猪胆囊收缩素与生长抑素的融合表达与鉴定  

Fusion expression and identification of porcine cholecystokinin and somatostatin

作　　者：赵婷芳 赵铖 黄嘉瑶 刘婷[1] 曾沛暄 黄晶 唐青海[1] 唐斯萍 Zhao Tingfang;Zhao Cheng;Huang Jiayao;Liu Ting;Zeng Peixuan;Huang Jing;Tang Qinghai;Tang Siping(Hunan Key Laboratory for Conservation and Utilization of Biological Resources in the Nanyue Mountainous Region,College of Life Sciences,Hengyang Normal University,Hunan Hengyang 421008;College of Chemistry and Materials Science,Hengyang Normal University,Hunan Hengyang 421008)

机构地区：[1]衡阳师范学院生命科学学院、南岳山区生物资源保护与利用湖南省重点实验室,湖南衡阳421008 [2]衡阳师范学院化学与材料科学学院,湖南衡阳421008

出　　处：《现代畜牧兽医》2024年第10期1-6,共6页Modern Journal of Animal Husbandry and Veterinary Medicine

基　　金：大学创新创业训练项目(S202210546015、cxcy2022002、S202310546001);湖南省教育厅科学研究项目重点项目(21A0442);衡阳市科技创新项目(202250045230);中央引导地方科技发展资金项目(2022ZYC091);湖南省普通高等学校教学改革研究项目重点项目(HNJG-20230871)。

摘　　要：研究旨在运用原核表达系统制备重组猪胆囊收缩素与生长抑素融合蛋白(rpCCK-SS)。人工合成猪胆囊收缩素与生长抑素融合基因(pCCK-SS),分别克隆至pET28a和pCold GST载体中,转化BL21 (DE3)或Transetta (DE3),构建重组表达菌株,采用双酶切和测序鉴定;并通过进行SDS-PAGE检测和Western blot鉴定优化诱导剂浓度和诱导时间。结果显示,pCCK-pSS基因全长693 bp,共编码231 aa;BL21 (DE3)-pET28apCCK-SS表达目的蛋白分子量25 kDa,以包涵体形式存在;Transetta (DE3)-pCold-pCCK-SS表达目的蛋白分子量50 kDa,在上清与沉淀中均有表达,以沉淀中的包涵体形式为主,最优表达条件为15℃、0.25 mmol/L IPTG浓度诱导8 h。研究表明,试验成功运用两个表达载体表达了rpCCK-SS,且pCold GST的表达效果优于pET28a,为进一步开发新型促生长制剂奠定了基础。The objective of this study was to prepare recombinant porcine cholecystokinin and somatostatin fusion protein(rpCCK-SS)using a prokaryotic expression system.The synthetic porcine cholecystokinin and somatostatin fusion gene(pCCK-SS)was cloned into pET28a and pCold GST vectors,transformed into BL21(DE3)or Transetta(DE3),and the recombinant expression strains were constructed,and identified by double enzyme digestion and sequencing.The concentration and induction time of the inducer were optimized by SDS-PAGE and Western blot.The results showed that the total length of pCCK-pSS gene was 693 bp,encoding 231 aa.BL21(DE3)-pET28a-pCCK-SS expresses the target protein with a molecular weight of 25 kDa and exists in the form of inclusion bodies.Transetta(DE3)-pCold-pCCK-SS was expressed with a molecular weight of 50 kDa in both supernatant and precipitate,mainly in the form of inclusion bodies in precipitate.The optimal expression conditions were 15℃and induced by 0.25 mmol/L IPTG concentration for 8 h.The results showed that rpCCK-SS was successfully expressed by two expression vectors,and the expression effect of pCold GST was better than pET28a,which laid a foundation for further development of new growth promotion agents.

关 键 词：猪胆囊收缩素 猪生长抑素 低温诱导 融合表达 

分 类 号：S828[农业科学—畜牧学]