大鼠Clara细胞分泌蛋白CC16基因的克隆和原核表达及纯化  

Gene-cloning,prokaryotic expression and purification of rat Clara cell secretory protein

作　　者：庞敏[1] 杜永成[1,2] 蒋毅[1] 刘志宏[1] 张彩苹[1] 陈丽丽[1] 王海龙[3] 

机构地区：[1]山西医科大学第一医院呼吸科,山西太原030001 [2]山西省人民医院呼吸科 [3]山西医科大学基础医学院

出　　处：《环境与健康杂志》2013年第4期290-294,共5页Journal of Environment and Health

基　　金：国家自然科学基金(81200032);山西省卫生厅科技攻关计划项目(2011029);山西医科大学青年科技基金(02201120);山西医科大学第一医院青年科研基金(YQ1102)

摘　　要：目的克隆大鼠Clara细胞分泌蛋白(CC16)基因,原核表达并纯化重组的CC16蛋白,为进一步研究CC16蛋白的功能及对炎症性气道疾病的治疗作用奠定基础。方法制备大鼠肺组织总RNA,根据大鼠CC16基因全长编码序列(GenBank登录号:NM_013051)的开放阅读框设计引物并进行逆转录PCR(RT-PCR)扩增,扩增产物经双酶切后连接入pET30a(+)载体,重组质粒转化大肠埃希菌(E.coli DH5α),阳性菌落经PCR和双酶切鉴定并测序。将重组质粒pET30a(+)-rCC16转化至E.coli Rosetta(DE3)并加入异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结合考马斯亮蓝染色检测表达产物,重组融合蛋白经Ni2+琼脂糖凝胶亲和纯化。分别以抗His标签抗体和山羊抗CC16抗体进行免疫印迹(Western blotting)分析并鉴定重组蛋白。结果 RT-PCR扩增产物约为290 bp。菌落PCR、双酶切及测序结果显示,重组质粒pET30a(+)-rCC16构建成功。SDS-PAGE结果显示,经IPTG诱导获得相对分子质量约16 kDa的可溶性重组蛋白。Western blotting结果表明,诱导表达的蛋白质为带His标签的重组融合蛋白,且能被山羊抗CC16抗体识别。结论成功构建基因重组体pET30a(+)-rCC16,并制备出可溶性大鼠重组CC16蛋白。Objective To explore the cloning,prokaryotic expression and purification of the rat Clara cell secretory protein （CC16）, which paves the way to study the function of CC16 protein and its intervention effect on chronic inflammatory airway diseases. Methods Total RNA was extracted from the lung tissue of Wistar rat. The open reading frame of CC16 gene was amplified with a pair of specific primers, which was designed according to the coding sequence of CC16 gene （GenBank accession No： NM_013051）,the product of RT-PCR was digested with double restrict enzyme and ligated into a pET30a（＋） vector. The recombinant pET30a（＋）-rCC16 plasmid was transferred into E. coli DH5α and the positive clones were selected through the colony-PCR and confirmed by the double restrict enzyme digestion and sequencing. The successful pET30a（＋）- rCC16 construct was transformed into E. coli Rosetta （DE3） and induced with IPTG to express. The products of expression were analyzed through SDS-PAGE followed by coomassie blue staining and the recombinant rCC16 was purified using Ni2＋-NTA affinity chromatography. Western blotting assay with His primary antibody and goat anti-CC16 antibody was used to confirm the expression of rCC16. Results The product of RT-PCR was 290 bp. The recombinant plasmid pET30a（＋）-rCC16 was confirmed successfully by colony-PCR,double restrict enzyme digestion and sequencing. A recombinant protein with a rough molecular weight of 16 kDa was analyzed by SDS-PAGE followed by coomassie blue staining. Meanwhile the rCC16 was detected efficiently by Western blotting analysis with the His primary antibody and with the goat anti-CC16 primary antibody respectively. Conclusion The recombinant pET30a（＋）-rCC16 is successfully established and the purified dissoluble rat CC16 protein is obtained.

关 键 词：大鼠Clara细胞分泌蛋白 原核表达 亲和纯化 免疫印迹 

分 类 号：R994.6[医药卫生—毒理学]