抗猪囊尾蚴头节特异性单链抗体的构建及克隆

首先对猪囊尾蚴头节特异性单链抗体重链可变区基因(Vh)和轻链可变区基因(Vl)进行酶切鉴定,PCR鉴定以及测序分析,然后采用重叠延伸PCR(SOE PCR)技术,将重链可变区基因和轻链可变区基因,通过linker链连接成Vh-linker-Vl单链抗体基因(ScFv)。将此基因与pMD18-T载体连接,转化感受态大肠杆菌J M109,从而进一步克隆并对重组载体pMD-ScFv进行鉴定。结果:成功构建并克隆了抗猪囊尾蚴头节单链抗体,为重组免疫毒素ScFv-PE40的构建?表达及活性测定奠定了良好的基础。     

猪囊尾蚴特异性单克隆抗体的制备及其识别抗原成分的鉴定

通过SDS-PAGE方法对猪囊尾蚴囊液抗原成分进行了分离鉴定,并分别分离纯化了其中的16 000和10 000特异性蛋白质成分;将16 000和10 000纯化蛋白质成分分别做成佛氏佐剂苗,分组免疫BALB/c小鼠,超免后,无菌取脾脏制备脾细胞,分别与NS0骨髓瘤细胞进行融合,经阳性筛选和特异性鉴定获得2株分泌猪囊尾蚴特异性单抗的杂交瘤细胞,命名为TSCF-1611H12B8和TSCF-1012G5B5。免疫学鉴定结果表明,单抗TSCF-1611H12B8和TSCF-1012G5B5与猪细颈囊尾蚴、旋毛虫、住肉孢子虫和蛔虫抗原间不存在交叉反应;单抗TSCF-1611H12B8识别囊液中16 000和10 000蛋白质抗原条带,而单抗TSCF-1012G5B5识别囊液中的10 000蛋白质条带。     

猪囊尾蚴头节单链抗体-PE40重组载体的构建及表达

从绿脓杆菌ATCC27853菌株中提取基因组,PCR扩增PE40基因,与pMD18-T栽体连接并转化感受态大肠杆菌JM109中,构建重组质粒pMD-PE40;经鉴定正确后将PE40基因插入抗猪囊尾蚴头节单链抗体基因重组质粒pMD-ScFv中,构建重组质粒pMD-ScFv-PE40,经酶切、电泳回收目的片段,并亚克隆到表达栽体pET-28a中,构建重组表达载体pET-ScFv-PE40;鉴定后转化E.coli BL21,IPTG诱导后进行SDS-PAGE和Western blotting验证.结果表明,试验成功构建了重组表达载体pET-ScFv-PE40,目的片段约为1800 bp;Western blotting证明重组蛋白具有一定的反应原性,为重组免疫毒素的制备及动物保护性试验奠定了基础.     

抗猪支原体共同抗原单克隆抗体的制备与鉴定

以猪肺炎支原体（Mycoplasma hyopneumoniae,Mhp)168菌株F332作为抗原，免疫8周龄BALB/c小鼠，利用淋巴细胞杂交瘤技术，获得两株能稳定分泌特异单克隆抗体的杂交瘤细胞株。两株单抗与Mhp和猪鼻支原体（M.hyorhinis,Mh)等反应，而不与鸡支原体，对照血清等反应。结果表明两株单抗可能是针对猪支原体共同抗原的单抗，用SDS-PAGE电泳和Western-blot分析猪支原体的膜蛋白成分。结果显示，猪支原体的共同抗原是80kd和30kd蛋白，两株单抗均与Mhp和Mh的30kd蛋白条带反应，表明这两株单抗特异地针对猪支原体的共同抗原成分30kd蛋白，可以看出，这两株单抗在Mhp的抗原分析，血清学诊断和疫苗质量监测以及猪源支原体污染细胞检测等方面有重要应用价值。     

猪囊尾蚴的细胞培养与染色体制备

本研究采取自然病例的猪囊尾蚴(又称猪囊虫),进行细胞培养,现已传至20代。猪囊尾蚴细胞培养的成功,将对猪囊虫病的免疫防治与低等动物细胞生物学研究,提供了实验材料,并具有一定的理论价值和实践意义。     

猪囊尾蚴头节抗原的制备及间接ELISA方法的建立

采用Sephadex G-200层析技术纯化猪囊尾蚴头节抗原,用纯化抗原包被ELISA板,建立间接ELISA方法。通过各种条件优化,最终确定最佳试验条件为:抗原最适包被量为30mg/L;血清稀释度为1∶800;血清最佳反应时间为60min。结果表明,该方法具有较好的稳定性和重复性,可用于分泌抗猪囊尾蚴头节单克隆抗体杂交瘤细胞株的筛选。     

抗氯霉素单克隆抗体的制备及鉴定

用人工合成的氯霉素-人血清白蛋白(CAP-HSA)作抗原免疫BALB/c小鼠,通过杂交瘤技术建立了1株分泌抗CAP单克隆抗体(McAb)的杂交瘤细胞1F9.经检测,其分泌的抗体亚类为IgG1,杂交瘤染色体数目84～96条,间接ELISA检测细胞培养上清效价为1256,诱生小鼠腹水的抗体效价可达16×105.该细胞连续培养生物学性状稳定,竞争间接酶联免疫吸附试验(ciELISA)显示,其与供试抗生素交叉反应小,表明该单抗具有较大的应用价值.     

猪囊尾蚴头节单链抗体-PE40重组蛋白的表达及抗虫活性分析

将含有抗猪囊尾蚴头节单链抗体基因（ScFv）和绿脓杆菌外毒素基因（PE40）的重组质粒pET28．ScFv-PE40转化到大肠杆菌BL21中,经IPTG诱导,表达产物通过SDS-PAGE和westernblot鉴定,其表达蛋白的相对分子量约为68ku,表达量约占菌体总蛋白的13．8％。将融合蛋白通过金属Ni2^＋亲和层析柱纯化后,目的蛋白纯度在95％以上,可溶性蛋白的回收率约为50％,回收量为5mg／L。本研究所进行的纯化重组蛋白的体外杀伤六钧蚴试验表明,重组蛋白对六钩蚴具有较强的杀灭作用,而且与其剂量呈正相关性。     

抗猪脂肪细胞特异膜蛋白单克隆抗体的制备及生物学特性鉴定

以纯化的猪脂肪细胞特异膜蛋白 (40 0 0 0 )为抗原免疫 BAL B/ c小鼠 ,制备了 2 A3、3 H7和 3 A6等 3株单克隆抗体 ,分别属于 Ig A、Ig M和 Ig G1亚类 ,其最佳稀释倍数分别为 1∶ 1 6 0 0、1∶ 3 2 0 0和 1∶ 6 4 0 0。经 Western blotting鉴定 ,3株单抗均能与 4 0 0 0 0的抗原结合 ,且具有较强的抗原亲和力 ,亲和常数分别为 4 .86× 1 0 8、1 .86× 1 0 9和 2 .4 6× 1 0 9;对皮下脂肪细胞具有很强的特异性 ,经 Western blotting鉴定仅与猪和人脂肪细胞的 4 0 0 0 0膜蛋白发生特异反应 ,而与牛、羊、兔、鸡、鼠等动物的脂肪细胞膜蛋白均无交叉反应 ;能与滇玉、滇昆、滇陆、长撒、长白、DL Y、约长撒和撒坝等品种猪皮下脂肪细胞的 4 0 0 0 0膜蛋白发生特异反应 ,而与皮下脂肪细胞的其他膜蛋白无交叉反应 ;仅与长撒猪皮下脂肪和腹部脂肪细胞的 4 0 0 0 0膜蛋白发生特异反应 ,与心、肝、脾、肺、肾、肌肉、肠系膜脂肪和肾周脂肪细胞膜蛋白均无交叉反应     

抗猪瘟病毒单克隆抗体的制备及特性鉴定

将纯化后的猪瘟病毒免疫4只SPF级BALB/c小鼠,无菌取脾细胞与SP2/0骨髓瘤细胞融合,经半固体培养基克隆化和间接ELISA筛选,最终获得了4株稳定分泌抗猪瘟病毒单克隆抗体的杂交瘤细胞株。ELISA结果显示4株单克隆抗体效价均较高,并与其他病毒及PK-15细胞培养物无交叉反应。Western blotting结果证实3株单克隆抗体可特异性识别猪瘟病毒。间接免疫荧光试验可在细胞膜内观察到特异性绿色荧光,表明3株单克隆抗体与猪瘟病毒具有良好的反应性。该研究为猪瘟病毒新型诊断试剂开发奠定了基础。     

抗金玉兰酶制剂(β-内酰胺酶)单克隆抗体的研制与特性分析

以金玉兰酶制剂(β-内酰胺酶)为抗原,制备出16株能稳定分泌抗β-内酰胺酶特异单克隆抗体的杂交瘤细胞株.对其中的高效价单克隆抗体进行了特性和表位分析,结果显示,抗原金玉兰酶制剂分子表面至少有两个相同或相近的表位,且它们的距离较远,采用4F11、2H5或1B10中的任一株单抗,就能实现对β-内酰胺酶的双抗夹心ELISA检测;4F11、2H5和1B10的免疫球蛋白亚型鉴定都是IgG1,亲和力常数在1×107L/mol～1×1010L/mol之间;4F11的检测灵敏度为19 ng/mL.证实该3株单克隆抗体可用于研制检测牛奶中金玉兰酶制剂的免疫学试剂盒.     

抗鹅γ-干扰素单克隆抗体的制备及鉴定

IFN-γ也称免疫IFN,主要由CD4+ Th1细胞、CD8+T细胞和NK细胞产生,是体内具有抗病毒和免疫调节功能的重要细胞因子之一[1].IFN-γ不但具有抗病毒、抗肿瘤活性,也可作加强和改善机体 免疫应答的免疫佐剂,与病毒的保护性抗原基因共 表达可加强和改善机体免疫应答.在禽类IFN-γ的研究中,鸡、鸭等IFN-γ的相关性研究较深入,有 关鹅IFN-γ的研究报道较少[2-3].     

两株抗猪伪狂犬病毒的单克隆抗体制备与鉴定

采用猪伪狂犬病毒(PRV)ZJ01株纯化病毒免疫BALB/c小鼠,利用融合细胞技术和间接ELISA抗体筛选技术,制备并获得2株能稳定分泌抗PRV单克隆抗体的杂交瘤细胞株2B3和5C10,其中,2B3单抗为Ig G2a亚类,5C10为Ig G1亚类,轻链均为κ链。间接免疫荧光检测结果表明,2株单克隆抗体均能与PRV发生特异性反应。Western-blot结果表明,2B3单抗针对PRV g C蛋白,5C10单抗针对PRV g E蛋白。本研究为建立快速检测伪狂犬病毒感染的免疫学方法奠定了基础。     

Preparation and characterization of monoclonal antibodies against Mycoplasma bovis.

Monoclonal antibodies (mabs) against Mycoplasma (M.) bovis were prepared for use in diagnosis of bovine mastitis. From the original 32 hybridomas actively secreting mabs against M. bovis, 6 stable lines were cloned. Two of them, Mb 5D8 and Mb 4F6, recognized M. bovis antigens of estimated molecular weights of 33 and 26 kDa, respectively. They showed no cross-reaction to other bovine mycoplasmas, thus rendering them useful for specific detection of this pathogen. All mabs investigated cross-reacted with M. agalactiae which is known to be closely related to M. bovis, but does not occur in cattle. Two other mabs, Mb 5D4 and Mb 1F6, exhibited further cross-reactions to a number of bovine mycoplasma species. Finally, mabs Mb 5D5 and Mb 2G5 reacted with all mycoplasmas tested. The possibility that they recognized constituents of the broth culture medium is discussed.     

Preparation and characterization of monoclonal antibodies against an avian reovirus

Thirteen monoclonal antibodies against avian reovirus strain Uchida were derived. Of the 13 antibodies, three (MAb1, MAb2, and MAb3) had the ability to neutralize the infectivity of the virus. MAb1 neutralized strains Uchida, CS-108, and TS-142 equally. MAb2 neutralized the same three strains, but the activity of neutralization was 10 times higher against Uchida than against CS-108 and TS-142. MAb3 neutralized only strain Uchida. It seems that MAb1 and MAb2 have a rather broad neutralization activity and that MAb3 has a type-specific activity. These data indicate that there are both type-specific and more broadly specific neutralization epitopes in avian reovirus particles, as in mammalian reoviruses. In Western blot analysis, MAb1 bound to a sigma protein of strain Uchida, so it was suggested that this protein carries the epitope of the less specific neutralizing activity.     

抗鸡传染性支气管炎病毒N蛋白单克隆抗体制备与鉴定

为建立抗鸡传染性支气管炎病毒(IBV)的单克隆抗体(MAb),利用浓缩的IBV致弱株CK/CH/LDL97Ⅰ F115病毒免疫BALB/c小鼠,采用杂交瘤技术,经间接ELISA和有限稀释法,经筛选和克隆后获得了一株抗IBV N蛋白MAb的杂交瘤细胞系(2D2).经鉴定,其MAb的重链为lgG<.1>亚型,轻链为κ链.ELISA和westernblot试验结果表明,所获得的这株2D2 MAb可特异性识别IBV N蛋白.2D2 MAb可与多种血清型IBV发生反应,表明该MAb识别的表位可能位于N蛋白的保守区域.为进一步鉴定IBV的表位及诊断试剂的研究奠定了基础.     

Preparation and characterization of polyclonal and monoclonal antibodies against Lawsonia intracellularis.

Proliferative enteropathy is an intestinal infectious disease caused by the obligate intracellular bacterium Lawsonia intracellularis. Immunohistochemistry staining has superior sensitivity over hematoxylin and eosin and silver staining for detecting L. intracellularis in histological sections. A L. intracellularis-specific monoclonal antibody (MAb) produced in the UK (IG4 MAb) has been described in the literature. However, no monoclonal or polyclonal antibodies are commercially available. Therefore, the objective of this study was to produce and characterize new polyclonal and monoclonal antibodies against L. intracellularis that are suitable for diagnostic use. The new monoclonal (2001 MAb) and polyclonal antibodies (1999 PAb) were compared with the IG4 MAb using Western blot analysis of outer membrane proteins (OMPs) of 6 L. intracellularis isolates, Bilophila wadsworthia and Brachyspira hyodysenteriae and using immunohistochemistry of known positive and negative histologic samples and pure cultures of L. intracellularis, B. wadsworthia, B. hyodysenteriae, Salmonella choleraesuis, S. typhimurium, and Escherichia coli K88. Immunogold staining using 2001 MAb was performed to show the specificity of the antibody against an L. intracellularis surface protein. Western blot analysis showed that the 2001 MAb targeted an OMP of 77 kD, which made it different from the IG4 MAb that targeted an 18-kD OMP. The immunogold stain demonstrated the specificity of the 2001 MAb to a surface protein of L. intracellularis. The polyclonal antibody (1999 PAb) targeted 5 OMPs (77, 69, 54, 42, and 36 kD). Both the 2001 MAb and 1999 PAb stained known positive, but not negative, histologic samples. Both the 2001 MAb and 1999 PAb reacted with a pure culture of L. intracellularis but not with any other common enteric pathogens. These two new antibodies will be useful for immunodiagnosis of L. intracellularis.