日本血吸虫胰岛素样多肽的真核表达及其多克隆抗体的制备  

Eukaryotic expression and polyclonal antibody preparation of insulin-like peptide of Schistosoma japonicum

作　　者：岳生赟 王帅[2] 何伟[2] 王力[2] 刘仲藜[1] 曾巧英[1] 才学鹏[1,2] YUE Sheng-yun WANG Shuai HE Wei WANG Li LIU Zhong-li ZENG Qiao-ying CAI Xue-peng(College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China State Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences ,Lanzhou 730046, China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2017年第7期805-809,共5页Chinese Veterinary Science

基　　金：国家重点基础研究发展计划(973)项目(2015CB150300)

摘　　要：为克隆和表达日本血吸虫胰岛素样多肽基因(Sjilp)并制备特异性多克隆抗体,根据基因组注释信息,设计特异性引物,利用RT-PCR技术从日本血吸虫成虫m RNA中成功扩增出长度为588 bp的特异性c DNA序列,其CDS序列长393 bp。基于毕赤酵母密码子偏好性,对Sjilp基因密码子进行优化,构建p PIC9KSjilp重组表达载体,并在毕赤酵母GS115中进行诱导表达。SDS-PAGE与Western-blot检测结果显示,重组蛋白Sj-ILP获得高效表达,其大小约为15 ku,与预期结果一致。利用合成肽技术,设计并合成得到2条特异性的肽段,偶联KLH,免疫新西兰大白兔,制备多克隆抗体,间接ELISA检测其效价达到1︰51 200,Western-blot鉴定表明这2条特异性多肽具有良好的免疫原性。本试验为后续验证Sj-ILP与其结合蛋白的功能奠定了良好的基础。In order to clone and express the insulin-like peptide gene（Sjilp） of Schistosoma japonicum and obtain its specific polyclonal antibodies,the primers were designed according to the genome database annotation information,and the cDNA with a length 588 bp from adult worm m RNA was successfully amplified by using RT-PCR.The CDS sequence of Sjilp was 393 bp in length.Sjilp gene codon was optimized based on the codon bias in Pichia pastoris.The recombinant expression vector p PIC9K-Sjilp was constructed and induced in P.pastoris GS115.Sj-ILP recombinant protein was highly expressed in eukaryotic expression system with a molecular weight of about 15 ku,which was evidenced by SDS-PAGE and Western-blot analysis.Two specific small polypeptides of Sj-ILP were synthesized and linked to KLH,followed by immuniza tion of the New Zealand white rabbits for preparing polyclonal antibodies.The titer of the polyclonal antibodies could reach to 1∶51 200 by indirect ELISA analysis.Furthermore,Western-blot analysis showed that the polyclonal antibodies had specifical immunogenicity.This study provided the important foundation for further functional studies between Sj-ILP protein and insulin-like growth factor-binding protein.

关 键 词：日本血吸虫 胰岛素样多肽 真核表达 多克隆抗体 

分 类 号：S852.735[农业科学—基础兽医学]