紫藤脉花叶病毒cp基因在大肠杆菌中的表达及抗血清的制备

采用RT-PCR方法自紫藤脉花叶病毒北京分离物(WVMV-BJ)的基因组中分离出其CP基因,连接到原核表达载体pET22b(+)上.获得的重组子pET-WVMVCP转化大肠杆菌BL21(DE3)后,用IPTG进行诱导表达.SDS-PAGE和Western blot分析表明,cp基因在大肠杆菌中获得了高效表达,融合蛋白分子量约为34.4 kDa.将融合蛋白纯化后免疫兔子,获得了特异性较高的抗血清.微量免疫沉淀法测定该抗血清的效价为1/1024,酶联法(enzyme-linkedimmunosorbant assay,ELISA)测定的效价为1/8192.     

侵染节瓜的小西葫芦黄花叶病毒CP基因的克隆、表达及其抗血清的制备

通过鉴别寄主反应、病毒部分序列测定确定了采自广州白云区表现花叶、斑驳症状的节瓜上的病毒为ZYMV。采用RT PCR方法扩增和克隆了该病毒的外壳蛋白基因 ,连接到原核表达载体pET 2 2b( )上。获得的重组子pET ZCP转化大肠杆菌BL2 1(DE3)后 ,用IPTG进行诱导表达。SDS PAGE和Westernblot分析表明 ,CP基因在大肠杆菌中获得了高效表达 ,融合蛋白分子量约为 33 0kD。将融合蛋白纯化后免疫兔子 ,获得了特异性较高的抗血清。ELISA测定其效价为 1 4 0 96     

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。     

猪FcγRIII的原核表达及多克隆抗体的制备

目的：构建猪FcγRIII 基因的原核表达载体,诱导表达重组蛋白,制备鼠抗猪 FcγRIII 抗血清。方法：从质粒pTG19-T-FcγRIII中用PCR方法克隆到编码完整猪FcγRIII蛋白分子的基因片段,将其插入到原核表达载体pET-32a中,构建了猪FcγRIII 原核表达载体pET-FcγRIII ,转化大肠杆菌BL21 (DE3) ,IPTG诱导蛋白表达,经尿素洗涤纯化后,以纯化后的融合蛋白FcγRIII-His 为抗原免疫小鼠,获得抗血清。Western blotting、ELISA 法鉴定获得的抗血清,ELISA 结果显示抗体效价为1∶16000,具有高度特异性,免疫印迹结果显示制备的多抗可以与重组猪FcγRIII蛋白特异性结合。结果：成功构建猪FcγRIII原核表达载体,纯化到融合蛋白FcγRIII-His,用纯化的融合蛋白免疫小鼠制备了多克隆抗体,Western blotting、ELISA 法证实多克隆抗体制备成功。结论：成功获得了猪 FcγRIII 多克隆抗体,为进一步研究猪FcγRIII 蛋白的功能奠定了基础。     

犬卵透明带3蛋白原核表达和抗血清的制备

在研发控制流浪犬卵透明带3(CZP3)免疫不育疫苗的过程中,需要制备特异性的CZP3抗血清进行免疫检测。本研究克隆获得CZP3的c DNA(全长1 281 bp)和除去跨膜区和信号肽的核心片段(CZP3C,984 bp),通过构建原核表达载体p ET28a-CZP3C,转化大肠杆菌BL21(DE3)。在28℃下以0.6 mmol·L~(-1)异丙基硫代β-D-半乳糖苷诱导表达4 h,获得CZP3C融合蛋白。利用CZP3C融合蛋白免疫新西兰大白兔,制备特异性的抗血清,并对抗血清的特异性和效价进行评价。SDS-PAGE结果表明,融合蛋白以包涵体形式存在。经过镍柱亲和纯化及8 mmol·L~(-1)尿素复性后,获得了高纯度的CZP3C融合蛋白。用0.8μg·μL~(-1)的CZP3C融合蛋白免疫新西兰大白兔,免疫后的兔抗血清用Western blot及间接免疫荧光实验检测,结果证明CZP3C兔抗血清具有较高的特异性,ELISA检测效价为1∶409600。本研究所制备的抗血清能够为流浪犬免疫不育疫苗的研制提供检测材料。     

侵染辣椒的黄瓜花叶病毒CP基因的序列分析、原核表达及抗血清制备

从吉林长春感病辣椒上获得一黄瓜花叶病毒(Cucumber mosaic virus,CMV)分离物(CMV-CC),根据GenBank中已登录的CMV外壳蛋白(coat protein,CP)基因核苷酸序列设计简并引物,通过RT-PCR的方法克隆到了长度为657 bp的目的片段。序列分析表明,CMV-CC与CMVI组各分离物核苷酸同源性为93.2%-97.9%。根据完整CP基因核苷酸序列构建的系统进化树显示:38个CMV分离物可分为3个组,CMV-CC属于CMV的IB亚组。将CMV-CC CP基因与原核表达载体pET-22b(+)连接,在大肠杆菌BL21(DE3)诱导表达出分子量约27 kD的融合蛋白。表达的融合蛋白经树脂纯化后免疫家兔制备了抗血清。用间接ELISA测定抗血清效价为1/4 096。Western blotting分析表明制备的抗血清对CP有高度特异性,为准确、快速地检测CMV奠定了基础。     

沙门菌外膜蛋白D的原核表达及多克隆抗体制备

目的:在大肠杆菌中表达沙门菌外膜蛋白(OMP)D,纯化后制备兔抗OMPD抗体。方法:用PCR方法从鼠伤寒沙门菌中扩增出ompD基因,并插入融合表达载体pET-28a(+)的多克隆位点,构建重组表达质粒pET28a(+)-ompD;以重组质粒转化大肠杆菌BL21(DE3),筛选阳性重组菌株,经IPTG诱导目的蛋白表达,在变性条件下对目的蛋白进行亲和层析纯化;以表达的OMPD蛋白免疫家兔,制备抗OMPD的多克隆抗体并进行鉴定。结果:扩增了ompD基因,测序证实正确后亚克隆于表达载体pET-28a(+)中,经PCR筛选和酶切鉴定获得阳性克隆,经诱导在大肠杆菌中表达出相对分子质量为40×103的目的蛋白并进行纯化;纯化的OMPD免疫家兔后,能有效地刺激特异性抗体的产生,抗血清的效价达到1∶10000以上,且具有良好的特异性。结论:构建ompD基因的原核表达载体,并在大肠杆菌中获得高效表达;制备出兔抗OMPD抗体,效价及特异性均良好,为进一步制备肠黏膜高亲和力疫苗奠定了基础。     

番茄环纹斑点病毒Gn蛋白的原核表达及多抗血清制备

通过生物信息学预测,番茄环纹斑点病毒(Tomato zonate spot virus,TZSV)Gn蛋白为跨膜蛋白,有3个跨膜结构域,抗原表位预测发现非跨膜区包含有较高的抗原指数区域,因此选择Gn蛋白的非跨膜区进行原核表达及多抗血清制备。用特异性引物,通过RT-PCR从感染TZSV的番茄中扩增出部分Gn基因片段,将获得的片段构建到原核表达载体pET-30a中,获得原核表达载体pET-30a-Gn,转化大肠杆菌BL21(DE3),用IPTG诱导表达。SDS-PAGE电泳分析显示,高效诱导表达了40 kD融合蛋白。用回收纯化的融合蛋白免疫家兔,获得多抗血清。间接ELISA测定多抗血清的效价为1/16 384。利用制备好的抗血清对感染TZSV的病样进行Western blotting检测,能检测到特异性条带。结果表明该抗血清特异性良好,可用于TZSV Gn相关的免疫反应实验。     

猪FcγRⅢ的原核表达及多克隆抗体的制备

目的:构建猪FcγRIII基因的原核表达载体,诱导表达重组蛋白,制备鼠抗猪FcγRIII抗血清。方法:从质粒pTG19-T-FcγRIII中用PCR方法克隆到编码完整猪FcγRIII蛋白分子的基因片段,将其插入到原核表达载体pET-32a中,构建了猪FcγRIII原核表达载体pET-FcγRIII,转化大肠杆菌BL21(DE3),IPTG诱导蛋白表达,经尿素洗涤纯化后,以纯化后的融合蛋白FcγRIII-His为抗原免疫小鼠,获得抗血清。Western blotting、ELISA法鉴定获得的抗血清,ELISA结果显示抗体效价为1∶16000,具有高度特异性,免疫印迹结果显示制备的多抗可以与重组猪FcγRIII蛋白特异性结合。结果:成功构建猪FcγRIII原核表达载体,纯化到融合蛋白FcγRIII-His,用纯化的融合蛋白免疫小鼠制备了多克隆抗体,Western blotting、ELISA法证实多克隆抗体制备成功。结论:成功获得了猪FcγRIII多克隆抗体,为进一步研究猪FcγRIII蛋白的功能奠定了基础。     

Production of Polyclonal Antibodies to a Recombinant Coat Protein of Potato mop-top virus

The coat protein (CP) coding regions of two Czech Potato mop‐top virus (PMTV) isolates were sequenced and shown to be identical. One, the Korneta isolate CP gene, was cloned in several expression vectors. The recombinant PMTV‐CP was expressed in Escherichia coli and the purified recombinant protein was used to produce PMTV‐specific polyclonal antibodies. The antiserum had a titre of 1 : 2000 in an indirect enzyme‐linked immunosorbent assay (ELISA) and reacted specifically in immunoblotting and IPTA‐ ELISA (indirect plate‐trapped antigen (PTA)‐ELISA).     

甘蔗花叶病毒E株系外壳蛋白基因原核表达载体的构建与表达

目的:用原核表达的方法获取大量带6个His标记的甘蔗花叶病毒E株系(ScMV-E)外壳蛋白(CP)。方法:用带有BamHⅠ和SalⅠ酶切位点的特异引物,以带有多个基因的重组质粒pNUSCP为模板,扩增出片段长度为942bp的ScMV-E外壳蛋白基因,亚克隆到pMD18-T载体上,转化E.coliDH5α,经双酶切检测获得阳性克隆。BamHⅠ和SalⅠ双酶切阳性克隆质粒,回收目的片段ScMV-E的CP基因。把目的片段插入表达载体pET29a( ),转化E.coliBL21(DE3),测序。结果:阳性质粒pET29a-CP在E.coliBL21(DE3)中得到大量特异表达。SDS-PAGE分析表明,该蛋白的相对分子质量约36000,与预测一致。结论:以上方法可以得到带6个His标记的目的蛋白,有利于纯化并获取高纯度的ScMV-E的外壳蛋白。     

Location of the cell-binding domain of CP65, a 65kDa cysteine proteinase involved in Trichomonas vaginalis cytotoxicity

The cysteine proteinase (CP) of 65 kDa, CP65, binds to the surface of HeLa cells and is involved in Trichomonas vaginalis cellular damage. To identify and locate the CP65 cellular-binding domain, we enriched the CP65 protein band by ammonium sulfate fractionation and ion-exchange chromatography and the N-terminal sequence was obtained. A 618 bp gene fragment was obtained by PCR using genomic DNA as template and primers derived from the N-terminal sequence of CP65 and the Asn papain-catalytic conserved region. This gene fragment encodes for 206 amino acid (aa) residues corresponding to the N-terminal region of a mature CP with 67–76% identity to the reported trichomonad cathepsin-L-like CPs. This gene fragment was expressed in a bacterial system for antibody production and functional analysis. Antibodies against the native trichomonad CP65 recognized the recombinant protein, referred to as rCP65, confirming its relationship with the CP65 gene. The rCP65 protein was bound to the surface of HeLa cells and competed with the native CP65 for binding. Antibodies to the rCP65 (-rCP65) reacted with the trichomonad CP65 located on the parasite surface, and inhibited trichomonal cytotoxicity in a concentration-dependent manner. These data strongly suggest that this gene fragment encodes for the putative cell-binding domain (CBD) of CP65 located at its N-terminal region.     

非洲猪瘟病毒CP204L基因的原核表达与单抗制备

由非洲猪瘟病毒(ASFV)引起的非洲猪瘟(ASF)给我国养猪业带来了不可估量的经济损失,严重阻碍了我国养猪业的发展,研发ASFV快速诊断试剂是目前最重要的内容之一。CP204L基因编码ASFV结构蛋白p30。本研究以克隆ASFV的CP204L基因为基础,通过基因重组技术,加入His标签,将构建的重组质粒命名为pET-28a-CP204L。将重组质粒转化至大肠杆菌BL21(DE3)感受态细胞,37℃经1mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达6h,表达蛋白进行SDS-PAGE鉴定和Western Blot检测。重组蛋白纯化后免疫小鼠制备筛选单克隆抗体,Western Blot和IFA验证单抗的结合特异性。结果表明,重组的pET-28a-CP204L诱导后表达蛋白为30kD,以不可溶性包涵体形式存在;表达蛋白利用His标签进行纯化,获得纯化蛋白2mg,单克隆抗体筛选获得5株IgG亚型的ASFV p30蛋白的单抗,且均具有良好的结合活性。本研究为发展ASFV检测方法提供了基础。     

Production of Polyclonal Antibodies to the Recombinant Potato virus M (PVM) Non‐structural Triple Gene Block Protein 1 and Coat Protein

The genes encoding the coat protein (CP) and triple gene block protein 1 (TGBp1) of Potato virus M (PVM) were cloned into expression vector pET‐45b(+) (N‐terminal 6xHis tag) and expressed in E. coli Rosetta gami‐2(DE3). The purified recombinant antigens were used for raising polyclonal antibodies. The antibodies against recombinant CP were successfully used in Western blot analysis, plate‐trapped ELISA and DAS‐ELISA as a coating for PVM detection in infected potato leaf samples. The antibodies against recombinant non‐structural protein detected the TGBp1 only in Western blot analysis. This is the first report of the production of polyclonal antibodies against recombinant coat protein and TGBp1 of PVM and their use for detecting the virus.     

甜菜坏死黄脉病毒75kDa通读蛋白基因构建与表达

利用DNA重组技术,将甜菜坏死黄脉病毒(BNYVV)内蒙分离物的CP基因和54kDa通读区片段拼接。构建了BNYVV 75kDa通读蛋白基因。序列分析表明,构建的75kDa通读蛋白基因与野生型相比．只有4个核苷酸发生了改变(包括将CP基因的终止密码子TAG改造为ATG),相应地2个氨基酸也发生了改变。将75kDa通读蛋白基因及其54kDa片段分别克隆到pJw2上,构建了这两十基因的原核表达载体。SDS—PAGE和western blotting检测结果表明,75kDa通读蛋白基因在E coli BL21(DE3)中经温度(42℃)诱导后除可特异地表达75kDa蛋白外。还产生两种小蛋白。75kDa通读蛋白基因的54kDa片段只表达出37kDa的蛋白。     

Expression of Coat Protein of an Ordinary Strain of Potato virus Y in Escherichia coli and Production of Polyclonal Antibodies for Diagnosis

The coat protein gene (CP) of an ordinary strain of Potato virus Y (PVY^O) was cloned into the expression vector, pET‐28a(+). The insert was sequenced and analysis showed that the CP gene was in frame with intact N‐terminal 6X histidine tags. An approximately 35 kDa recombinant fusion protein was observed in inclusion bodies of induced Escherichia coli BL21 cells. This fusion protein was purified and used as antigen to raise polyclonal antibodies in rabbits. In Western blot and dot blot immuno‐binding assay (DIBA), both PVY^O‐CP IgG and PVY^O IgG strongly reacted with the recombinant CP. The PVY^O‐CP IgG could detect PVY^O in infected samples up to 1 : 3200 dilutions. A PVY^O‐CP ELISA kit was prepared and compared with conventional ELISA kit based on purified virus particles (PVY^O ELISA kit). The PVY^O‐CP ELISA kit consistently detected the PVY^O in DAS‐ELISA of field samples and was as effective as PVY^O ELISA kit.