-昏IL-2。同样采用PCR扩增汉坦病毒H8205株G1基因片段，将回收的G1片段经双酶切插入到pcDNA3．1／His-B-IL-2，构建成新的质粒pcDNA3．1／His-B-IL-2-G1。采用脂质体介导包裹，转入NIH3T3细胞中进行瞬时表达；采用原位杂交观察细胞内的基因表达，SDS-PAGE法作重组质粒pcDNA3．1／His-B-IL-2-G1瞬时表达产物的鉴定。结果 融合基因可转录并表达-分子量为78kD左右的蛋白，对照组则未见电泳条带出现。结论 成功构建pcDNA3．1／His-B-IL-2-G1融合基因，融合基因能在真核细胞中瞬时表达。     

人成熟转化生长因子β1蛋白的克隆、表达、纯化及鉴定

目的构建人成熟转化生长因子β1（hmTGF-β1）的原核表达载体,表达并纯化hmTGF-β1蛋白。方法应用RT-PCR方法获得hmTGF-β1基因,连接至自带6His标签表达载体pET23b,构建原核表达质粒pET23b-hmTGF-β1,诱导表达hmTGF-β1融合蛋白,镍亲合层析法纯化融合蛋白,并对纯化产物进行纯度分析。结果成功构建了hmTGF-β1原核表达质粒pET23b-hmTGF-β1,IPTG诱导蛋白表达,SDS-PAGE电泳分析,在约15kD处出现了一条新的蛋白条带,WesternBlot结果显示该条带能够与His抗体特异性结合。应用Ni-NTA亲和层析法纯化目的蛋白,分析纯度达95%。结论成功构建了hmTGF-β1蛋白的原核表达质粒,并成功表达与纯化hmTGF-β1蛋白,为进一步复性、获得有活性的hmTGF-β1打下基础。     

SARS病毒S1蛋白N端片段在大肠杆菌中的表达、纯化与鉴定

目的:获得纯化的重组SARS病毒S1蛋白N端部分,研究其与机体产生针对SARS病毒免疫应答的规律和机制.方法:将编码SARS病毒S1蛋白N端334个氨基酸残基的基因进行克隆,并在原核表达系统中表达,获得了纯化的重组蛋白.利用SARS患者恢复期已知的SARS抗体阳性血清,鉴定纯化的重组S1蛋白片段.结果:克隆表达的重组蛋白基因序列与公布的SARS病毒S1蛋白N端的基因序列相同,所表达的重组蛋白相对分子质量约为64 000.3份SARS患者恢复期的血清均与重组蛋白反应,在相对分子质量64 000处形成特异性的反应条带,而来自SARS流行前的正常人对照血清则不能与重组蛋白反应.结论:获得的重组蛋白与SARS病毒抗体呈现特异性的反应,为进一步研究SARS病毒感染免疫应答机制和制备SARS病毒的重组疫苗奠定基础.     

小鼠PGRP-L分子PGRP结构域基因的克隆及其在大肠杆菌中的表达

目的 克隆小鼠长型肽聚糖识别蛋白（PGRP—L）的PGRP结构域基因并在大肠杆菌中表达。方法采用RT-PCR技术。从Balb／C小鼠肝组织总RNA中扩增PGRP—L的PGRP结构域基因片段,将其克隆人pUCm-T载体,以PCR、酶切和测序进行鉴定。以PCR从含PGRP结构域基因的重组质粒中扩增目的基因片段,插入表达质粒pQE-30构建重组表达质粒,转化大肠杆菌M15,诱导表达并纯化目的蛋白。结果RT—PCR扩增得到约500bp的基因片段,将其与pUCm-T载体连接构建成重组质粒prnPGRPd,其酶切图谱与计算机分析结果一致,该基因片段长518bp,序列与Genbank中相应基因序列相同。构建成重组表达载体pQE-PGRPd,在大肠杆菌M15中表达,表达产物主要存在于菌体裂解液上清中,为一相对分子质量约29000的可溶性蛋白。结论成功克隆并原核表达了小鼠PGRP—L分子PGRP结构域基因,为PGRP—L分子的研究奠定了一定基础。     

SARS冠状病毒PUMC_2株N蛋白cDNA的克隆、表达和纯化

目的原核表达并纯化SARS冠状病毒N蛋白。方法用RT-PCR技术克隆SARS冠状病毒PUMC2株N蛋白全长cDNA,cDNA经序列分析鉴定后,克隆到pET32a表达载体,转化E.coliBL21进行原核表达并纯化出SARS冠状病毒N蛋白。结果实现了SARS冠状病毒N蛋白的表达及纯化。结论用基因重组方法表达的SARS冠状病毒N蛋白可为其进一步功能研究提供条件。