犬DEA1.1血型犬源单链抗体库构建及筛选

发布时间：2018-06-20 22:59

本文选题：犬DEA.血型 + 单链抗体(scFv)　；参考：《农业生物技术学报》2017年06期

【摘要】：在我国缺乏用于犬(Canis lupus familiaris)血型鉴定的检测试剂,本研究目的在于制备犬DEA1.1血型鉴定抗体,为宠物犬输血前血型鉴定提供材料。本研究以比格犬为研究对象,以外周血淋巴细胞为抗体基因来源,设计犬抗体可变区基因简并引物,用反转录聚合酶链反应(reverse transcriptionpolymerase chain reaction,RT-PCR)合成cDNA,扩增抗体重链可变区(heavy chain variable region gene,VH)和轻链可变区(light chain variable region gene,VL)基因,用重叠延伸PCR将VH和VL通过Linker连接重组单链抗体(single chain antibody variable fragment,scFv)的基因片段,将scFv基因连接至噬菌体载体pCANTAB 5E,构建犬源单链抗体scFv文库,用斑点酶联免疫吸附测定(dot enzyme-linked immunosorbent assay,Dot-ELISA)方法和犬红细胞抗原1.1(dog erythrocyte antigen 1.1,DEA1.1)血型免疫库为基础,进行3轮筛选。以鉴定阳性的克隆为基础,构建了原核表达重组单链抗体蛋白。结果表明,犬抗体可变区简并引物有效可用,并成功构建犬源单链抗体scFv文库。构建的免疫库实际库容量为7×10~5,具有多样性和足够大的库容量。最终筛选到3株能够与犬DEA1.1血型抗原相结合的单链抗体。检测鉴定原核表达的Clone16蛋白为可溶蛋白。亲和纯化获得Clone16单链抗体进一步用临床样本验证,具有较强结合特性。本研究为制备犬DEA1.1血型鉴定试剂提供了理论依据。
[Abstract]:The purpose of this study was to prepare DEA1.1 blood group identification antibody in dogs and to provide materials for blood group identification before blood transfusion in pet dogs. In this study, the degenerate primers of variable region gene of dog antibody were designed with peripheral blood lymphocytes as the source of antibody gene. CDNAs were synthesized by reverse transcriptionpolymerase chain reactionin reverse polymerase chain reaction (RT-PCR). The heavy chain variable region gene (VH) and the light chain variable region (LVC) gene were amplified. VH and VL were linked to the recombinant single chain antibody variable fragment FV by overlapping extension PCR. The scFv gene was ligated to the phage vector pCANTAB5E, and a canine scFv library was constructed. Three rounds of screening were carried out on the basis of dot-ELISA-dot enzyme-linked immunosorbent assay and 1.1(dog erythrocyte antigen 1.1 DEA1.1) blood group immunological library. Based on the positive clones, prokaryotic expression of recombinant scFv protein was constructed. The results showed that the degenerate primers of canine antibody variable region were effective and the canine scFv library was successfully constructed. The actual capacity of the constructed immune library is 7 脳 10 ~ (5), with diversity and large enough capacity. Finally, three single chain antibodies (scFv) which could bind to DEA1.1 blood group antigen were screened. The prokaryotic expression of Clone16 protein was identified as soluble protein. Clone16 scFV was obtained by affinity purification and further validated by clinical samples, with strong binding properties. This study provides a theoretical basis for the preparation of DEA1.1 blood group identification reagent in dogs.
【作者单位】：青岛农业大学动物科技学院;
【基金】：国家自然科学基金(No.31502138和No.31572590) 山东省自然科学基金(No.BS2015NY001) 山东省高校计划项目(No.J15LF03)
【分类号】：S852.4

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