鹅FSH基因原核表达  

Prokaryotic expression of goose FSH gene

作　　者：甄霆[1] 俞钦明[2] 王彬[1] 朱振[1] 徐琪[1] 陈国宏[1] 

机构地区：[1]扬州大学动物科技学院,江苏扬州225009 [2]江西农业大学动物科技学院,江西南昌330045

出　　处：《中国兽医学报》2014年第11期1805-1812,共8页Chinese Journal of Veterinary Science

基　　金：现代农业产业技术体系建设专项基金资助项目(CARS-43-3);江苏高校优势学科建设工程资助项目(苏政办发【2011】137号)

摘　　要：促卵泡激素(follicle stimulating hormone,FSH)作为调节家禽卵泡发育重要的候选基因,但FSH在生物体内的半衰期非常短,本试验将具有延长基因半衰期作用的CTP序列添加到鹅FSH基因各亚基的C末端进行基因融合,以期获得pET32a-FSHα-CTP、pET32a-FSHβ-CTP和pET32a-FSHβ-CTP-α重组蛋白。设计去除目的基因信号肽和含有酶切位点的特异性引物,经融合PCR扩增获得了目的基因FSHα-CTP、FSHβ-CTP和FSHβ-CTP-α,通过构建原核表达载体pET32a-FSHα-CTP、pET32a-FSHβ-CTP和pET32a-FSHβ-CTP-α,将重组质粒转化到大肠杆菌BL21(DE3)中,经IPTG诱导,SDS-PAGE检测,蛋白纯化。结果显示,3个融合蛋白FSHα-CTP、FSHβ-CTP和FSHβ-CTP-α的IPTG最佳诱导时间分别为4、5和5h,最佳诱导浓度分别为0.2、0.8和1.5mmol/L,且都是以包涵体形式存在,相对分子质量分别为32 000、35 000和46 000,与预期大小一致。结果表明,本试验成功构建了鹅pET-32a-FSHα-CTP、pET-32a-FSHβ-CTP和pET-32a-FSHβ-CTP-α的原核表达载体,并对它们的重组蛋白进行了纯化,为开展鹅FSH的基因免疫研究和鹅rFSH生物制剂研发奠定基础。Follicle-stimulating hormone （FSH） is the main candidate gene that regulates the growth of avian follicle,but its half-life period is very short in biosome. We inserted the CTP sequence which could prolong the half-life of gene into the end of each FSH subunit sequence by fu- sion PCR in order to constructed fusion proteins, such as pET32a-FSHα-CTP, pET32a-FSHβ-CTP and pET32a-FSHβ-CTP-α. We designed specific primers with restriction enzyme sites and without signal peptides, then acquired target genes FSHa-CTP, FSHI3-CTP and FSHI3-CTP-a by fusion PCR and constructed expression vectors pET32a-FSHa-CTP, pET32a-FSHI3-CTP and pET32a- FSH^3-CTP-a and transferred them into E. COLI BL21（DE3）. Proteins were purified after induced by IPTG and checked by SDS-PAGE. The results confirmed that the best inducing time of IPTG of three fusion proteins FSHa-CTP, FSHβ-CTP and FSHβ-CTP-a are 4,5 and 5 h, respectively, and the best inducing concentrations of IPTG are 0.2 ,0.8 and 1.5 mmol/L respectively,all fusion proteins are existed in the form of inclusion body. As expected, the molecular weight of FSHα-CTP, FSHβ-CTP and FSHβ-CTP-β is 32 000,35 000 and 46 000, respectively. Prokaryotic ex- pression vectors pET-32a-FSHα-CTP, pET-32α-FSHβ-CTP and pET-32a-FSHβ-CTP-a of goose were constructed successfully, and their fusion proteins were purified, the data lay the foundation for immunity study of goose FSH gene and development on biologics of goose rFSH.

关 键 词：鹅 FSH基因 原核表达 蛋白纯化 

分 类 号：S835[农业科学—畜牧学]