重组结核分枝杆菌PPE17蛋白原核可溶性表达纯化和血清学诊断研究

目的利用原核系统可溶性表达结核分枝杆菌PPE17蛋白并进行纯化,研究其免疫学特性,并评价该重组蛋白在结核病血清学诊断方面的价值。方法从结核分枝杆菌H37Rv基因组中利用PCR扩增PPE17核酸序列然后克隆至融合表达载体pET-DsbC中,转入大肠埃希菌BL21(DE3)进行诱导、表达和纯化,用Western Blot和EL ISA方法进行抗原性初步评价。结果融合蛋白Ds-bC-PPE17在原核系统内经IPTG诱导表达后,主要以可溶性形式表达存在,经镍柱层析获得了纯的重组蛋白,纯度达95%以上。Western Blot和EL ISA方法结果证明重组DsbC-PPE17蛋白具有较强的抗原活性。用纯化的PPE17蛋白做抗原,临床诊断结核病人血清,阳性率达60%。结论融合蛋白DsbC-PPE17在大肠埃希菌中以可溶性表达形式存在,高纯度的重组融合蛋白有可能成为结核病的血清学诊断抗原。     

日本血吸虫乳酸脱氢酶原核表达、纯化及鉴定

目的原核表达日本血吸虫乳酸脱氢酶（SjLDH）,对表达产物进行纯化及生物活性鉴定。方法将SjLDH编码序列克隆入pET-28a原核表达载体并转染大肠埃希菌,异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达后亲和层析法对表达产物进行纯化,并用聚丙烯酰胺变性凝胶电泳（SDS-PAGE）、蛋白印迹（Western blot）、酶联免疫吸附实验（ELISA）、酶活性染色及活性测定进行鉴定。结果成功构建pET28a-SjLDH重组质粒并在大肠埃希菌中获得可溶性表达,亲和层析纯化表达产物。SDS-PAGE及Western Blot结果显示,表达及纯化产物分子量约36kDa,与SjLDH理论分子量相符。ELISA结果表明,重组蛋白具有良好的免疫活性,酶活性染色表明重组蛋白具有乳酸脱氢酶活性,活性单位为379U/mg。结论成功进行SjLDH的原核表达并获得纯化的重组蛋白,重组蛋白具有良好的免疫原性及酶活性,为研究SjLDH的功能奠定了基础。     

结核分枝杆菌Rv3914抗原的克隆表达与纯化

目的构建结核分枝杆菌Rv3914蛋白的重组质粒,并在大肠埃希菌中表达及纯化,为结核病血清学诊断提供候选抗原。方法用PCR方法从结核分枝杆菌H37Rv基因组中扩增Rv3914基因片段,插入到pET-28b(+)载体中,构建pET28b-Rv3914重组质粒,转化大肠埃希菌E.coli BL21plysS(DE3),经IPTG诱导表达后,应用Ni-NTA亲和柱纯化重组蛋白。结果 PCR扩增出Rv3914基因序列,重组质粒经测序并经BLAST分析后发现无点突变。pET28b-Rv3914重组质粒在大肠埃希菌E.coli BL21plysS(DE3)中主要以可溶性形式表达,重组蛋白占细胞蛋白总表达量的30%以上,经Ni-NTA柱纯化后,得到纯度超过90%、相对分子质量约为14.7×103的目的蛋白质。结论高纯度结核分枝杆菌Rv3914重组蛋白的获得为研究结核互补特异性抗原组合在结核病血清诊断中的价值奠定了基础。     

解脲脲支原体的N端保守部分MB优化基因在大肠埃希菌中的高效表达

目的 制备N-端保守部分多条带抗原(MB),克隆并在大肠埃希菌中表达其基因,并鉴定重组MB的抗原性.方法分析14个不同血清型解脲脲支原体(Uu)的基因排列,根据大肠埃希菌偏爱密码子,优化、设计合成DNA序列编码的氨基酸保守序列,合成的基因克隆到表达质粒,重组蛋白诱导和亲和层析纯化.结果 通过对Uu 14个血清型MB抗原氮端的氨基酸序列的分析、比对,根据大肠埃希菌的偏好密码子优化、设计并合成碱基序列,目的基因片段与pET-41a构建重组质粒并转化大肠埃希菌后,IPTG诱导表达,SDS-PAGE分析,得到了分子量约为45 kd的重组蛋白,纯化后的MB抗原蛋白能够较特异地与特异性抗体结合,敏感性为85.7％,特异性为87.8％.结论 Uu N-端保守部分的MB蛋白已成功表达、纯化并被证明具有良好的免疫原性.     

恙虫病东方体56kDa抗原基因片段在不同载体的表达

目的 构建恙虫病东方体56kDa表面抗原基因(sta56)片段在不同载体的重组表达质粒，在E．coli中表达sta56重组抗原并比较不同表达系统对sta56的表达效果。方法 从含有恙虫病东方体Karp株sta56基因的重组克隆，扩增出不同长度的截短的sta56片段．定向插入pPROEX HTb及pET30a载体，转化大肠埃希菌DH5a或BL21(DE3)，IPTG诱导表达，应用十二烷基磺酸钠—聚丙烯酰胺凝胶电泳(SDS-PAGE)观察重组蛋白表达情况。应用蛋白印迹(WB)方法分析重组抗原的活性。结果 成功构建含sta56不同长度片段的重组表达质粒pHTbOt957、pHTbOt498、pHTbOt342和pETOt957、pETOt498、pETOt342，各重组子均可在E．coli中以融合蛋白的形式有效表达，SDS-PAGE显示各表达重组子表达不同分子量的重组蛋白，WB证实各重组蛋白均能被恙虫病患者阳性血清所识别。结论 恙虫病东方体Karp株sta56基因可在大肠埃希菌获得高效表达，pET30a对sta56的表达效果优于pPROEX HTb；重组蛋白具有免疫反应性，纯化后可用作免疫诊断抗原。     

梅毒密螺旋体Tp47重组蛋白的表达及其应用

目的：在大肠埃希菌中表达梅毒密螺旋体（treponema pallidum,Tp）Tp47重组蛋白，用其建立诊断梅毒的酶联免疫法（enzyme immunoassay,EIA）。方法：应用聚合酶链反应法（PCR）从Tp全基因组中扩增目的片段Tp47，并将其克隆到表达载体pET-30a中，构建重组质粒pET-30a-Tp47。在大肠埃希菌中表达重组蛋白Tp47，亲和层析柱纯化，建立EIA法，检测血清中抗－Tp抗体。结果：获得了Tp47基因重组蛋白的高效表达，该重组蛋白存在于细菌上清中，占全菌体蛋白12％左右，分子量约39kDa。蛋白印迹法（western blot,WB）显示，Tp47重组蛋白具有良好的抗原性。用Tp47重组蛋白建立EIA法，检测11份梅毒血凝试验阳性和28份阴性血清，灵敏度为100％（11／11），特异性为96．4％（27／28）。结论：Tp47重组蛋白EIA可用于梅毒的诊断。     

亚洲带绦虫包虫诊断抗原P-29基因克隆表达

目的 识别亚洲带绦虫包虫诊断抗原P-29(Hydatid disease diagnostic antigen P-29)的已知序列并行克隆和蛋白表达及免疫学研究.方法 利用在线生物信息学工具序列分析后以亚洲带绦虫成虫包虫诊断抗原P-29基因克隆到原核表达质粒pET-28a(+)中,在大肠埃希菌BL21/DE3中诱导表达,重组后的蛋白用His-镍蛋白纯化柱纯化,纯化的重组蛋白用蛋白印迹进行免疫学分析.结果 重组体构建成功并以包涵体形式存在,破包涵体纯化蛋白并得到高纯度的蛋白,且该重组蛋白可被亚洲带绦虫及牛带绦虫病人血清识别,表明其具有免疫反应性.结论 亚洲带绦虫成虫包虫诊断抗原P-29可在原核表达系统中获得具有免疫活性的高效表达.     

PTD-Lmp-3融合蛋白的纯化、表达和鉴定

目的构建融合蛋白PTD-Lmp-3的表达质粒,并进行诱导表达、纯化及鉴定。方法利用基因重组技术构建pET43.1a-PTD-Lmp-3融合蛋白表达载体,并在大肠埃希菌BL21（DE3）中表达融合蛋白,经Ni2＋-NTA层析纯化,SDS-PAGE和Western blot鉴定目的蛋白。结果成功构建了表达质粒pET43.1a-PTD-Lmp-3,表达的融合蛋白经SDS-PAGE分析,经IPTG诱导表达的总菌体蛋白在融合蛋白的相应位置PTD-LMP-3约为22 kD,可溶性分析发现融合蛋白主要以上清形式存在,纯化后得到了目的蛋白。Western blot鉴定显示表达蛋白具有抗原性。结论成功构建了PTD-Lmp-3的重组表达载体,使融合蛋白在大肠埃希菌BL21（DE3）中高效表达,亲和层析后获得了纯化目的蛋白,为进一步研究奠定了基础。     

肺炎支原体渗透压诱导蛋白C的分子生物克隆及生物活性分析

目的:用生物克隆的方法表达肺炎支原体(Mycoplasmsa pneamoniae,Mp)渗透压诱导蛋白C(Osmotically inducible protein C,Osm C)并测定其生物活性活性。方法:根据Mp的Osm C基因序列设计特定引物,PCR扩增Osm C基因。通过构建重组质粒p ET28a-Mp Osm C,并转化入大肠埃希菌BL21(DE3),来诱导目的蛋白表达。我们采用镍柱亲和层析法对Osm C蛋白进行纯化,再用氧化铁二甲酚橙试剂(FOX)测定Osm C蛋白的活性。结果:我们用生物克隆的方法得到Mp的Osm C基因片段,并成功构建重组质粒p ET28a-Mp Osm C。重组质粒转化入大肠埃希菌BL21(DE3)后经过表达我们得到重组Mp Osm C蛋白,再经亲和层析得到高纯度的目的蛋白,且具有分解H2O2的活性。结论:重组Mp Osm C蛋白具有过氧化物酶的活性,为研究肺炎支原体的抗氧化机制提供了理论依据。     

麻疹病毒Edmonston疫苗株血凝素基因克隆及表达

目的 构建麻疹病毒(MV)血凝素蛋白(H蛋白)基因的重组质粒，使其在大肠埃希菌中大量表达H蛋白，为进一步研究奠定基础。方法从MV Edmonston株减毒活疫苗中提取基因组RNA，RT-PCR扩增H蛋白基因。用限制性内切酶Barn-HI和Hind-Ⅲ双酶切H基因片段和pGEMEX-1载体，连接后获得重组质粒MV-H-pGEMEX-1。然后，用IPTC诱导重组质粒在大肠埃希菌JM109(DE3)和BL21(DE3)中表达。结果 RT-PCR获得的片段长度约为1900bp，与MV-H基因相符。MV-H-pGEMEX．1测序结果显示：MV-H基因对码正确，核苷酸序列符合率达99．4％。转化重组质粒的大肠埃希菌，其表达产物经SDS-PAGE蛋白凝胶电泳，出现与目的蛋白分子量相一致的条带。结论 成功构建MV-H蛋白表达载体MV-H-pGEMEX-1，并在大肠埃希菌中获得表达。     

A recombinant 63-kDa form of Bacillus anthracis protective antigen produced in the yeast Saccharomyces cerevisiae provides protection in rabbit and primate inhalational challenge models of anthrax infection

Infection by Bacillus anthracis is preventable by prophylactic vaccination with several naturally derived and recombinant vaccine preparations. Existing data suggests that protection is mediated by antibodies directed against the protective antigen (PA) component of the anthrax toxin complex. PA is an 83-kDa protein cleaved in vivo to yield a biologically active 63-kDa protein. In an effort to evaluate the potential of yeast as an expression system for the production of recombinant PA, and to determine if the yeast-purified rPA63 can protect from a lethal inhalational challenge, the sequence of the 63-kDa form of PA was codon-optimized and expressed in the yeast Saccharomyces cerevisiae. Highly purified rPA63 isolated from Saccharomyces under denaturing conditions demonstrated reduced biological activity in a macrophage-killing assay compared to non-denatured rPA83 purified from Escherichia coli. Rabbits and non-human primates (NHP) immunized with rPA63 and later challenged with a lethal dose of B. anthracis spores were generally protected from infection. These results indicate that epitopes present in the 63-kDa from of PA can protect rabbits and non-human primates from a lethal spore challenge, and further suggest that a fully functional rPA63 is not required in order to provide these epitopes.     

脆性X智力低下蛋白在大肠杆菌中的表达及纯化

目的:在大肠杆菌中对脆性X智力低下蛋白(FMRP)进行重组表达,获得高产量及高纯度的可溶性重组蛋白。方法:以人脑cDNA文库为模板,PCR扩增FMR1基因后将其克隆至融合表达载体pGEX-6P-1中,重组载体转化进入大肠杆菌BL21(DE3)pLysS,并用IPTG进行诱导表达。应用谷胱甘肽-琼脂糖凝胶4B进行亲和层析纯化GST-FMRP融合蛋白,并以SDS-PAGE电泳和Westernblot对融合蛋白的表达进行检测。结果:亲和纯化后的融合蛋白经FMRP及GST单抗分别进行Westernblot鉴定为GST-FMRP融合蛋白。结论:大肠杆菌中FMRPISO10的重组表达产量高,纯化得到可溶性重组蛋白,为FMRP抗体的制备及FXS检测试剂盒的开发奠定基础。     

HCCR重组蛋白的表达及其单克隆抗体的制备

目的:克隆人宫颈癌基因(human cervical cancer oncogene,HCCR)的C末端截短序列(膜外区序列),重组表达并纯化HCCR-C端蛋白,制备和初步鉴定针对HCCR-C端蛋白的单克隆抗体。方法:从人肝癌细胞株HepG2中提取总RNA,RT-PCR扩增出HCCR-C端,构建其原核表达质粒,表达HCCR-C融合蛋白,以纯化的融合蛋白免疫BALB/c小鼠,常规细胞融合和选择性培养,亚克隆筛选出分泌特异抗体的杂交瘤细胞株,制备单抗腹水,Western blot、细胞组化初步鉴定抗HCCR-C端单克隆抗体。结果:成功克隆了HCCR-C末端截短序列,原核表达并纯化了HCCR-C端重组蛋白;经ELISA双筛,获得3株能稳定分泌抗HCCR-C端单克隆抗体的杂交瘤细胞株;Western blot及细胞免疫荧光结果显示,其分泌抗体效价高且均能特异识别HCCR蛋白。结论:成功制备并初步鉴定了针对HCCR-C端的单克隆抗体,为深入研究HCCR蛋白的生物学功能和建立新的肝细胞癌早期诊断方法奠定了可靠基础。     

梅毒螺旋体重组蛋白Tp0965的表达与鉴定

目的：研究梅毒螺旋体（Tp）重组蛋白Tp0965（r Tp0965）的表达并鉴定其免疫反应性,为进一步探讨其在梅毒血清学诊断与疫苗中的应用价值奠定基础。方法：构建Tp0965原核表达重组体p ET-28a（＋）/Tp0965,诱导转化宿主菌表达重组蛋白,Ni-NTA亲和层析法纯化蛋白,Western blot鉴定其免疫反应性。结果：成功构建了原核表达重组体p ET-28a（＋）/Tp0965,经诱导高效表达了一分子量约为43 KDa的可溶性重组蛋白,纯化蛋白纯度〉95%;Western blot显示该纯化蛋白能被梅毒患者血清特异性识别。结论：高效表达了可溶性r Tp0965,该重组抗原有良好的免疫反应性。     

Towards a human oral vaccine for anthrax: the utility of a Salmonella Typhi Ty21a-based prime-boost immunization strategy

We previously demonstrated the ability of an orally administered attenuated Salmonella enterica serovar Typhimurium strain expressing the protective antigen (PA) of Bacillus anthracis to confer protection against lethal anthrax aerosol spore challenge [Stokes MG, Titball RW, Neeson BN, et al. Oral administration of a Salmonella enterica-based vaccine expressing Bacillus anthracis protective antigen confers protection against aerosolized B. anthracis. Infect Immun 2007;75(April (4)):1827-34]. To extend the utility of this approach to humans we constructed variants of S. enterica serovar Typhi Ty21a, an attenuated typhoid vaccine strain licensed for human use, which expressed and exported PA via two distinct plasmid-based transport systems: the Escherichia coli HlyA haemolysin and the S. Typhi ClyA export apparatus. Murine immunogenicity studies confirmed the ability of these constructs, especially Ty21a expressing the ClyA-PA fusion protein, to stimulate strong PA-specific immune responses following intranasal immunization. These responses were further enhanced by a subsequent boost with either parenterally delivered recombinant PA or the licensed US human alum-adsorbed anthrax vaccine (AVA). Anthrax toxin neutralizing antibody responses using this prime-boost regimen were rapid, vigorous and broad in nature. The results of this study demonstrate the feasibility of employing a mucosal prime with a licensed Salmonella Typhi vaccine strain followed by a parenteral protein boost to stimulate rapid protective immunity against anthrax.     

肺炎嗜衣原体蛋白酶样活性因子重组蛋白的表达与临床应用

目的探讨肺炎嗜衣原体(CPN)蛋白酶样活性因子(CPAF)重组蛋白在CPN感染实验室诊断中的应用价值。方法构建CPAF免疫优势区基因重组质粒,诱导表达并纯化重组蛋白,分析其抗原特异性;间接ELISA法检测CPN参考血清、临床血清标本中的特异性IgM和IgG抗体,以及沙眼衣原体(Ct)阳性血清。结果高效表达和有效纯化出一相对分子质量(Mr)约为5.13×103的特异性重组蛋白;间接ELISA法测定用其免疫兔血清中的特异性IgG和IgM抗体效价,分别高达1∶16 000和1∶8 000;检测Cpn IgM和IgG参考血清,阴性和阳性结果符合率均为100.0%;与"金标准"方法MIF比较,检测300份呼吸道感染患者抗血清,IgMI、gG符合率分别为98.3%、99.0%;检测Ct阳性参考血清和阳性临床血清标本,无阳性结果。结论表达的CPN CPAF免疫优势区重组蛋白具有良好的抗原性,在CPN感染的实验室诊断中具有较高的应用价值。     

Comparative vaccine efficacy of different isoforms of recombinant protective antigen against Bacillus anthracis spore challenge in rabbits

The next-generation human anthrax vaccine developed by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) is based upon purified Bacillus anthracis recombinant protective antigen (rPA) adsorbed to aluminum hydroxide adjuvant (Alhydrogel). In addition to being safe, and effective, it is important that such a vaccine be fully characterized. Four major protein isoforms detected in purified rPA by native PAGE during research and development were reduced to two primary isoforms in bulk material produced by an improved process performed under Good Manufacturing Practices (GMP). Analysis of both rPA preparations by a protein-isoaspartyl-methyl-transferase assay (PIMT) revealed the presence of increasing amounts of iso-aspartic acid correlating with isoform content and suggesting deamidation as the source of rPA charge heterogeneity. Additional purification of GMP rPA by anion exchange chromatography separated and enriched the two principal isoforms. The in vitro and in vivo biological activities of each isoform were measured in comparison to the whole GMP preparation. There was no significant difference in the biological activity of each isoform compared to GMP rPA when analyzed in the presence of lethal factor using a macrophage lysis assay. Vaccination with the two individual isoforms revealed no differences in cytotoxicity neutralization antibody titers when compared to the GMP preparation although one isoform induced more anti-PA IgG antibody than the GMP material. Most importantly, each of the two isoforms as well as the whole GMP preparation protected 90-100% of rabbits challenged parenterally with 129 LD50 of B. anthracis Ames spores. The equivalent biological activity and vaccine efficacy of the two isoforms suggests that further processing to separate isoforms is unnecessary for continued testing of this next-generation anthrax vaccine.     

Production of recombinant rat hepatic histidase

Rat liver histidase was expressed in E. coli by using a PCR product of the coding sequence obtained from the rat liver cDNA of histidase cloned in the expression vector pRSET. The construct (pRSET-HAL) produced a fusion protein containing a tail of polyhistidine. The expression product was purified with a resin containing Ni+ that retains proteins with polyhistidine fragments. pRSET-HAL was analyzed by restriction enzyme mapping and by sequencing confirming the correct orientation and nucleotide sequence. Native rat liver histidase was also purified and it had a Mr of 72 kDa. An antiserum against native histidase was obtained in rabbit. Western blot analysis revealed one band of 72 kDa observed in membranes containing purified histidase or rat liver high speed supernatants. The same antiserum also detected in cell lysates of E. coli transformed with the pRSET-HAL plasmid a single band of 74 kDa of the recombinant histidase before cleavage with enterokinase. After the proteolysis, the Western blot analysis showed a single band of approximately 72 kDa. Kinetic analysis of the recombinant histidase showed similar Km and Vmax compared with native histidase.     

日本血吸虫核糖体蛋白S4基因的克隆、表达、纯化及免疫诊断价值的初步研究

目的 体外重组日本血吸虫核糖体蛋白S4(SjRPS4),并评价其在日本血吸虫病免疫诊断中的应用价值.方法 从日本血吸虫尾蚴cDNA文库获得了SjRPS4蛋白相关基因,将该段基因克隆入pQE30表达载体并转化到大肠杆菌M15中,异丙基-β-D-硫代半乳糖苷(IPTG)诱导融合蛋白表达.融合蛋白经Ni2+-NTA树脂柱进行亲和层析纯化,并通过十二烷基磺酸钠一聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定蛋白反应,通过Western blot和ELISA对其免疫原性进行鉴定.结果 成功构建了pQE30/SjRPS4重组菌.重组菌诱导培养后,SDS-PAGE电泳分析可见在相对分子质量约30×103处有SjRPS4融合蛋白的表达,经纯化获得纯度达90%以上的表达蛋白.Western blot检测结果表明该重组蛋白能被日本血吸虫病患者血清识别.用ELISA法检测疑似血吸虫病患者血清,敏感性为90.91%(70/77),特异性为92.59%(25/27),SjRPS4重组表达蛋白阳性率为67.30%(70/104),与肺吸虫病患者血清无交叉反应.结论 成功克隆表达了SjRPS4蛋白,初步证实了SjRPS4在诊断血吸虫病中具有良好的应用价值.     

亚洲牛带绦虫26kDa GST基因表达及免疫学分析

目的对亚洲牛带绦虫成虫26kDa谷胱甘肽S-转移酶基因(glutathione S-transferase,GST)进行克隆、表达和免疫学初步分析研究,为深入研究其生物学功能提供依据。方法将亚洲牛带绦虫成虫26kDa GST克隆到原核表达质粒pET-30a(+)中,在大肠埃希菌BL-21/DE3中用异丙硫代-β-D半乳糖苷(IPTG)诱导表达,表达产物通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行鉴定,用镍离子金属螯合剂亲和层析柱进行纯化,纯化的重组蛋白用蛋白印迹(Western Blotting)进行免疫学分析。结果PCR、双酶切及DNA测序结果均表明重组质粒pET-30a(+)-TaGST构建成功。SDS-PAGE结果表明,目的基因在大肠埃希菌BL-21/DE3中获得高效表达,经亲和层析获得了高纯度蛋白。重组蛋白可被其免疫的SD大鼠血清识别,表明其具有免疫原性;并且能识别感染了亚洲牛带绦虫的猪血清和亚洲牛带绦虫患者血清,表明其具有免疫反应性。结论亚洲牛带绦虫成虫26kDa谷胱甘肽S-转移酶基因可在原核表达系统中获得具有免疫学活性的高效表达。