产肠毒素大肠埃希菌CfaB亚单位的免疫原性分析

目的表达并纯化产肠毒素大肠埃希菌（Enterotoxigenic Escherichia coli,ETEC）CFA／I定居因子的CfaB亚单位,分析其免疫原性。方法将不含信号肽序列的cfaB基因克隆到pQE一30上,构建重组质粒pQE-30-cfaB并转化EcoliM15,表达融合蛋白6xHis—CfaB,将表达的蛋白纯化和复性后免疫BALB／c小鼠,制备抗CfaB的抗血清,用ELISA法检测抗血清效价。结果6×His—CfaB融合蛋白高效表达,相对分子质量为15．5kD,纯化复性后免疫小鼠所得抗血清效价为1：125000。结论成功构建了高效表达6×His-CfaB融合蛋白的重组质粒,表达的融合蛋白免疫小鼠后获得了高效价的抗血清,为进一步研制以双歧杆菌为表达系统的新型ETEC亚单位口服疫苗奠定了基础。     

人免疫缺陷病毒早期蛋白Nef的表达、纯化及免疫原性研究

目的：通过原核细胞表达人免疫缺陷病毒（HIV）Nef抗原,制备特异抗血清,为Nef抗原检测提供技术方法。方法：以HIVBotswana毒株基因组为模板,用PCR法获得Nef蛋白编码基因,将其克隆到pET30a载体中,在大肠杆菌中表达Nef融合蛋白;用纯化的融合蛋白免疫BALB／c小鼠获得抗血清,用真核表达的Nef抗原对其特异性进行分析。结果：构建的Nef融合基因在大肠杆菌中获得表达,相对分子质量约为36x103,免疫BALB／c小鼠获得针对融合蛋白的高效价抗血清,ELISA抗体滴度为1：6400;免疫荧光和Westemblot检测表明,该抗血清能特异地与重组痘苗病毒表达的Nef抗原反应。结论：在大肠杆菌中表达了HIVNef融合蛋白,制备了Nef融合蛋白的高效价小鼠免疫血清,该血清能特异性识别HIVNef抗原,为HlVNef抗原检测提供了技术方法。     

盐穗木HcDMC1的原核表达和抗血清的制备

DMC1是减数分裂过程中同源染色体配对和重组修复所必需的减数分裂特异蛋白。根据盐胁迫下盐穗木转录组数据库,克隆获得的盐穗木DNA损伤修复基因命名为HcDMC1。为深入分析盐穗木HcDMC1基因的耐盐功能,通过原核表达获得盐穗木HcDMC1的融合蛋白,用于免疫小鼠制备特异性的HcDMC1抗血清。结果表明,利用pET30a-HcDMC1能够在大肠杆菌BL21（DE3）中诱导表达融合蛋白His-HcDMC1,纯化融合蛋白His-HcDMC1的含量为1.0 mg·mL^-1。每次每只小鼠免疫接种50 μg融合蛋白,三次免疫接种后进行抗体效价及特异性检测。ELISA检测抗血清滴度约为1：400 000,Western Blot检测证明了抗血清的特异性。本研究制备的小鼠抗血清能够为盐穗木HcDMC1蛋白的功能鉴定和免疫检测提供实验材料。     

幽门螺杆菌HP0762蛋白的原核表达纯化及多克隆抗体制备

目的:表达和纯化幽门螺杆菌HP0762蛋白,并制备该蛋白的多克隆抗体。方法:从幽门螺杆菌SS1中经PCR扩增得到了hp0762基因,将其克隆至含有6×His编码序列的原核表达载体pET-28a(+)中,再将重组质粒转化大肠杆菌BL21(DE3),在IPTG诱导下进行蛋白表达;用HiTrap Chelating HP亲和柱纯化重组蛋白,Western印迹进一步鉴定;以纯化后的蛋白为抗原免疫新西兰大耳白兔,制备该蛋白的多克隆抗体;用ELISA和Western印迹检测抗血清。结果:目的蛋白在大肠杆菌BL21(DE3)中获得了可溶性表达,纯化后纯度可达90%以上;制备了针对HP0762重组蛋白的抗血清,抗体ELISA效价为1:256000,Western印迹分析表明该抗体能特异性识别内源性HP0762。结论:完成了HP0762蛋白的原核高效表达与纯化,并制备了其高效价的多克隆抗体,为进一步对其进行疫苗研制与基因功能研究奠定了基础。     

DNA损伤检查点蛋白调节子1片段的表达纯化及抗体制备

目的：原核表达、纯化DNA损伤检查点蛋白调节子1（MDC1）片段,并制备其多克隆抗体。方法：设计特异引物,通过RT-PCR扩增编码MDC1 N端194个氨基酸残基的基因片段,测序正确后插入含GST基因的原核表达载体pGEX-KG中,以IPTG诱导表达,并经谷胱甘肽琼脂糖珠纯化融合蛋白;用纯化的蛋白免疫小鼠制备多克隆抗体,用ELISA测定抗体的效价,Western印迹鉴定抗体的特异性。结果：原核表达并纯化了MDC1 N端片段,并获得了抗MDC1的多克隆抗体,抗体效价达到1∶12800,Western印迹显示该抗血清能特异识别原核及真核细胞表达的MDC1。结论：MDC1 N端片段能够诱导小鼠产生具有较高效价和特异性的多克隆抗体,为进一步研究MDC1在Fhit特异信号通路中的作用奠定了基础。     

人NDRG1的融合表达、纯化及抗体制备

NDRG1是在N myc缺陷的小鼠胚胎组织中发现的一异常高表达的新基因 .在研究高同型半胱氨酸血症引起动脉粥样硬化的机制时 ,在培养的人脐静脉内皮细胞 (HUVEC)中发现了人NDRG1 .为了研究人NDRG1的功能以及结构与功能之间的关系 ,用RT PCR技术 ,从人脑总RNA中克隆NDRG1cDNA ,进行序列测定后 ,将NDRG1插入pPROEXHTb表达载体中 ,转化E .coliDH5α感受态细胞 ,并在LB培养基中获得高效可溶表达 .表达的 6His NDRG1融合蛋白经Ni2 + NTA偶联的琼脂糖珠纯化 ,通过圆二色性分析其二级结构 ,结果为 :α螺旋 :2 3 6 ,β片层 :1 8 6 ,转角 :2 5 7,无规卷曲 :32 0 .用此融合表达的蛋白免疫家兔 ,制备得到高效价的抗体 ,利用固定于硝酸纤维素膜上的NDRG1抗原亲和吸附纯化抗血清提高了抗体的特异性 ,为进一步研究NDRG1的功能打下了良好的基础     

水稻Qb-SNARE蛋白OsNPSN11多克隆抗体制备、鉴定与应用

在真核生物细胞囊泡运输过程中的膜融合主要是由SNARE蛋白介导的, OsNPSN11是从水稻中克隆的Qb-SNARE家族基因, 文章将OsNPSN11构建到原核表达载体pET-30a中与6个His标签融合, 重组质粒pET-OsNPSN11转化BL21(DE3)0.5 mmol/L IPTG诱导4 h后获得了高效表达。用镍离子亲和树脂(Ni²⁺-NTA His Bind Resin) 纯化融合蛋白, 以纯化后的蛋白为抗原免疫新西兰家兔制备多克隆抗体, Western blotting结果显示, 该抗体能特异识别在原核系统表达的抗原, 以及水稻不同组织质膜组分中的OsNPSN11, 可用于转基因水稻中目标蛋白的表达分析。     

登革2型病毒非结构蛋白NS4B的原核表达、纯化及多克隆抗体制备

目的：原核表达、纯化登革2型病毒非结构蛋白NS4B,并制备其多克隆抗体,以研究其结构与功能。方法：扩增编码登革2型病毒NS4B的24-238位氨基酸残基的基因序列,并将其克隆到原核表达载体pGEX-4T-1,转化大肠杆菌BL21（DE3）,IPTG诱导表达;采用蛋白浸提方法从SDS-PAGE胶中回收融合蛋白;用纯化后的融合蛋白免疫BALB／c鼠制备多克隆抗体,采用间接免疫荧光法检测抗体效价。结果：原核表达了NS4B-GST融合蛋白,并获得了其多克隆抗体,抗体效价为1：800。结论：登革2型病毒NS4B的24-238位氨基酸残基可诱导小鼠产生具有较高效价和特异性的多克隆抗体,这为研究NS4B的结构与功能奠定了基础。     

昆虫神经毒素LqhIT2的表达、抗血清制备及活性分析

根据毕赤酵母密码子偏爱性,不改变毒素蛋白质一级结构,设计合成了昆虫神经毒素LqhIT2基因,并分别克隆至大肠杆菌融合表达载体pPET30-a( )和毕赤酵母分泌表达载体pPIC9K.在IPTG的诱导下,神经毒素在大肠杆菌中融合表达,表达产物经镍亲和层析纯化后,用于免疫BALB/c小鼠,制备了特异性较高的抗血清,抗体滴度超过1:128 000.利用制备的抗血清,采用斑点杂交,筛选得到了较高水平分泌表达重组LqhIT2的酵母转化子,摇瓶条件下,毒素表达量约9 mg/L.大肠杆菌表达产物没有生物活性,酵母表达产物经注射蝗虫表现出杀虫活性.     

Purification and immunogenicity of a recombinant Bordetella pertussis S1S3FHA fusion protein expressed by Streptococcus gordonii

Acellular pertussis vaccines typically consist of antigens isolated from Bordetella pertussis, and pertussis toxin (PT) and filamentous hemagglutinin (FHA) are two prominent components. One of the disadvantages of a multiple-component vaccine is the cost associated with the production of the individual components. In this study, we constructed an in-frame fusion protein consisting of PT fragments (179 amino acids of PT subunit S1 and 180 amino acids of PT subunit S3) and a 456-amino-acid type I domain of FHA. The fusion protein was expressed by the commensal oral bacterium Streptococcus gordonii. The fusion protein was secreted into the culture medium as an expected 155-kDa protein, which was recognized by a polyclonal anti-PT antibody, a monoclonal anti-S1 antibody, and a monoclonal anti-FHA antibody. The fusion protein was purified from the culture supernatant by affinity and gel permeation chromatography. The immunogenicity of the purified fusion protein was assessed in BALB/c mice by performing parenteral and mucosal immunization experiments. When given parenterally, the fusion protein elicited a very strong antibody titer against the FHA type I domain, a moderate titer against native FHA, and a weak titer against PT. When given mucosally, it elicited a systemic response and a mucosal response to FHA and PT. In Western blots, the immune sera recognized the S1, S3, and S2 subunits of PT. These data collectively indicate that fragments of the pertussis vaccine components can be expressed in a single fusion protein by S. gordonii and that the fusion protein is immunogenic. This multivalent fusion protein approach may be used in designing a new generation of acellular pertussis vaccines.     

带Tat标记质粒载体的构建及其转导融合蛋白进入细胞的研究

采用点突变技术构建了带 6×His、Tat和Flag多个标记的pET HTF的质粒载体 ,利用基因重组技术构建pET HTF EGFP融合蛋白载体 .酶切和DNA测序证明 ,所构建的pET HTF和pET HTF EGFP载体正确 .BL2 1(DE3)表达融合蛋白 ,用Ni2 + 分离柱纯化His Tat Flag EGFP蛋白 ,并加入培养的NIH3T3细胞 .荧光显微镜观察显示 ,His Tat Flag EGFP融合蛋白进入细胞 .带His、Tat和Flag标记的质粒载体pET 14b HTF表达的融合蛋白能够进入细胞 ,该载体为进行蛋白质功能研究和基因治疗研究提供了一个重要工具     

HPV16 L1蛋白在大肠杆菌中的表达、纯化及免疫原性研究

用IPTG诱导目的工程菌pQE31-HPV16L1/M15(pREP4),对表达产物进行SDSPAGE和Western blot分析;用表达的L1蛋白免疫BAL B/C小鼠得到抗血清后,利用真核源性的VLP粗提物验证小鼠抗血清的特异性.利用IMAC金属亲和层析柱纯化L1蛋白.SDSPAGE结果显示表达产物在约57 ku处有蛋白条带;Western blot结果证实此条带可与HPV16 L1蛋白的单克隆抗体反应;纯化后的L1蛋白也同样保留免疫特异性;小鼠抗血清可与HPV16L1 VLP(病毒样颗粒)发生特异性反应,证实重组表达的L1蛋白具有免疫原性.本实验表明HPV16 L1蛋白在工程菌M15(pREP4)中高效表达,为研制HPV16预防性基因工程疫苗和感染的诊断试剂提供了物质基础和技术方法.     

Cloning, sequencing and prokaryotic expression of cDNAs for antifreeze protein family from beetle Tenebrio molitor

Partial cDNA sequences coding for antifreeze proteins in Tenebrio molitor were obtained by RT-PCR.Sequence analysis revealed nine putative cDNAs with a high degree of homology to Tenebrio molitor antifreeze protein genes published in GenBank.The recombinant pGEX-4T-l-tmafp-XJ430 was introduced into E.coli BL21 to induce a GST fusion protein by IPTG.SDS-PAGE analysis for the fusion protein shows a band of 38 kDa.pCDNA3- tmafp-XJ430 was injected into mice to generate antiserum which was later detected by indirect ELISA.The titer of the antibody was 1:2000.Western blot-ting analysis shows that the antiserum was specifically against the antifreeze protein.Our results laid the founda-tion for further studies on the properties and functions of insect antifreeze proteins.     

Purification and Immunogenicity of a Recombinant Bordetella pertussis S1S3FHA Fusion Protein Expressed by Streptococcus gordonii

Acellular pertussis vaccines typically consist of antigens isolated from Bordetella pertussis, and pertussis toxin (PT) and filamentous hemagglutinin (FHA) are two prominent components. One of the disadvantages of a multiple-component vaccine is the cost associated with the production of the individual components. In this study, we constructed an in-frame fusion protein consisting of PT fragments (179 amino acids of PT subunit S1 and 180 amino acids of PT subunit S3) and a 456-amino-acid type I domain of FHA. The fusion protein was expressed by the commensal oral bacterium Streptococcus gordonii. The fusion protein was secreted into the culture medium as an expected 155-kDa protein, which was recognized by a polyclonal anti-PT antibody, a monoclonal anti-S1 antibody, and a monoclonal anti-FHA antibody. The fusion protein was purified from the culture supernatant by affinity and gel permeation chromatography. The immunogenicity of the purified fusion protein was assessed in BALB/c mice by performing parenteral and mucosal immunization experiments. When given parenterally, the fusion protein elicited a very strong antibody titer against the FHA type I domain, a moderate titer against native FHA, and a weak titer against PT. When given mucosally, it elicited a systemic response and a mucosal response to FHA and PT. In Western blots, the immune sera recognized the S1, S3, and S2 subunits of PT. These data collectively indicate that fragments of the pertussis vaccine components can be expressed in a single fusion protein by S. gordonii and that the fusion protein is immunogenic. This multivalent fusion protein approach may be used in designing a new generation of acellular pertussis vaccines.     

棉铃虫病毒gp41基因的克隆表达及抗体制备

根据棉铃虫单核衣壳核多角体病毒(Helicoverpa armigerasingle nucleocapsid nucleopolyhedrovirus,HaSNPV)gp41基因的序列,设计引物,引入适当的酶切位点,通过PCR的方法扩增目的片段。将扩增出的基因片段克隆至原核表达载体pET-28a,构建重组质粒并转化至大肠杆菌中,经IPTG诱导表达。纯化蛋白产物并免疫家兔产生抗血清。该抗血清可与原核表达的His-GP41融合蛋白及在感染的昆虫细胞中表达的GP41蛋白发生特异性免疫反应。该抗体的获得为深入研究GP41的功能提供了基础。     

Immunodiagnosis of Parietaria mottle virus in Tomato Crops Using a Polyclonal Antiserum against its Coat Protein Expressed in a Bacterial System

Recombinant DNA technology was used to raise a polyclonal antiserum against the coat protein (CP) of Parietaria mottle virus (PMoV). The CP gene was expressed in Escherichia coli as a fusion to a 6xHis tag and purified by affinity chromatography. Recombinant purified protein was used as antigen to raise a polyclonal antiserum. This polyclonal antiserum consistently detected PMoV specifically infected tomato plants from different commercial tomato crops by indirect enzyme-linked immunosorbent assay (I-ELISA) and direct tissue-printing immunoassay (DTBIA).     

青鳉prdm14的原核表达、多克隆抗体制备及其应用

青鳉(Oryzias latipes)是研究遗传发育和细胞多能性的重要模式鱼类, 为探究prdm14同源基因的潜在作用, 实验将青鳉prmd14经原核表达后制备了兔抗Prdm14多克隆抗体。首先, 将prdm14基因的部分编码区连接到pET32a质粒中, 构建重组表达载体pET32a-prdm14?600。随后将重组载体转化至大肠杆菌(Escherichia coli)Rosetta(DE3), 经异丙基-β-d-硫代半乳糖苷(Isopropyl-β-d-thiogalactoside, IPTG)诱导表达, 获得分子量为60 kD的Prdm14重组蛋白。接着大量诱导蛋白表达并切胶纯化, 免疫家兔(Oryctolagus cuniculus), 6周后获得阳性抗体, 最后通过ELISA和Western blot检测抗体效价及其特异性。结果显示, 在37℃、0.6 mmol/L IPTG、诱导3h的条件下, 可获得Prdm14重组蛋白的高效表达; 制备的兔抗青鳉Prdm14多克隆抗体能够特异性识别青鳉组织中表达的Prdm14蛋白以及在HepG2细胞中过表达的青鳉Prdm14: EGFP融合蛋白。综上所述, 研究首次制备了一种能有效识别青鳉Prdm14的多克隆抗体, 该抗体的获得为后续研究prdm14基因在鱼类多能性干细胞中的作用提供了有力工具。     

Characterization of molecular markers for specific and sensitive detection of Botrytis cinerea Pers.: Fr. in strawberry (Fragariaxananassa Duch.) using PCR

Part of the gyrase A gene (gyrA) of Acholeplasma laidlawii was cloned and incorporated directly downstream from a 6 x His tag segment of the pQE expression vector. The 23-kDa fusion protein was expressed as a 6 x His-tagged protein in Escherichia coli. The fusion protein was purified and used as an antigen for rabbit immunization. Western immunoblot analysis revealed that the antiserum raised against the gyrase A fragment had a specific affinity for a 108-kDa protein of A. laidlawii and cross-reacted with a 107.5-kDa protein of Acholeplasma axanthum, a 107-kDa protein of Acholeplasma granularum, and 95-97-kDa proteins of several phytoplasma-infected plants. The antiserum could also detect phytoplasmas in infected plant sap. These results demonstrate that the gyrase A protein (GyrA) of A. laidlawii shares antigenicity with the GyrA of other Acholeplasma species and also with those of phytoplasmas including some from a few groups with unrelated 16S rRNAs.     

Production of the polyclonal anti-human metallothionein 2A antibody with recombinant protein technology

Metallothionein 2A(MT2A)is a small stress response protein that can be induced by exposureto toxic metals.It is highly expressed in breast cancer cells.In this study,the cDNA encoding the humanMT2A protein was expressed as glutathione S-transferase(GST)fusion protein in Escherichia coli.Recombinant MT2A proteins were loaded onto 12% sodium dodecylsulfate-polyacrylamide gel and separatedby electrophoresis,the recombinant protein was visualized by Coomassie blue staining and the 33 kDarecombinant GST-MT2A fusion protein band was cut out from the gel.The gel slice was minced and used togenerate polyclonal antisera.Immunization of rabbit against MT2A protein allowed the production of hightiter polyclonal antiserum.This new polyclonal antibody recognized recombinant MT2A protein in Westernblot analysis.This low-cost antibody will be useful for detection in various immuno-assays.     

Expression of the gene of interest fused to the EGFP-expressing gene in transgenic mice derived from selected transgenic embryos

The present paper describes the expression of a target fusion gene, WAP/hGH fused to the EGFP-expressing gene in transgenic mice derived from the transfer of transgenic embryos selected because of their expression of enhanced green fluorescent protein (EGFP). The 6.7-kb fusion gene was microinjected as a single cassette gene construct into the pronuclei of mouse zygotes. The surviving embryos were cultured and were classified according to the EGFP expression patterns at the morula or blastocyst stage. After the transfer of embryos with uniform-expression or mosaic-expression of EGFP, transgenesis occurred in 85.7% to 86% or 44.1% to 44% of the pups, respectively. No transgenic pups were derived from EGFP negative embryos. In the transgenic females, EGFP was ubiquitously expressed under the control of the CAG promoter, and hGH was expressed under the control of the WAP promoter in an appropriate fashion: hGH was secreted into the milk of lactating transgenic females. The presence or absence of the expression of EGFP coincided with that of the hGH gene in the transgenic mice. The present cassette gene construct is a useful example for circumventing the routine analyses of DNA and RNA required for the generation and maintenance of transgenic lines.