Expression of the Fused Pili and Ovine IL-2 Gene in Pseudomonasaeruginus

用改进的 Mg Cl2 法制备宿主菌株PAK/ 2 pfs感受态细胞 ;由 JM10 5工程菌中提取纤毛蛋白与绵羊白细胞介素 2 ( Pilin-IL-2 )融合基因表达载体 ,转化宿主细胞 PAK/ 2 pfs,筛选出具有 Pilin-IL-2融合基因表达载体的阳性克隆菌株 ;在营养肉汤中进行 Pilin-IL-2融合基因的表达。培养 16～ 18h后 ,离心 ,向上清液中加入饱和 ( NH4) 2 SO4提取 Pilin-IL-2融合基因表达蛋白。用羊抗兔 E型节瘤拟杆菌抗血清与提取的融合蛋白进行对流免疫电泳 ,证明该融合蛋白具有特异性The pmPilin IL-2 was transformed into the host competent cell PAK/rpfs and the recombinant pmPilin IL-2 was expressed in the supernatant of the culture of the transformed cell PAK/rpfs.The recombinant pmPilin IL-2 was purified by (NH_4)_2_SO_4 from the supernatant of the culture of the transformed PAK/rpfs. The specific reaction of the recombinant pmPilin IL-2 and antiserum of B.nodosus serotype E pilin was demonstrated by cross electrophoresis.国家科技部研究所技术开发研究专项资金项目[国科财字(1999) 592号

Protective Antigens from Virulent Isolates of Fusobacterium necrophorum FN(A)

通过胰蛋白酶裂解坏死梭杆菌FN(A)型毒力菌株菌体,分别以菌体裂解物上清和沉淀作为抗原免疫家兔制备血清。利用SDS-PAGE/Western-blot技术在菌体中筛选出4种具有免疫原性的组分,通过免疫后的攻毒试验,筛选出1种具有免疫保护性的抗原。抗原分离纯化后,进行N端氨基酸序列测定、免疫试验及生物学特性研究,据试验结果可初步判定该抗原为溶血素类似物或一种新发现的抗原物质。该抗原不仅能使机体产生保护性免疫反应,而且不同菌株中此种抗原间可产生明显的交叉免疫。A virulent isolate of the bacterium FN(A) was lysed with trypsin.Supernatants and precipitates of the bacterial lysates were used to prepare anti-FN serum by immunizing rabbits.Four immunogenic components were isolated using SDS-PAGE/Western-blot.Immunized rabbits were challenged,and one protective antigen was screened.After antigen isolation and purification,the N-terminal amino acid sequence was determined and an immunological test and hemolysis test were performed.Results suggested that this antigen is either a hemolysin analogue or a new antigenic substance.The antigen was able to induce a protective immune response as well as generate a marked cross-immune reaction among different bacterial strains.国家科技部奶牛专项(2002BA518A04);; 中国农业科学院特产研究所科研基金项目(tcs-20041

Expression and Identification of the fused Dichelobacter Pili and Ovine Interleukin 2 Gene in Pseudomonasaeruginus

将前期工作中筛选出的含有腐蹄病节瘤拟杆菌纤毛蛋白与绵羊白细胞介素-2(Pilin-IL-2)融合基因表达载体的PAK/2pfs工程菌[1]接种于营养肉汤,40℃培养16～18h,离心,向上清液中加入饱和(NH4)2SO4提取Pilin-IL-2融合基因表达蛋白。用羊抗兔E型节瘤拟杆菌抗血清与提取的融合蛋白进行对流免疫电泳,证明该融合蛋白具有特异性,且有较高的表达量。经SDS-PAGE电泳和Westernblot确证其为Pilin-IL-2融合蛋白。The pmPilinIL2 was transformed into the host competent cell PAK/rpfs and the recombinant pmPilinIL2 was expressed in the supernatant of the coltures of the transformant cell PAK/rpfs.The recombinant pmPilinIL2 was purified by (NH_4)_2SO_4 from the supernatant of the colture of the transformed PAK/rpfs. The specificreaction of the recombinant pmPilinIL2 and antiserum of D.nodosus serotype E pilin was demonstrated by cross electrophoresis.33000 Expression protein was found by SDS-PAGE.Through Western-biotting,the expression protein was recognized by antiserum of D.nodosus serotype E pilin.国家科技部研究所技术开发研究专项资金项目[国科财字(1999)592号

Cloning and Construction Expressing Vector of Ovine Interleukin-2 Gene

从绵羊血中分离白细胞,提取绵羊白细胞RNA,利用RT-PCR、PCR扩增绵羊白细胞介素2(OvineIL-2)基因。将OvineIL-2PCR产物与TE载体定向连接,用SaLI和BamHI双酶切重组TE,利用低溶点胶回收500bp大小的片段;将其补平后与PPSD质粒连接,用EcoRI酶切鉴定,找出正向连接重组质粒。用BamHI酶切重组PPSD质粒,低熔点胶回收2500bp大小的片段;将其与经BamHI酶切去磷酸化的PME290表达质粒连接,筛选出阳性重组PME290,即为OvineIL-2基因表达载体。IL2 is known to be as an adjuvant in many areas. The ovine IL2 gene with synthesized recognition sequence at 5` and 3` end respectivecy was amplified and cloned from the peripheral blood of ovine. Ovine IL2 gene was amplified with ovine RNA as template by RTPCR and PCR. The amplified fragment was subjected to restriction digest and coined into the TE and PPSD vector. An expression plasmid was constructed by cloning the IL2 gene into PME290. The PME290SDIL2 was transformed into the host competent cell PAK/2pfs and the recombinant IL2 was expressed in the supernatant of the cultures of the transformant cell PAK/2pfs.国家科技部科研院所技术开发专项资金项目(国科发财字(1999)592号

Cloning and Expression of D.nodosus Serotype C Pili Gene in Pseudomonas

用 PCR从腐蹄病 C型节瘤拟杆菌扩增出具有免疫保护性抗原 0 .85 kb纤毛蛋白基因 (pili基因 ) ,并构建了该基因的表达载体。转化宿主细胞 PAK/2 pfs,在营养肉汤中进行 pili基因的表达。培养 18～ 2 4h后 ,收集培养液上清 ,加入 0 .1mol/L Mg Cl2 ,离心提取重组纤毛蛋白。用羊抗兔 C型节瘤拟杆菌抗血清与提取的纤毛蛋白进行对流免疫电泳试验 ,结果表达的重组纤毛蛋白有特异性 ;用 SDS- PAGE和 Western- blot证明表达的蛋白是 C型节瘤拟杆菌纤毛蛋白。The pili gene that dominates the main protective immunogen was amplified and cloned from D.nodosus serorype C by PCR.An expression plasmid was constructed by cloning the pili gene into PME290.The plasmid harbouring the pili sequence was designated PME290-pili.The PME290-pili was transformed into the host competent cell PAK/2pfs and the recombinant pili was expressed in the supernatant of the cultures of the transformant cell PAK/2pfs.The recombinant pili was purified by MgCl 2 from the supernatant of the culture of the transformed PAK/2pfs.The specific reacion of the recombinant pili and antiserum of D.nodosus serotype C pili was demonstrated by cross electrophoresis.The recombinant pili was expressed at high level in PAK/2pfs.国家科技部研究所技术开发研究专项基金项目〔国科财字 (1999) 5 92号

杆状病毒表达EV71病毒样颗粒的装配、制备纯化与鉴定

将EV71P1和3CD基因片段克隆入同一杆状病毒穿梭质粒Bacmid中,构建出重组杆状病毒表达质粒Bac-mid-P1-3CD;脂质体介导其转染Sf9昆虫细胞获得共表达P1和3CD的重组杆状病毒(AcMNPV-P1-3CD)。用IFA和Western-blot法对表达产物进行鉴定和分析。电镜结果显示P1经3CD切割装配成了大小约为27nm的类球形颗粒(即EV71VLPs)。进一步分析影响杆状病毒表达系统的因素以对表达条件进行优化,结果显示MOI值和时间均可影响目的蛋白的表达,其中时间是主要因素。选择优化后条件利用无血清培养基对贴壁Sf9细胞在多层细胞培养器中进行VLPs的大量表达,密度梯度离心法纯化,SDS-PAGE结果可见三条大小约为39kD、34kD和26kD的VP1、VP0和VP3特异性条带。纯化后EV71VLPs颗粒结构完好,为下一步EV71蛋白结构的基础研究和基因工程疫苗的研究奠定了基础

甲烷化催化剂及反应机理的研究进展

概述了甲烷化反应在工业生产中的应用,重点介绍了甲烷化催化剂中活性组分、载体、助剂的种类及催化剂制备方法、条件对其催化性能的影响;分析了甲烷化催化剂失活的原因及甲烷化反应的机理,指出床层飞温和积碳是造成催化剂失活的主要因素,必须从甲烷化催化剂和工艺技术两方面予以改进;并对甲烷化催化剂研究进行了展望,提出高比表面复合载体的研制、稀土元素的添加、新型耐硫、高热稳定性甲烷化催化剂的开发及流化床甲烷化工艺技术的改进是甲烷化研究的主要方向

甲烷化催化剂及反应机理的研究进展

概述了甲烷化反应在工业生产中的应用,重点介绍了甲烷化催化剂中活性组分、载体、助剂的种类及催化剂制备方法、条件对其催化性能的影响;分析了甲烷化催化剂失活的原因及甲烷化反应的机理,指出床层飞温和积碳是造成催化剂失活的主要因素,必须从甲烷化催化剂和工艺技术两方面予以改进;并对甲烷化催化剂研究进行了展望,提出高比表面复合载体的研制、稀土元素的添加、新型耐硫、高热稳定性甲烷化催化剂的开发及流化床甲烷化工艺技术的改进是甲烷化研究的主要方向

甲烷化催化剂及反应机理的研究进展

概述了甲烷化反应在工业生产中的应用,重点介绍了甲烷化催化剂中活性组分、载体、助剂的种类及催化剂制备方法、条件对其催化性能的影响;分析了甲烷化催化剂失活的原因及甲烷化反应的机理,指出床层飞温和积碳是造成催化剂失活的主要因素,必须从甲烷化催化剂和工艺技术两方面予以改进;并对甲烷化催化剂研究进行了展望,提出高比表面复合载体的研制、稀土元素的添加、新型耐硫、高热稳定性甲烷化催化剂的开发及流化床甲烷化工艺技术的改进是甲烷化研究的主要方向