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

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.