荷包猪SLA-3-HB01基因四聚体前体链的构建及表达

为了构建荷包猪SLA-3-HB01基因四聚体前体链原核表达载体,并获得SLA-3-HB01表达蛋白,试验以SLA-3-HB01/pMD18-T为模板进行PCR扩增四聚体前体链SLA-3-HB01-BSP,并克隆至pMD19-T载体中,经NdeⅠ和XhoⅠ双酶切筛选阳性克隆并测序,目的基因连接至表达载体pET-21a（+）,转化大肠杆菌BL21（DE3）感受态细胞,经IPTG诱导表达,SDS-PAGE检测目的蛋白大小及表达情况,提取包涵体并进行检测。结果显示,PCR成功扩增得到SLA-3-HB01-BSP,大小为896 bp左右。酶切鉴定证实,目的基因成功克隆至pMD19-T载体中,插入片段大小为876 bp,阳性克隆经测序后所获序列与原序列一致,并在3'端带有BSP标签序列。酶切鉴定进一步证实成功构建SLA-3-HB01-BSP/pET-21a（+）重组表达载体,经转化及诱导表达,SDS-PAGE检测显示目的蛋白分子质量在33.5 ku左右。包涵体蛋白分子质量约33.5 ku,与菌体中目的蛋白大小一致,经凝胶成像系统UVP扫描分析,包涵体蛋白纯度接近于90%,符合进行相关结构和功能研究的要求。本研究成功构建了荷包猪SLA-3-HB01基因四聚体前体链的pET-21a（+）重组表达系统,并获得了一定纯度的包涵体蛋白。     

荷包猪SLA-3-HB01基因四聚体前体链的构建及表达

为了构建荷包猪SLA-3-HB01基因四聚体前体链原核表达载体,并获得SLA-3-HB01表达蛋白,试验以SLA-3-HB01/pMD18-T为模板进行PCR扩增四聚体前体链SLA-3-HB01-BSP,并克隆至pMD19-T载体中,经NdeⅠ和XhoⅠ双酶切筛选阳性克隆并测序,目的基因连接至表达载体pET-21a(+),转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导表达,SDS-PAGE检测目的蛋白大小及表达情况,提取包涵体并进行检测。结果显示,PCR成功扩增得到SLA-3-HB01-BSP,大小为896bp左右。酶切鉴定证实,目的基因成功克隆至pMD19-T载体中,插入片段大小为876bp,阳性克隆经测序后所获序列与原序列一致,并在3′端带有BSP标签序列。酶切鉴定进一步证实成功构建SLA-3-HB01-BSP/pET-21a(+)重组表达载体,经转化及诱导表达,SDS-PAGE检测显示目的蛋白分子质量在33.5ku左右。包涵体蛋白分子质量约33.5ku,与菌体中目的蛋白大小一致,经凝胶成像系统UVP扫描分析,包涵体蛋白纯度接近于90%,符合进行相关结构和功能研究的要求。本研究成功构建了荷包猪SLA-3-HB01基因四聚体前体链的pET-21a(+)重组表达系统,并获得了一定纯度的包涵体蛋白。     

大约克猪SLA-1原核表达载体的构建及表达

为构建大约克猪SLA-1胞外区的原核表达载体及表达目的蛋白,试验设计1对引物,经PCR扩增获得大约克猪SLA-1胞外区基因（命名为SLA-1-DYKe）,将此片段克隆至pMD^®19-T Simple Vector,转化大肠杆菌TOP10感受态细胞,经Nde Ⅰ和Xho Ⅰ双酶切筛选阳性克隆菌并测序,将目的基因插入到原核表达载体pET-28a（+）中,转化至宿主菌BL21（DE3）进行诱导表达,用SDS-PAGE检测目的蛋白的表达情况,大量诱导提取包涵体并检测。结果显示,PCR成功扩增SLA-1-DYKe的胞外区,得到大小为837 bp的目的基因,目的基因成功克隆至pMD^®19-T Simple Vector,并获得序列正确的重组质粒。以得到的重组质粒成功构建了SLA-1-DYKe/pET-28a（+）表达载体,目的蛋白大小约为34 ku。本研究成功构建了大约克猪SLA-1原核表达载体,获得了表达蛋白,为今后研究大约克猪SLA-1的空间结构和基因功能奠定了基础。     

中国2个地方品系猪SLA-3原核表达载体构建及表达

为构建中国地方品系荷包猪及莱芜黑猪SLA-3(命名为SLA-3-HB和SLA-3-LW)原核表达载体及表达目的蛋白,试验通过PCR扩增获得SLA-3胞外区基因,并克隆至pMD19-T Simple载体,转化大肠杆菌Top10感受态细胞,酶切及测序筛选阳性重组质粒;重组质粒经酶切回收,目的片段进一步连接pET-21a(+)表达载体,并转化大肠杆菌BL21感受态细胞,IPTG诱导目的基因的表达;SDS-PAGE检测目的蛋白。结果显示,PCR成功扩增SLA-3-HB及SLA-3-LW胞外区,大小约850 bp,目的基因成功克隆至pMD19-T Simple载体,并筛选序列正确的阳性重组质粒。进一步研究证实,SLA-3-HB及SLA-3-LW成功连接到表达载体pET-21a(+),插入片段长度均为831 bp。经诱导后,SLA-3-HB及SLA-3-LW均成功表达,表达蛋白大小为31 ku,相对表达含量达到40%。本研究成功构建了中国地方品系荷包猪及莱芜黑猪SLA-3原核表达载体,为进一步研究其结构和功能奠定基础。     

烟台黑猪SLA-2-YTH原核表达载体的构建及表达

为构建烟台黑猪SLA-2-YTH基因原核表达载体,本研究设计引物PCR扩增SLA-2-YTH胞外区,将其克隆至pMD 19-T Simple 载体,筛选阳性克隆。阳性克隆经酶切后,进一步与表达载体pET-28a(+)连接,转化BL21（Rosseta）感受态细胞并进行诱导表达,SDS-PAGE检测蛋白表达情况。结果显示,SLA-2-YTH胞外区亚克隆大小为834 bp,酶切鉴定证实其成功插入pET-28a（+）表达载体。SDS-PAGE结果显示,SLA-2-YTH基因导入宿主菌后成功表达,蛋白大小约31.0 ku,与预期结果相符,优化后蛋白相对表达量达25%以上。本研究成功构建了烟台黑猪SLA-2-YTH原核表达载体,获得了表达蛋白,为今后进一步的结构和功能研究奠定基础。     

托佩克猪SLA-2基因克隆与表达

为提高托佩克猪SLA-2-TPK基因胞外区在pET-21a的表达量,对其5′端进行密码子优化,并设计表达引物,PCR扩增SLA-2-TPK胞外区,然后克隆入pMD○R19-T Simple Vector,经酶切鉴定后连接至pET-21a载体,转化BL21(Rosseta)进行诱导表达,SDS-PAGE检测目的蛋白。PCR结果显示,SLA-2-TPKe大小约为850bp,并成功克隆入pMD○R19-T Simple Vector,双酶切后大小为834bp。酶切后的SLA-2-TPKe成功与pET-21a链接,重组菌经诱导后目的蛋白大小为30.9ku,与密码子优化前重组菌相比,目的蛋白相对表达含量提高约40%。研究证明,密码子优化可明显提高蛋白的表达量,为进行其他蛋白表达研究提供了参考。     

小尾寒羊β2微球蛋白的表达及其二级结构预测分析

为研究小尾寒羊绵羊白细胞抗原Ⅰ（Ovis aries leukocyte antigen classⅠ,OLAⅠ）轻链四聚体前体链β₂微球蛋白（β₂-microglobulin,β₂m）的结构和功能,根据GenBank中公布的绵羊β₂m基因设计特异性引物,提取小尾寒羊全血中的RNA并运用RT-PCR方法扩增绵羊β₂m基因,将扩增的绵羊β₂m基因克隆到pMD18-T载体,筛选出阳性克隆菌pMD18T-OLAⅠ-β₂m,经双酶切后与表达载体pET-28a（+）连接,再转化大肠杆菌BL21（DE3）感受态细胞中构建pET-28a（+）-OLAⅠ-β₂m重组表达菌,经IPTG诱导表达,SDS-PAGE检测目的蛋白大小及表达情况;运用SOMPA在线软件预测OLAⅠ-β₂m蛋白的二级结构。PCR扩增结果显示,目的基因大小为357 bp,与理论值相符,扩增片段成功克隆到pMD18-T载体,经EcoR Ⅰ和Hind Ⅲ双酶切筛选及测序,成功获得阳性克隆菌株pMD18-T-OLAⅠ-β₂m,插入的目的片段大小为357 bp。阳性克隆菌株与表达载体pET-28a（+）经EcoR Ⅰ和Hind Ⅲ双酶切后连接,转化大肠杆菌BL21（DE3）感受态细胞后获得pET-28a（+）-OLAⅠ-β₂m重组表达菌,经IPTG诱导表达,Western blotting检测目的蛋白大小为17.3 ku,目的蛋白在大肠杆菌中主要以包涵体的形式表达,经洗涤、变性、纯化、初步获得SDS-PAGE纯化的OLAⅠ-β₂m蛋白;PORTER在线软件预测OLAⅠ-β₂m蛋白的二级结构元件α-螺旋（Hh）、β-折叠（Ee）和无规则卷曲（Cc）分别占22.03%、22.03%和55.93%。本研究成功构建了小尾寒羊β₂m基因的pET-28a（+）重组表达体系,运用SOMPA在线软件预测OLAⅠ-β₂m的二级结构,为下一步绵羊OLAⅠ类分子四聚体的构建奠定基础。     

O型口蹄疫病毒VP1基因在2种原核表达载体上的表达

设计1对引物将口蹄疫病毒(foot and mouth disease virus,FMDV)VP1基因克隆到T载体上,通过NdeⅠ和Xho Ⅰ双酶切VP1基因和pET-28(a),在T4连接酶的作用下构建重组表达载体pET-28a-VP1;然后用EcoR Ⅰ和Xho Ⅰ双酶切VP1基因与pET-41(a),在T4连接酶的作用下构建重组表达载体pET-41a-VP1;然后将这2个新构建的载体分别通过热冲击法转化BL21(DE3),37℃不同IPTG浓度和32℃、0.01 mmol/L IPTG诱导表达,SDS-PAGE结果显示pET-28a-VP1的表达量高于pET-41a-VP1,在DTT和乙醇的作用下,二者均没有可溶性蛋白出现.     

小尾寒羊β_2微球蛋白的表达及其二级结构预测分析

为研究小尾寒羊绵羊白细胞抗原Ⅰ(Ovis aries leukocyte antigen classⅠ,OLAⅠ)轻链四聚体前体链β_2微球蛋白(β_2-microglobulin,β_2m)的结构和功能,根据GenBank中公布的绵羊β_2m基因设计特异性引物,提取小尾寒羊全血中的RNA并运用RT-PCR方法扩增绵羊β_2m基因,将扩增的绵羊β_2m基因克隆到pMD18-T载体,筛选出阳性克隆菌pMD18T-OLAⅠ-β_2m,经双酶切后与表达载体pET-28a(+)连接,再转化大肠杆菌BL21(DE3)感受态细胞中构建pET-28a(+)-OLAⅠ-β_2m重组表达菌,经IPTG诱导表达,SDS-PAGE检测目的蛋白大小及表达情况;运用SOMPA在线软件预测OLAⅠ-β_2m蛋白的二级结构。PCR扩增结果显示,目的基因大小为357 bp,与理论值相符,扩增片段成功克隆到pMD18-T载体,经EcoRⅠ和HindⅢ双酶切筛选及测序,成功获得阳性克隆菌株pMD18-T-OLAⅠ-β_2m,插入的目的片段大小为357 bp。阳性克隆菌株与表达载体pET-28a(+)经EcoRⅠ和HindⅢ双酶切后连接,转化大肠杆菌BL21(DE3)感受态细胞后获得pET-28a(+)-OLAⅠ-β_2m重组表达菌,经IPTG诱导表达,Western blotting检测目的蛋白大小为17.3 ku,目的蛋白在大肠杆菌中主要以包涵体的形式表达,经洗涤、变性、纯化、初步获得SDS-PAGE纯化的OLAⅠ-β_2m蛋白;PORTER在线软件预测OLAⅠ-β_2m蛋白的二级结构元件α-螺旋(Hh)、β-折叠(Ee)和无规则卷曲(Cc)分别占22.03%、22.03%和55.93%。本研究成功构建了小尾寒羊β_2m基因的pET-28a(+)重组表达体系,运用SOMPA在线软件预测OLAⅠ-β_2m的二级结构,为下一步绵羊OLAⅠ类分子四聚体的构建奠定基础。     

猪SLA—DRB基因的克隆及在大肠杆菌的表达

试验旨在通过基因工程方法获得重组猪SLA-DRB蛋白。根据基因库猪SLA-DRB基因序列设计合成特异性引物,引物两端分别加上BamHⅠ和XhoⅠ酶切位点及保护碱基;提取长白猪肠系淋巴结RNA,用RT-PCR的方法扩增猪SLA-DRB基因cDNA,并将其克隆到pMD18-T载体上,测序后经BamHⅠ和XhoⅠ双酶切后克隆到pET-32a( )中,构建原核表达载体pET-32a-DRB;将重组表达质粒转化至宿主菌Rosseta中,诱导表达。结果表明,成功扩增出猪SLA-DRB基因cD-NA,经DNA序列测定,所得基因与国外报道的序列99.9%相同;成功构建原核表达载体pET-32a-DRB;经IPTG诱导,表达出了6×His-DRB融合蛋白,对表达的蛋白用SDS-PAGE电泳分析,得到了约50kDa左右蛋白,与预期大小相符,表明猪SLA-DRB基因在大肠杆菌中成功的进行了表达。     

Construction and Expression of the Heavy Chain Tetramer Precursor Chain of SLA-1 in Hebao Pig

To construct tetramer precursor chain of swine lymphocyte antigen 1 (SLA-1) heavy chain in Hebao pig and study its protein expression in the pET-21a (+) vector,the SLA-1 complete genome sequence was referenced with the characteristics of the expression vector,and a pair of primers was designed to integrate the BirA substrate peptide (BSP) sequence at the C-terminus of the SLA-1-HB01 and the SLA-1-HB01-BSP was amplified by PCR. Then,the products were cloned into the pEASY T1 vector and the positive clones of SLA-1-HB01-BSP/pEASY T1 was selected. After the double digestion,the interest of the gene in positive clone was further ligated into the pET-21a (+) expression vector and transformed into E.coli BL21 to construct the recombinant strain of pET-21a (+)/SLA-1-HB01-BSP. After induction with IPTG,the target protein were detected by SDS-PAGE. Finally,the inclusion body of the SLA-1-HB01-BSP was isolated and detected to evaluate its purity. The PCR results showed that the length of SLA-1-HB01-BSP was about 898 bp,which was consistent with the theoretical value. The amplified fragment was successfully cloned into pEASY T1 vector, and the positive clones were identified by Nde Ⅰ and Xho Ⅰ digestion. The size of inserted fragment was 876 bp. The interest of gene was also inserted into pET-21a (+) and transformed into E.coil BL21 successfully. After induction,SDS-PAGE detection results showed that the target protein was 31.4 ku. Further detection showed that the target protein was mainly expressed as inclusion bodies,and the purity of the protein was about 80%. In this study,the recombinant tetramer precursor of SLA-1-HB01 heavy chain was constructed in pET-21a (+) expression line successfully, which would lay a foundation to detect the tetramer of SLA class Ⅰ molecular.     

Construction and Expression of SLA-1 Prokaryotic Expressing Vector in Yorkshire Pig

In order to construct the prokaryotic expressing vector of SLA-1 derived form Yorkshire pig and express the interest of protein, a pair of primers was designed to amplify the extracellular domain of SLA-1 gene from Yorkshire pig (named SLA-1-DYKe) by PCR. Then the PCR product was cloned into pMD^®19-T Simple Vector and transformed into Escherichia coli TOP10. After cleaved by Nde Ⅰ and Xho Ⅰ, the positive clones were selected to be sequenced. Analyzing by biological soft, the fragment from positive clone with correct sequence was inserted into pET-28a (+) and transformed into E.coli BL21(DE3). After induction and expression, the interest of protein was detected by SDS-PAGE. The results showed that the extracellular domain of SLA-1-DYKe was successfully amplified with the fragment length of 837 bp. The interest of SLA-1 gene was successfully cloned into pMD^®19-T Simple Vector and the positive recombinant plasmids with correct sequences were obtained. The SLA-1-DYKe from positive recombinant plasmids was further inserted into pET-28a(+). After transformed into E.coli BL21(DE3) and induction, the SLA-1-DYKe was successfully expressed. The molecular weight of the protein was about 34 ku. It was concluded that the prokaryotic expressing vector of SLA-1 was constructed successfully from Yorkshire pigs and then the expressed protein was obtained, which would lay a base for studying on the structure and function of SLA-1 from Yorkshire pig in the future.     

家蚕G蛋白γ1亚基(BmGγ1)的克隆及其谷胱甘肽硫转移酶融合蛋白的表达与纯化

异三元G蛋白是真核细胞感知外界信号后将信号传递到胞内的重要分子,参与生物体广泛的信号转导。为了研究家蚕体内G蛋白的生理功能及其作用机制,运用生物信息学方法预测了家蚕G蛋白γ1亚基(Gγ1)的序列,设计引物验证预测序列后,克隆了家蚕Gγ1的序列,再通过酶切克隆至表达载体pET-41b(+)后,导入E.coliBL21宿主菌中,经异丙基β-D-硫代半乳糖苷(IPTG)诱导表达重组谷胱甘肽硫转移酶(glutathione s-transferase,GST)融合蛋白,并亲和层析纯化表达产物。家蚕Gγ1重组GST融合蛋白经SDS-PAGE电泳和Western blot分析,在分子质量约36 kD处出现特异性蛋白条带,重组蛋白经GST亲和层析柱纯化后,得到了高纯度的融合蛋白,说明已经成功克隆到家蚕Gγ1基因,并在E.coliBL21中高效表达。     

猪脑心肌炎病毒GXLC株VP1基因的原核表达及鉴定

根据猪脑心肌炎病毒(EMCV)GXLC株的基因组序列设计一对特异性引物,应用RT-PCR方法扩增EMCV VPl基因目的片段,将其克隆至原核表达载体pET-32a(+),构建了EMCV VPl基因重组表达质粒pET-32a-VP1.将pET-32a-VP1转化BL21(DE3)株感受态细胞,并用IPTG进行诱导表达.结...     

奶牛载脂蛋白B100基因克隆及原核表达

本研究根据GenBank已收录奶牛ApoB100基因序列,设计特异性引物获得目的基因。经pGM-T载体克隆,对重组质粒进行BamHⅠ和EcoRⅠ双酶切鉴定并测序,序列同源性达100%;将目的基因连接到pET-28a表达载体中,提取质粒,转化到Rosetta(DE3)中,筛选的阳性克隆经IPTG诱导。SDS-PAGE初步分析表明,成功获得分子质量为35 ku的蛋白质;Western blotting结果呈阳性,表明通过本试验成功获得了目的蛋白。