抗真菌肽CGA-N46基因多顺反子表达载体的构建与鉴定

Construction and Identification of Polycistronic Expression Plasmids of Antifungal Peptide CGA-N46 Gene

目的:为提高抗真菌肽CGA-N46表达量,对该基因多顺反子表达进行研究。方法:以pEASY-Blunt为克隆载体,以"pET-30a rbs序列-起始密码子-CGA-N46编码序列-终止密码子"为外源片段,利用同尾酶Nhe I、Spe I和Xba I,构建了含有上述外源片段1、3、5、8拷贝的重组载体pT-CAN46、pT-3CAN46、pT-5CAN46和pT-8CAN46;将pEASY-Blunt上的外源基因片段亚克隆至pET-30a的rbs序列下游,构建了CGA-N46基因1、3、5、8顺反子的重组表达载体pET-CAN46、pET-3CAN46、pET-5CAN46和pET-8CAN46;利用感受态转化法转化大肠杆菌Rosetta,进行了表达研究。结果:在IPTG诱导下,CGA-N46基因1、3、5、8顺反子重组表达载体均能表达CGA-N46单体,各重组表达载体表达CGA-N46的量在总蛋白中的百分含量分别为3.4%、5.0%、12.4%、17.1%。结果表明随着CGA-N46基因顺反子个数的增加,外源蛋白表达量也越来越多。其中,8顺反子表达量最高,实现了CGA-N46的高效表达。结论:多顺反子表达可以克服融合表达和串联表达肽类蛋白的不足;创建的多顺反子表达载体构建方法简化了传统基因工程构建方法的步骤,为基因工程有效提高小肽表达量奠定了基础。

Objective： To improve the expression of antifungal peptide CGA-N46, the polycistronic expression of CGA-N46 were studied. Methods： The recombinant cloning plasmids pT-CAN46, pT-3CAN46, pT- 5CAN46 and pT-8CAN46 were constructed firstly, in which one copy, three copies, five copies and eight copies of heterogeneous gene fragment which was pET-30a rbs sequence-initiation codon-CGA-N46 encoding sequencetermination codon was recombined with cloning plasmid pEASY-Blunt respectively. And then the recombinant expression plasmids pET-CAN46, pET-3CAN46, pET-5CAN46 and pET-8CAN46 were constructed, in which one cistron, three cistrons, five cistrons and eight cistrons of CGA-N46 gene were recombined with expression plasmid pET-30a at the down stream of rbs sequence of it respectively. The recombinant expression plasmids were transformed into the competent strains of E. coli Rosetta. The expression efficiency of CGA-N46 was researched. Results： Under the induction of IPTG, CGA-N46 gene cistrons expressed the monomers of CGA-N46 and transferred into the periplasm space of host strains, The percentage of each expression cassete of CGA-N46 in total protein was 3.4%, 5.0%, 12. 4%, 17.1%. The results showed that the production of CGA-N46 gene increased with the icreasing of its cistrons in expression vector, the production of 8 cistrons was the most. Conclusions ： Polycistronic expression can overcome the shortage of fusion expression and tandem expression. The technology of construction of polycistronic expression plasmid simplified the traditional gene engineering construction method by using the same end of endonucleases Nhe I, Spe I and Xba I, and sets up a sound foundation for efficient gene engineering expression of peptides.