樱桃谷鸭CD3ε胞外区多肽原核表达、抗体制备及初步应用

Prokaryotic expression,antibody preparation and prelimary application of extracellular region of CD3ε of cherry valley duck

应用PCR技术从樱桃谷鸭总RNA中克隆CD3ε基因ORF序列（DuCD3ε）,通过生物学软件分析,切除信号肽和跨膜区,将胞外区cDNA（DuCD3ε）插入表达载体pET-28a,构建了E.coliBL21（pET-28a/DuCD3ε）原核表达系。经IPTG诱导表达、蛋白纯化,制备兔抗DuCD3ε多克隆抗体。应用免疫组织化学SABC方法在樱桃谷鸭外周血液涂片上进行初步应用。结果显示,DuCD3ε胞外区序列长252 bp,编码83个氨基酸。SDS-PAGE和Western-blot分析证实重组DuCD3ε（rDuCD3ε）在BL21大肠杆菌中表达,表达蛋白相对分子质量约为14 000,经3次免疫后,通过间接ELISA测定兔抗rDuCD3ε蛋白多克隆抗体效价为1：12 800。在鸭瘟病毒感染后不同时间,对照组CD3＋T淋巴细胞数占淋巴细胞总数的百分比为28%~47%,试验组CD3＋T淋巴细胞百分比为32%~77%,在感染后1~3 d外周血液中CD3＋T淋巴细胞比值急剧升高,3 d以后比值缓慢下降。结果表明,本试验成功表达CD3ε胞外区多肽,制备的兔抗樱桃谷鸭CD3ε多克隆抗体可用于鸭外周血中CD3＋T淋巴细胞标记染色,具有较好的特异性。

ORF sequence of CD3ε gene from total RNA of cherry valley duck was cloned by PCR.After being analysised by biological software,signal peptide and transmembrane domain of CD3ε ORF gene were removed.Then extracellular region cDNA（DuCD3ε）was inserted into expression vetor pET-28a to make up system of prokaryotic expression of E.coli BL21（pET-28a/DuCD3ε）.Protein was expressed by inducing of IPTG and purified to prepare polyclonal antibody of rabbit ant-DuCD3ε.The antibody was applied in cherry valley duck blood smear by immunohistochemistry（SABC）.The consequence indicated that the length of extracellular region of DuCD3ε is 252 bp,coding 83 amino acids.SDS-PAGE and Western-blot analysis confirmed the recombinate plasmid（rDuCD3ε） was expressed in colibacillus（BL21）,and molecular weight of expressed protein is 14 000.The antibody titer of rabbit ant-DuCD3ε is 1∶12 800 which was detected by indirect ELISA after the third time innoculation.During different phases of post-infected duck plague virus,the percentage of CD3 positve lymphocyte in control group is 28%-47% which the infected group is 32%-77%.The rate of peripheral blood CD3 positve lymphocyte sharply raise from the first day to the third day after infected,and then regularly decrease.The result confirned extracellular region of DuCD3ε was expressed successfully.The prepared antibody of rabbit ant-DuCD3ε is specific in marker dyeing of duck peripheral blood CD3 positive T lymphocyte.