串联鹿茸多肽基因在单一蛋白生产系统中的表达

Expression of the tandem repeat antler polypeptide in single protein production system

目的利用单一蛋白生产(SPP)系统可溶性表达串联鹿茸多肽(AP)。方法设计并合成由凝血酶Ⅹa序列间隔的5倍串联AP基因(5×AP),并将其克隆至冷休克表达载体中构建pColdⅡ(sp-4)-5×AP质粒,然后与pMazF质粒共转化入大肠杆菌E.coli BL21(DE3)中建立SPP系统,表达后经凝血酶Ⅹa水解获得AP。结果 5倍串联AP基因的碱基数为570 bp,在30℃下利用1 mol/L IPTG诱导表达的串联AP的分子量为18 500,水解后获得的AP的分子量为3700。结论利用SPP系统可成功表达纯度较高的串联AP,并可被凝血酶Ⅹa水解获得AP,为进一步研究基因工程方法制备AP的生物学性质奠定基础。

Objective To express the tandem repeat antler polypeptide（ AP） solublely by the single protein production（ SPP） system. Methods The 5-fold tandem repeat AP gene separated by the thrombin Ⅹa sequence was designed,synthesized and cloned into the cold shock expression vector to constructpColdⅡ（ sp-4）-5 × AP plasmid. Then it was co-transformed with the pMazF plasmid into Escherichia coli（ E. coli） BL21（ DE3） to establish the SPP system. Finally,the APwas obtained viahydrolysis oftandem repeat APexpressed in the SPP system bythe thrombin Ⅹa.Results The base number of 5-fold tandem repeat AP genewas 570 bp,and the molecular weight of the tandem repeat AP induced by 1 mol/L IPTG at 30 ℃ was 18 500,and the molecular weight of AP hydrolyzed from tandem repeat AP was 3700. Conclusion High purity tandem repeat AP could be expressed successfully by SPP system and could be hydrolyzed by the thrombin Ⅹa to obtain AP,which lay the foundation for further research on the biological properties of AP prepared by gene engineering methods.