蚯蚓抗菌肽EP基因的克隆、表达和纯化  

Gene cloning,expression and purification of antibacterial peptide EP gene of earthworm

作　　者：许国芹 李密杰 蒋成砚[1] 鲁海菊[1] 孔琼[1] 何超[1] 沈登荣[1] 谢昆[1,2] XU Guoqin;LI Mijie;JIANG Chengyan;LU Haiju;KONG Qiong;HE Chao;SHEN Dengrong;XIE Kun(College of Life Science and Technology,Honglie University,Mengzi 661199,China;Yunnan Province Key Laboratory of Agricultural Crops High Quality and Efficient Cultivation and Safety Control,Mengzi 661199,China)

机构地区：[1]红河学院生命科学与技术学院,云南蒙自661199 [2]云南省高校农作物优质高效栽培与安全控制重点实验室,云南蒙自661199

出　　处：《黑龙江畜牧兽医》2021年第2期137-140,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(31760638);红河学院生物学重点建设学科资助项目(SZ1520)。

摘　　要：为了获得蚯蚓抗菌肽EP的基因序列并进行蛋白质表达,试验根据大肠杆菌密码子偏爱性原则推测EP基因核苷酸序列,设计3条互补引物,采用PCR技术扩增EP基因,构建pET32a-EP融合表达质粒,并对蚯蚓抗菌肽EP融合蛋白进行IPTG诱导和钴离子亲和层析纯化。结果表明:扩增得到大小约50 bp的目的条带,与预期大小一致,且测序结果与推测的EP基因序列完全一致。构建的pET32a-EP融合表达质粒表达出大小为18.315 ku的EP融合蛋白,且IPTG诱导5 h表达量最高,表达的融合蛋白存在于上清液中。说明成功表达和纯化出蚯蚓抗菌肽EP重组蛋白。In order to obtain the gene sequence of earthworm antibacterial peptide EP for protein expression,the predicted EP nucleotide sequence were designed in this experiment according to the principle of E.coli codon preference.Three special complementary primers were designed and EP gene was amplified by PCR method to construct fusion expression plasmid pET32 a-EP.The earthworm antimicrobial peptide EP fusion protein was induced by IPTG,and was purified by cobalt ion affinity chromatography.The results showed that the amplified target band was about 55 bp in size,which was consistent with the expected size,and the sequencing result was completely consistent with the predicted EP gene sequence.The constructed pET32 a-EP fusion expression plasmid obtained the EP fusion protein with a size of 18.315 ku,and the highest expression level was induced by IPTG for 5 h,and the expressed fusion protein existed in the supernatant.The results suggested that the earthworm EP recombinant protein was successfully expressed and purified.

关 键 词：蚯蚓 抗菌肽 EP基因 克隆 表达 

分 类 号：S859.797.9[农业科学—临床兽医学] Q78[农业科学—兽医学]