文献类型：期刊文献

中文题名：禽源CCL19蛋白的表达纯化和抗体的制备

英文题名：Expression of avian origin chemokine CCL19 and preparation of its polyclonal antibodies

作者：王秋霞[1];李鹏祥[1];李茵[1];刘兴友[1,2];余燕[1];张艳红[1];姜金庆[1];马金友[1];欧长波[1]

第一作者：王秋霞

机构：[1]河南科技学院动物科技学院,河南新乡453003;[2]新乡学院,河南新乡453003

第一机构：河南科技学院动物科技学院,河南新乡453003

年份：2018

卷号：48

期号：2

起止页码：155-160

中文期刊名：中国兽医科学

外文期刊名：Chinese Veterinary Science

收录：CSTPCD;;北大核心:【北大核心2017】；CSCD:【CSCD_E2017_2018】；

基金：国家自然科学基金项目(31402169);畜禽疫病防控河南省高校科技创新团队支持计划资助项目(18IRTSTHN019);河南科技学院2017年大学生创新创业训练项目(2017CX037)

语种：中文

中文关键词：趋化因子;CCL19;鸡;表达;多克隆抗体

外文关键词：chemokines;CCLl9 gene;gallus;expression;polyclonal antibody

摘要：为研究趋化因子CCL19在禽源疾病发生发展过程中的作用,利用RT-PCR技术,从攻毒后的鸡腔上囊组织克隆了禽源CCL19基因,测序结果显示,该基因ORF长300 bp,编码100个氨基酸,与Gen Bank中登录的禽源CCL19序列完全一致;将该基因亚克隆至原核表达载体p ET30a,IPTG诱导表达后,SDS-PAGE进行初步分析,在约13 ku处出现明显蛋白条带,与预期结果相符;经镍柱纯化后,获得了高纯度的重组融合蛋白,Western-blot分析显示,纯化后的重组融合蛋白能够与抗His标签单克隆抗体发生特异性反应。将纯化后的目的蛋白免疫新西兰大白兔进行多克隆抗体的制备,经Western-blot检测,制备的抗体能与CCL19蛋白发生特异性反应。本研究首次克隆并表达了禽源CCL19蛋白,并对该蛋白进行了纯化和抗体的制备,该基因的成功克隆、表达和纯化以及针对目的蛋白抗体的制备,为进一步研究趋化因子CCL19在禽源疾病发生发展过程中的作用奠定了基础。
To study the effects of CCL19 on the development of avian disease,CCLl9 was cloned from the bursae of IBDV-inoculated chickens by RT-PCR. Sequencing results showed that the cDNA of CCLI9 contained a 300 bp open reading frame（ORF）,which encoded 100amino acids and shared 100%nucleotide acid and amino acid similarity with avian CCLI9 deposited in GenBank. The cDNAof CCLI9 was then subcloned into pET30a vector,transformed into E coli BL21（DE3）and induced with IPTG. SDS-PAGEindicated that the molecular weight of the expressed fusion protein was approximately 13 ku,which was the same as the expected size. High purity recombinant protein was obtained by Ni-sepharose purification. Western-blot showed that the purified fusion protein could be recognized by the positive sera of mice against His-tag protein. The purified target protein was used to immunize New Zealand white rabbits and the obtained polyclonal antibodies were identified with CCLI9 protein by Western-blot. In all,the preparations of avian origin chemokine CeLl9 protein and its polyclonal antibodies have laid the foundation for studies of the development of avian disease.