猪白细胞介素15基因的克隆及在大肠杆菌中的表达  

Cloning and Expression of Porcine Interleukin-15

作　　者：江云波[1] 方六荣[1] 肖少波[1] 张辉[1] 潘永飞[1] 罗锐[1] 李彬[1] 陈焕春[1] 

机构地区：[1]华中农业大学动物病毒室农业微生物国家重点实验室,武汉430070

出　　处：《畜牧兽医学报》2007年第8期841-845,共5页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(30300257);国家973基础研究项目(2005CB523200)

摘　　要：本研究通过RT-PCR方法从用ConA刺激的猪外周血淋巴细胞中扩增出猪IL-15(pIL-15)编码序列的cD-NA,将其克隆至T载体pMD18-T后,序列测定表明pIL-15基因长度为489 bp,编码162个氨基酸。进一步通过PCR方法获得缺失其N端48 aa的信号肽序列的成熟pIL-15蛋白基因,将其亚克隆到原核表达载体pET-28a的6×His下游,构建重组表达质粒pET-pIL15,转化大肠杆菌BL21(DE3),IPTG诱导,重组菌体裂解物经SDS-PAGE可检测到分子量约为15 ku的重组蛋白,其表达量占总菌体蛋白量的17.5%。Western blot试验也证实表达的重组融合蛋白6×His-pIL15能够很好地与抗6×His单抗发生反应。猪IL-15基因的克隆、表达为进一步研究其功能和应用奠定了基础。Interleukin-15 （IL-15） is a multi-functional cytokine of IL-2-1ike cytokine family and exerts profound effects on the proliferation and differentiation of lymphocytes and NK cells and the secretion of immunoglobulin. In this study, the porcine IL-15 （pIL-15） full-length gene was cloned from porcine peripheral blood molecular cells （PBMCs） using RT-PCR and ligated into pMD18-T vector. Sequence analysis showed that the cDNA of pIL-15 is 486 base pairs in length and encodes a protein consisting of 162 amino acid （aa）. The gene of mature pIL-15,which was deleting a 48 aa leader sequence at N-terminus,was cloned by PCR and then was introduced into downstream of 6 His of pET-28a, resulting in the prokaryotic expression plasmid pET-pIL15. After transforming into E. coli BL21 （DE3） and inducing by IPTG, the results of SDS-PAGE showed that the 6× His-piL15 fusion protein about 15ku was acquired. Gel scanning analysis revealed expression quantity of the fusion expression in all tropina was 17.5 %. The result of Western blot demonstrated that the recombinant fusion protein 6 His-piL15 can be recognized by anti- 6 His monoclonal antibody. The cloning and expression of pIL-15 made a foundation for the study of function and application.

关 键 词：猪白细胞介素15 基因克隆 原核表达 

分 类 号：Q786[生物学—分子生物学]