抗红斑丹毒丝菌的rSpaA蛋白猪源单链抗体库构建及单链抗体淘选

从免疫过rSpaA的猪的脾淋巴细胞中提取总RNA,采用RT–PCR技术反转录合成cDNA。设计兼并引物扩增抗体重链可变区(VH)和轻链可变区(VL)基因片段,采用重叠延伸PCR方法,将VH和VL通过Linker连接成重组单链抗体(以下简写为sc Fv)的基因片段。将sc Fv的基因连接至噬菌粒载体p Comb3Xss,将重组载体电转化至宿主菌XL1-Blue,并经辅助噬菌体M13KO7拯救,获得猪源噬菌体单链抗体库,库容约为2.5×106。以rSpaA为靶抗原,经免疫亲和筛选,获得8株特异性较好的阳性克隆。本研究结果可为制备抗红斑丹毒丝菌的重组sc Fv提供新途径,并为猪丹毒的免疫检测和综合防制提供材料基础。     

Isolation of Goose-origin scFv Antibodies Against Goose Parvovirus from Bacterial Display Antibody Libraries

Goose parvovirus (GPV) can cause a highly contagious and fatal gosling plague (GP) disease in goslings and muscoy ducklings.Here,three goose-origin neutralizing single chain variable fragment (scFv) antibodies against GPV SYG-61 were isolated.The genes of scFv antibodies were derived from goslings immunized with GPV SYG-61,and scFvs were subcloned into a pBSD vector for the construction of pBSD-scFv libraries.The pBSD-scFv libraries were screened following three rounds using VP2 (protective antigen of GPV) as the bait by flow cytometry (FCM).After screening,the 15 clones with high mean fluorescence intensity (MFI) were isolated and sequenced.These 15 scFvs were expressed by pET-28a (+) in E.coli.The specificity and affinity of the 15 purified scFvs were successfully confirmed by ELISA.In the preliminary neutralization experiment on primary goose embryo fibroblast (GEF) in vitro,three of the 15 purified scFvs (named scFv-10,scFv-11 and scFv-50) showed significant neutralizing capacities.The study generated the first goose-origin neutralizing scFv against GPV and laid the foundation for the appearance of full-length goose-origin neutralizing monoclonal antibody against GPV.     

A Single-chain Fragment Variable Recombinant Antibody against F5 Fimbria of Enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> Inhibits Agglutination of Horse Red Blood Cells Induced by F5 Protein

Bovine colibacillosis caused by enterotoxigenic Escherichia coli (ETEC) is a worldwide problem. Adhesion of ETEC to intestinal cell receptors mediated by the surface protein F5 fimbriae is the initial step in the establishment of colibacillosis. Prevention of ETEC F5⁺ adhesion to enterocytes protects newborn calves against collibacillosis. On the enterocytes, the F5 fimbriae bind to a ganglioside that is also found on horse red blood cells. Thus, the presence of F5 fimbriae induces haemagglutination, which is useful as an indicator in a functional assay system. In this study, recombinant anti-F5 scFv antibody fragment produced in E. coli HB2151 reacted with F5 fimbriae in ELISA and Western immunoblot, and prevented haemagglutination induced by the binding of the F5 fimbriae to its natural host receptors on horse red blood cells. Given the ease with which recombinant antibodies can be mass-produced, the presently described scFv may hold promise as a prophylactic agent for colibacillosis.     

抗新城疫病毒HN蛋白单链抗体基因的克隆与序列分析

从分泌抗新城疫HN蛋白单克隆抗体的杂交瘤细胞中提取纯化mRNA,经RT-PCR扩增抗体轻、重链可变区基因,然后与L inker连接组装成单链抗体基因(scFv),其排列方式为VL-linker-VH。结果,成功地构建了scFv基因,序列分析表明,scFv基因长为744 bp,其中VH基因长为366 bp,VL基因长为333 bp。单链抗体基因的构建为抗新城疫HN蛋白基因工程抗体的制备奠定了基础。     

Improved Detection of Domoic Acid Using Covalently Immobilised Antibody Fragments

Antibody molecules, and antibody fragments in particular, have enormous potential in the development of biosensors for marine monitoring. Conventional immobilisation approaches used in immunoassays typically yield unstable and mostly incorrectly oriented antibodies, however, resulting in reduced detection sensitivities for already low concentration analytes. The 2H12 anti-domoic acid scFv antibody fragment was engineered with cysteine-containing linkers of two different lengths, distal to the antigen binding pocket, for covalent and correctly oriented immobilisation of the scFvs on functionalised solid supports. The Escherichia coli-produced, cysteine-engineered scFvs dimerised in solution and demonstrated similar efficiencies of covalent immobilisation on maleimide-activated plates and minimal non-covalent attachment. The covalently attached scFvs exhibited negligible leaching from the support under acidic conditions that removed almost 50% of the adsorbed wildtype fragment, and IC₅₀s for domoic acid of 270 and 297 ng/mL compared with 1126 and 1482 ng/mL, respectively, for their non-covalently adsorbed counterparts. The expression and immobilisation approach will facilitate the development of stable, reusable biosensors with increased stability and detection sensitivity for marine neurotoxins.     

‘Plantibodies’: a flexible approach to design resistance against pathogens

Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfecting plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal antibodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests.Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3-ends, but also the 5-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restriction sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins ofGlobodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (V_L) and heavy chain (V_H) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and intracellular location.     

抗克伦特罗单链抗体噬菌体展示文库的构建初步筛选及鉴定

构建抗克伦特罗(CBL)单链抗体噬菌体展示文库(scFv phage displaylibraries),从中筛选CBL特异性噬菌体单链抗体(phage scFv).提取经CBL免疫的小鼠的脾脏总RNA,RT-PCR扩增全套抗体轻链可变区(VL)及重链可变区(VH)基因,重叠延伸法将VL、VH片段拼接为单链抗体(scFv)基因片段.将scFv克隆到噬菌粒载体pCANTAB5E中,转化感受态大肠杆菌TG1,用辅助噬菌体M13KO7进行超感染,构建噬菌体单链抗体库.对抗体库进行亲合富集后,ELISA法筛选阳性克隆.扩增出抗CBL的VL、VH基因片段并拼接为全长的scFv,抗体库库容约为1.6×104,经4轮"吸附-洗脱-扩增"的富集,ELISA筛选到6个具有CBL结合活性的phage-scFv.成功构建抗CBL单链抗体噬菌体展示库,并初步筛选到具有CBL结合活性的噬菌体scFv,特异性好,为进一步大量表达CBL单链抗体奠定了基础.     

用于免疫治疗朊病毒病的抗体片段的研究进展

朊病毒是一种感染性蛋白质,能引起一系列的被称为传染性海绵状脑病(TSEs)或朊病毒病的致命性神经变性型疾病.在哺乳动物中,朊病毒通过招募普通的细胞蛋白(PrPc)并诱导其转化为具有致病性的PrPsc而实现自我复制.最近的研究结果表明,抗朊病毒的抗体能永久性地治愈被朊病毒感染的细胞.但是,全长的抗体和蛋白质不能通过血脑屏障(BBB),这妨碍了将其用于TSEs的体内治疗.采用另一种方式,即通过腺联病毒(AAV)转导将有朊病毒特异性的scFv传递到大脑内,尽管这不能完全保护其不受感染,但能延迟受感染小鼠的发病时间.通过直接向细胞培养物中加入羊痒病病毒或使用慢病毒和腺联病毒双转导载体使其感染,作者研究了重组的抗PrP(D18)单链可变片段(scFv)抗朊病毒的效果.结果表明,在感染细胞中重组的抗PrP scFv能够减少蛋白酶K的含量.另外,证实了与AAV相比,慢病毒能更有效的使抗PrP scFv基因转染并减少感染的神经元细胞中PrPsc的含量.最后,作者使用生物信息学的方法构建了D18scFv-PrP复合物的结构模型.有趣的是,根据反馈的结果,在D18scFv将PrPc锚定到抗体凹槽过程中,ArgPrP151(朊蛋白的第151位氨基酸)是一个关键的氨基酸残基.以上结果表明,将靶向PrP的被动和主动的免疫方法相结合,可能是治疗干预朊病毒病的一个可行策略.     

抗猪脂肪细胞膜单链抗体基因的构建及鉴定

应用噬菌体展示技术，从猪脂肪细胞免疫的小鼠脾细胞mRNA中构建出单链抗体(scFv)cDNA文库。cDNA文库克隆到噬菌粒载体pCANTAB5E，转化E.coli TG1,通过噬菌体表面展示，用猪脂肪细胞对表达的重组噬菌体单链抗体文库进行3轮亲和富集，筛选出了猪脂肪细胞膜scFv，为今后的应用奠定了基础。     

Expression of a single-chain antibody against GA24/19 in vascular tissues induces dwarf phenotype for rice plants

GA₁ and GA₄ are active gibberellis (GAs) in rice plants, which are synthesized from GA₁₉ and GA₂₄, respectively. However, the plant tissues where active GAs biosynthesis occurs and the route of their transport have not been fully elucidated. To study the importance of vascular tissues for the function of GAs, cDNA encoding anti-GA_24/19 single-chain variable fragment (scFv) was expressed under the control of the thioredoxin h promoter, which is active in companion cells of phloem tissues. Thetransgenic rice plants showed a dwarf phenotype with increased levels of GA₁₉. This indicated that the expression of the anti-GA_24/19 antibody in companion cells changed the synthesis and/or translocation of GAs in rice plants effectively, suggesting that the vascular tissues play an important role in the function of GAs in rice plants.