基于重组猪囊虫18 ku抗原的间接ELISA方法的建立

利用反转录聚合酶链式反应(RT-PCR)从猪囊尾蚴中克隆到18 ku蛋白基因,将扩增产物与pGEM-T Easy载体连接后测序分析.将目的基因亚克隆至表达载体,构建重组质粒pGEX-CE18,经转化大肠杆菌BL21 (DE3)后诱导表达,用SDS-PAGE和Western-blot分析表达产物.表达的目的蛋白纯化后作抗原建立检测猪囊虫抗体的重组蛋白间接ELISA方法.结果表明,18 ku蛋白基因在大肠杆菌中成功表达,表达产物约为35 ku的融合蛋白,并能被猪囊虫感染血清识别.经薄层扫描分析,表达量占菌体蛋白总量的28%.与商品化ELISA试剂盒平行检测178份阳性血清样品,二者的符合率为98.83%,说明建立的重组蛋白ELISA方法可用于猪囊虫病的诊断.     

边缘无浆体MSP5蛋白基因的克隆及原核表达

参照已发表的边缘无浆体主要表面蛋白5（MSP5）基因的核苷酸序列,设计了1对特异性引物,以边缘无浆体基因组DNA为模板,采用PCR技术扩增获得了MSP5蛋白基因.将其克隆到pGEM-T Easy载体,并进行测序分析.结果表明,克隆的MSP5基因与GenBank上登录的Florida株MSP5蛋白基因的序列同源性达98.6%,编码氨基酸的同源性为99.0%,并且该序列包含有完整的开放阅读框,大小为633 bp.将该基因亚克隆入原核表达载体pGEX-4T-1,构建了重组原核表达载体.将其转化到DH5a宿主菌中,用IPTG进行诱导表达,实现了融合表达,表达产物的分子质量为45 ku.Western-blot分析表明,此表达产物能够被抗边缘无浆体阳性血清所识别.     

猪IL-2基因在毕赤酵母中的高效表达及生物活性分析

本研究根据密码子偏爱原则设计猪IL-2引物,克隆其基因后构建pPIC9k-IL-2重组表达载体,线性化后电转化毕赤酵母GS115,获得优化密码子的重组酵母转化子;再用G418抗性梯度法筛选得到多拷贝重组菌株,不同条件下进行甲醇诱导表达。结果表明:经PCR鉴定IL-2基因已整合到酵母基因组中。表达产物经SDS-PAGE电泳分析发现相对分子质量为16、20ku两条目的蛋白表达带,脱糖基化分析表明目的蛋白得到了适度的糖基化修饰,Western blotting分析显示表达的蛋白具有良好的免疫反应性,分子筛纯化可以获得纯度为95%的蛋白质,淋巴细胞增殖活性分析表明所得的蛋白质具有促淋巴细胞增殖的活性。毕赤酵母表达系统可以高效地表达具有生物活性的重组猪IL-2蛋白分子。     

荣昌猪白细胞介素-2受体γ链基因克隆、表达与鉴定

运用RT-PCR技术从由刀豆蛋白(ConA)诱导培养的荣昌猪外周血单核淋巴细胞(PBMC)扩增出猪白细胞介素-2受体γ链基因的完整开放阅读框(ORF),长约1 107 bp,编码由368个氨基酸残基组成的相对分子质量为41 780的蛋白多肽.荣昌猪IL-2Rγ与人、猕猴、牛、犬、鼠在氨基酸水平上的同源性分别为81.6%,80.8%,85.1%,83.2%和68.8%.运用PCR技术从含荣昌猪IL-2Rγ,开放阅读框序列质粒中扩增其成熟蛋白编码基因,共1 020 bp.将其定向克隆于原核表达载体pET-32a(+)后在E.coli BL21中诱导表达,SDS-PAGE结果显示表达的融合蛋白约为52 000,重组蛋白以包涵体的形式表达,表达产物约占茵体总蛋白的37.6%;Western-blotting分析表明,在相对分子质量52 000处有一奈特异性的带;对γ诱导重组茵进行转录检测得到约1 020 bp的特异性片段.     

猪血管内皮细胞中分子伴侣Jiv9O基因的克隆与原核表达

克隆到猪Jiv90基因,构建原核表达载体并在大肠埃希菌中表达.从猪血管内皮细胞中克隆到Jiv90基因,将其克隆到pET-32a载体上,构建出重组表达载体,经IPTG诱导表达.结果表明,本试验成功克隆了大小为693 bp的基因,重组质载体pET-32a-Jiv90所表达的蛋白在大肠埃希菌中以包涵体的形式存在,表达产物经S...     

猪白细胞介素15基因的克隆及在大肠杆菌中的表达

本研究通过RT-PCR方法从用ConA刺激的猪外周血淋巴细胞中扩增出猪IL-15(pIL-15)编码序列的cD-NA,将其克隆至T载体pMD18-T后,序列测定表明pIL-15基因长度为489 bp,编码162个氨基酸。进一步通过PCR方法获得缺失其N端48 aa的信号肽序列的成熟pIL-15蛋白基因,将其亚克隆到原核表达载体pET-28a的6×His下游,构建重组表达质粒pET-pIL15,转化大肠杆菌BL21(DE3),IPTG诱导,重组菌体裂解物经SDS-PAGE可检测到分子量约为15 ku的重组蛋白,其表达量占总菌体蛋白量的17.5%。Western blot试验也证实表达的重组融合蛋白6×His-pIL15能够很好地与抗6×His单抗发生反应。猪IL-15基因的克隆、表达为进一步研究其功能和应用奠定了基础。     

猪γ干扰素的体外表达及其多克隆抗血清的制备

从健康仔猪前腔静脉无菌采血,分离外周血单个核细胞,提取细胞总RNA,用猪γ干扰素(IFN-γ)基因特异性引物通过RT-PCR扩增猪IFN-γ的全长cDNA序列,将其克隆到pMD18-T载体中,再亚克隆到pUC18和表达载体pET-30a中,经测序发现其序列与GenBank报道的IFN-γ基因序列基本一致.将重组质粒pET-30a-IFN-γ转化大肠杆菌BL21,于37℃经IPTG诱导表达,IFN-γ基因获得表达,表达产物以包涵体形式存在,占菌体总蛋白的39%.经SDS-PAGE及Westernblot分析表明,表达的融合蛋白分子量约为25 ku.将包涵体用8 mol/L脲变性,用ProBondTM Purification Systerm纯化,经透析复性,得到纯化的目的蛋白,含量可达0.3 mg/mL.将复性蛋白免疫新西兰白兔三次,制备了高滴度的猪IFN-γ抗血清.本实验表达和纯化了猪IFN-γ,并制备兔抗猪IFN-γ的抗血清,为下一阶段关于猪γ干扰素重组蛋白其应用奠定了基础.     

绵羊黏膜趋化因子CCL28基因的克隆、序列分析与原核表达

利用同源序列克隆原理设计1对克隆引物,从绵羊结肠黏膜组织提取mRNA,通过RT-PCR获得CCL28基因,将PCR产物克隆、测序,并进行序列分析;再根据克隆的序列设计1对表达引物,用PCR法从重组克隆载体中扩增出含BamHⅠ/XhoⅠ酶切位点的CCL28片段,双酶切后亚克隆至pGEX-4T-1载体,构建重组原核表达载体pGEX-CCL28,转化宿主菌E.coliBL21（DE3）,经IPTG诱导进行表达,SDS-PAGE鉴定。克隆的绵羊CCL28基因包含完整的开放阅读框,克隆片段长444 bp,ORF为387 bp,编码129个氨基酸,与人、小鼠、猪、牛该基因的同源性分别为76.4%、61.4%、84.3%和92.9%,推测的氨基酸序列与人、小鼠、猪、牛的同源性分别为76%、63%、84%和93%,表达的融合蛋白分子量约为39 ku,并以包涵体形式存在。为下一步研究绵羊CCL28的生物学功能奠定基础。     

猪IL-6基因在毕赤酵母中的表达及生物活性鉴定

为了快捷、大量地表达猪白细胞介素-6(IL-6)蛋白,试验采用PCR的方法,以含猪IL-6全基因的重组质粒p MD19-T-IL-6为模板扩增猪IL-6成熟蛋白基因,构建真核重组表达载体p PICZαΑ-IL-6,转化毕赤酵母野生株X-33进行分泌表达,并进行表达蛋白的初步处理和活性鉴定。结果表明:SDS-PAGE可检测到预期的蛋白条带,诱导表达24,48,72,96 h后,分泌IL-6蛋白分别占总蛋白的76.1%、83.4%、84.6%、89.6%。第96小时时,猪IL-6表达产物浓度约为0.94 g/L。Western-blot试验证实,重组猪IL-6蛋白与兔抗猪IL-6多克隆抗体发生特异性反应。猪IL-6表达产物浓度为10.5 mg/L时对猪外周血淋巴细胞具有促进增殖作用,浓度为105.0 mg/L时则具有抑制作用。     

荣昌猪白细胞介素15成熟肽基因的克隆、表达及其表达产物活性检测

运用RT—PCR技术从由刀豆蛋白（ConA）诱导培养的荣昌猪外周血淋巴细胞（PBMC）扩增出猪白细胞介素15（pIL-15）完整开放阅读框（ORF），共489bp，编码162aa。与已公布的2个猪IL-15基因核苷酸同源性均为99．4％。分子进化分析表明其与人及哺乳动物IL-15基因的进化关系较近，而与鸡的IL-15基因进化距离较远。运用PCR技术从含荣昌猪IL-15开放阅读框序列质粒中扩增其成熟蛋白编码基因，共345bp。将其定向克隆于原核表达载体pET-32a（＋）后在E．coli BL21中诱导表达，SDS-PAGE结果显示表达的融合蛋白约为34ku，重组蛋白以包涵体形式表达，表达产物约占菌体总蛋白的38．7％；Western blot分析表明，在相对分子质量34ku处有一条特异性带；对诱导重组菌进行转录检测得到约356bp的特异性片段。MTT法试验证实，表达产物初步纯化、透析复性后，可明显增强淋巴细胞的增殖。荣昌猪IL-15成熟肽成功在体外表达，获得的高效表达的产物具有一定的生物学活性。     

Cloning,Sequence Analysis and Prokaryotic Expression of Mink Interleukin-4 Gene

In order to obtain high purity of interleukin 4 (IL-4) protein,the specific primers were designed and synthesized according to the mink IL-4 gene sequence in GenBank. The IL-4 gene was amplified by RT-PCR using total RNA of the lymphocyte isolated from the peripheral blood that induced by phytohemagglutinin (PHA). The length of IL-4 gene sequence was 399 bp which encoded 132 amino acids. Phylogenetic analysis revealed that the amino acid sequences homology between mink and ferret was 99.2%,90.0% with Ailuropoda melanoleuca and Canis familiaris. Prokaryotic expression vector pProEX-HTb-IL-4 was constructed and induced by IPTG. The recombinant protein of 15 ku was isolated by SDS-PAGE and detected by Western blotting. Highly purified recombinant protein of mink IL-4 was obtained by His-Trap HP affinity columns method. This research laid the foundation for the further studies on the biological function of mink IL-4 gene.     

水貂白细胞介素-4基因的克隆、序列分析与原核表达

为了获得高纯度的白细胞介素-4（IL-4）蛋白,对分离得到的水貂外周血淋巴细胞经植物血凝素（PHA）诱导后,提取淋巴细胞总RNA,根据GenBank中登录的雪貂IL-4基因序列,设计并合成特异性引物,通过RT-PCR扩增获得了水貂IL-4基因序列全长399 bp,编码132个氨基酸,与雪貂氨基酸序列同源性高达99.2%,与熊猫、犬同源性均为90.0%。将编码成熟蛋白基因构建到原核表达载体pProEX-HTb,IPTG诱导后,SDS-PAGE及Western blotting结果显示,IL-4表达产物为15 ku的包涵体蛋白。通过His-Trap HP亲和层析预装柱变性、复性洗脱可获得高纯度的重组IL-4蛋白。本试验为水貂IL-4基因的进一步研究奠定了基础。     

猪白细胞介素-4重组蛋白的纯化及其生物学特性分析

用已构建好的重组表达载体pGEX-4T-IL-4转染E.coli,在最适条件下诱导表达,表达产物经SDS-PAGE分析,表达出38 ku的融合蛋白,表达量占菌体总蛋白的25%,表达产物主要以包涵体的形式存在;对表达产物经变性、复性及初步纯化后再结合饱和硫酸铵沉淀和Sephadex-G100凝胶层析柱进一步纯化,所得蛋白的纯度约在90%以上。在体外利用MTT法检测其生物学特性,结果表明其具有较好的生物学活性,本试验为获得猪IL-4重组蛋白奠定了基础。     

猪轮状病毒地方株JL94株VP4基因的克隆与序列分析

根据GenBank已发表的猪轮状病毒VP4基因保守序列,利用Oligo4.1软件设计一对引物扩增猪轮状病毒地方株JL94株VP4全基因序列;并将扩增产物与pMD-18T载体连接,经鉴定后将重组质粒进行测序并与国外分离株CRW-8株、BEN-307株进行核苷酸和氨基酸序列同源性比较分析.结果表明:JL94株和CRW-8株、BEN-307株VP4全基因片段核苷酸同源性和氨基酸同源性分别为96.98%和98.05%,说明JL94株与CRW-8株、BEN-307株属于同一VP4血清型.本研究为进一步研制诊断性抗原和病毒重组活载体疫苗奠定了基础.     

Molecular cloning, chromosomal location, and biological activity of porcine interleukin-21

A pig interleukin-21 (IL-21) cDNA was successfully cloned and sequenced from porcine peripheral blood lymphocytes (PBL) stimulated with 10 microg/ml concanavalin A (ConA), 10 microg/ml phytohemagglutinin P (PHA), 50 ng/ml phorbol 12-myristate 13-acetate (PMA), and 0.5 microg/ml anti-porcine CD3 antibody for 48 hr. The open reading frame of the porcine IL-21 cDNA is 459 base pairs in length and encodes 152 amino acids. The predicted amino acid sequence of the porcine IL-21 shows 86.2%, 77.7%, and 58.4% identity to the bovine, human, and murine IL-21, respectively. The porcine IL-21 gene was mapped to porcine chromosome 8 (8q22-->q23) by means of fluorescence in situ hybridization and radiation hybrid mapping, where the porcine IL-2 gene had been mapped nearby. The recombinant porcine mature IL-21 expressed by E. coli induced dose-dependent proliferation and IFN-gamma production from a human NK cell line, NK0. The porcine IL-21 identified in this study will be helpful for the enhancement of innate immune responses of pigs.     

Quantitative Analysis of Interleukin-6 Expression in Porcine Spleen Cells and Alveolar Macrophages using Real-Time PCR

Interleukin-6 (IL-6), a multifocal cytokine produced by lymphoid and non-lymphoid cells, regulates immune responses, acute-phase reactions against bacterial infections, and haematopoiesis. After cloning and sequencing of porcine IL-6, the expression pattern of porcine IL-6 mRNA was evaluated through real-time RT-PCR using porcine immune cells (spleen cells and alveolar macrophages) following stimulation with LPS. The sequence has been reported to GenBank with Accession no. AF 518322. The nucleotide sequence was different at the 89th and 205th positions in comparison with M80258, but only at the 205th with M86722. Comparison of porcine IL-6, Accession no. AF 518322, with IL-6 of human, canine, ovine, and mouse showed homologies of 78%, 81%, 82% and 73% in nucleotide sequence and 42%, 69%, 61% and 42% in amino acids. Expression of IL-6 mRNA was induced by stimulation with LPS. IL-6 mRNA expression in alveolar macrophages peaked at 2 h and decreased sharply to control levels at 4 h, whereas it peaked at 14 h and decreased at 24 h in spleen cells after stimulation with LPS (1 microg/ml). These results suggest that IL-6 mRNA expression in porcine immune cells is cell-type specific and the results of this study could be used as the basis for research on the porcine immune system.     

猪白介素4在CHO-K1细胞中的重组表达及生物学活性分析

将猪白介素4(Porcine interleukin4,PIL-4)基因亚克隆到真核表达载体pEGFP-N1中,构建重组表达质粒pEGFP-PIL-4,利用脂质体法转染CHO-K1细胞,采用荧光显微镜实时观察、RT-PCR和Western-blot分别检测CHO细胞转录表达目的分子的情况,通过MTT法检测所表达蛋白的生物学活性。结果在将重组质粒转染CHO-K1细胞中24、48 h后的均观察到绿色荧光;经G418筛选14~20 d后转染的细胞形成了阳性细胞集落,用RT-PCR扩增出约339 bp的目的基因片段;经Western-blot检测到约为39 000的特异蛋白分子条带,并证明在转染重组质粒的细胞中表达了高生物活性的重组蛋白。     

Cloning and Eukaryotic Expression Plasmid Construction of Porcine Interleukin-18 Gene

To obtain recombinant eukaryotic expression plasmid of porcine interleukin-18(IL-18), the whole gene of porcine IL-18 gene was amplified from porcine spleen, lung and lymph nodes by RT-PCR and cloned into eukaryotic expression vector pZJ-1. The recombinant expression plasmid pZJ-IL-18 were identified by enzyme digestion and sequencing analysis, and was transfected into 293T cells.The expression of IL-18 was detected by Real-time PCR and Western blotting in both gene and protein levels. The results showed that the eukaryotic expression plasmid of porcine IL-18 was constructed and could express transiently in 293T cells. Western blotting result confirmed that porcine IL-18 polyclonal antibody could react specifically with approximately 17 ku expression products,and indicated that IL-18 could express correctly and be responsive.This study constructed the eukaryotic expression plasmid of porcine IL-18 gene which could express transiently in 293T cells, and laid the foundation for studying function of IL-18.