鸭白细胞介素-2基因的克隆表达和单克隆抗体的制备  

Cloning and expression of duck interleukin-2 gene and preparation of the monoclonal antibody against duck IL-2

作　　者：刘瑞[1,2] 韩先干[2] 范红结[1] 王少辉[2] 刘海文[2] 丁铲[2] 于圣青[2] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国兽医科学》2013年第12期1262-1267,共6页Chinese Veterinary Science

基　　金：家禽病毒病基因工程疫苗创新项目(2011AA10A209)

摘　　要：为研制鸭IL-2单克隆抗体,运用RT-PCR技术从樱桃谷鸭脾淋巴细胞中扩增鸭IL-2基因,将其克隆至原核表达载体pCold TF DNA中,进行诱导表达。用纯化的IL-2重组蛋白免疫BALB/c小鼠,融合后筛选抗鸭IL-2的阳性杂交瘤细胞株。通过间接ELISA法测定腹水效价、Western-blot法检测其免疫反应性,并鉴定抗体的亚类。测序结果显示,去除信号肽后的鸭IL-2基因,其编码区全长为363bp,编码120个氨基酸;SDS-PAGE检测结果表明,获得了可溶性的65ku的IL-2融合蛋白;免疫小鼠后获得3株抗鸭IL-2的阳性杂交瘤细胞株,腹水效价均达到1∶512 000以上,为IgG2b亚类。Western-blot结果表明,制备的单克隆抗体具有良好的免疫反应性。上述结果表明,成功制备了抗鸭IL-2的单克隆抗体,为进一步建立鸭IL-2的检测方法奠定了基础。The objective of this study is to prepare monoclonal antibody against duck IL-2. Duck IL-2 gene was amplified by RT PCR from spleen lymphocytes of Cherry Valley Ducks, and the expression plasmid pCold-DuIL2 was constructed. The recombinant protein was purified and used for immunization of BALB/c mice. The monoclonal antibody was identified by indirect ELISA and Western-bolt. The sub-types of mAbs were analyzed. Sequence analysis indicated that the encoding gene of duck IL-2（excluded the sig- nal peptide sequence） was about 363 bp in length and encode 120 amino acids. SDS-PAGE analysis showed that the fusion protein was 65 ku. All three clones of mAbs in ascitic fluids were shown to be with the titer of more than 5.12 〉（ l0S in ELISA assay, and the isotype of the three mAbs belonged to IgGeb. Western-blot analysis showed that the three mAbs could react with duck IL 2. In conclusion, the high titer monoclonal antibody was prepared successfully,which will benefit for developing methods for detecting the IL-2 level in ducks.

关 键 词：鸭 白细胞介素-2基因 克隆 表达 单克隆抗体 

分 类 号：S852.42[农业科学—基础兽医学]