绵羊骨髓细胞抗菌肽基因的改造及其在原核生物中的表达  

Gene reformation and prokaryotic expression of sheep myeloid antibacterial peptides with 29 amino acids

作　　者：刘科鹏[1,2] 柴晓虹 韩跃武[1] 姚海燕[1] 张雪燕[1] 徐攀[1] 

机构地区：[1]兰州大学基础医学院生物化学与分子生物学研究所,甘肃兰州730000 [2]甘肃省第二人民医院检验科,甘肃兰州730000 [3]甘肃农业大学草业学院草业生态系统教育部重点实验室甘肃省草业工程实验室中-美草地畜牧业可持续发展研究中心,甘肃兰州730070

出　　处：《中国生物制品学杂志》2014年第9期1164-1167,共4页Chinese Journal of Biologicals

摘　　要：目的通过对绵羊骨髓细胞抗菌肽(sheep myeloid antibacterial peptides with 29 amino acids,SMAP-29)的活性片段SMAP-29(1-18)基因的改造,使其以C-端融合蛋白的形式在大肠埃希菌(E.coli)BL21(DE3)中表达。方法根据大肠埃希菌偏爱密码子表,将SMAP-29的活性片段SMAP-29(1-18)基因序列进行改造,应用ProtParam分析SMAP-29的理化特性,Anthorprot软件预测其螺旋轮结构,设计新的基因片段,插入质粒pET-30a中,构建重组表达质粒pET-30aSMAP-29[1-18,K2,4,L13],转化感受态E.coli BL21(DE3),IPTG诱导表达,表达产物过2次Ni2+NTA His Bind resin柱进行纯化。结果 SMAP-29基因序列经改造后,稳定性、等电点及半衰期均明显提高。重组质粒经PCR、双酶切及测序鉴定,证明构建正确。表达的重组蛋白相对分子质量约61 257,以包涵体形式存在,表达量占菌体总蛋白的41%,纯度为81%。结论已成功对SMAP-29基因进行改造,实现了其在E.coli BL21(DE3)中的高表达,获得了纯度较高的目的蛋白,为进一步规模化生产SMAP-29奠定了理论及实践基础。Objective To reform sheep myeloid antibacterial peptides with 29 amino acids（SMAP-29）（1-18）gene and express it as a C-terminal fusion protein in E.coli BL21（DE3）.Methods According to the E.coli-preferred codon,SMAP-29（1-18）gene was reformed,based on which the physicochemical characteristics of SMAP-29 was analyzed by ProtParam software,while the helical wheel by Anthorprot software.The synthesized gene was inserted into plasmid pET-30 a,and the constructed recombinant plasmid pET-30a-SMAP-29[1-18,K3,4,L13]was transformed to E.coli BL21（DE3）for expression under induction of IPTG.The expressed product was purified for 2 times by Ni2 ＋NTA His Bind resion column chromatography.Results The stability,isoelectric point and half-life of reformed SMAP-29 gene were improved significantly.Recombinant plasmid pET-30a-SMAP-29[1-18,K3,4,L13]was constructed correctly as proved by PCR,restriction analysis and sequencing.The expressed recombinant protein,with a relative molecular mass of about 61 257,existed in a form of inclusion body,contained 41% of total somatic protein,and reached a purity of 81%.Conclusion SMAP-29 gene was reformed successfully and highly expressed in E.coli BL21（DE3）,and the target protein with high purity was obtained,which laid a theoretical and practical foundation of large-scale production of SMAP-29.

关 键 词：绵羊骨髓细胞抗菌肽 基因改造 原核细胞 基因表达 

分 类 号：Q786[生物学—分子生物学]