中国对虾素在大肠杆菌(Escherichia coli)中的重组表达  

RECOMBINANT EXPRESSION OF CH-PENAEIDIN FROM CHINESE SHRIMP FENNEROPENAEUS CHINENSIS IN ESCHERICHIA COLI

作　　者：薛剑峰[1] 康翠洁[1] 王金星[1] 赵小凡[1] 相建海[2] 

机构地区：[1]山东大学生命科学学院,济南250100 [2]中国科学院海洋研究所,青岛266071

出　　处：《海洋与湖沼》2005年第3期235-240,共6页Oceanologia Et Limnologia Sinica

基　　金：国家重点基础研究发展规划项目;G19990120007号;国家高技术研究发展计划(863计划)项目;2001AA621120号;欧共体第五框架协议项目;ICA4CT200110023;国家自然科学基金项目;30371094号

摘　　要：中国对虾素是从中国对虾血细胞中克隆得到的一种抗菌肽。为了进一步研究中国对虾素(CHP)的功能并为制备特异性抗体作准备,采用大肠杆菌表达外源蛋白的方法,进行了对虾素原核表达的研究。根据从中国对虾血细胞中克隆得到的对虾素基因设计特异性引物扩增中国对虾素成熟肽基因(Chp),插入原核表达载体pGEX4T1中,在E.coliBL21表达融合蛋白GSTCHP。结果表明,不同表达菌株的融合蛋白表达量为30%—34%,同一菌株在诱导5h后能达到最高表达量。利用GST亲和层析纯化融合蛋白,将融合蛋白用凝血酶裂解以得到CHP,其分子量约为5.6kDa,N端测序结果与期望的成熟对虾肽序列一致。用液体生长抑制方法检测活性,表明重组GSTCHP蛋白及CHP均表现出对大肠杆菌的抑菌活性。Penaeidins, a unique antimicrobial peptide family, were first purified from haemocytes of the shrimp Litopenaus vannamei and their genes cloned from the haemocyte shrimp cDNA in 1997. Ch-penaeidin, a new antimicrobial peptide gene of the penaeidin family, was cloned from the hemocytes of Chinese shrimp, Fenneropenaeus chinensis and the gene expression and tissue distribution were studied by RT-PCR, Northern Blot and in situ hybridization. In order to further study Ch-penaeidin function and to prepare the anti-CHP polyclonal antibody, Escherichia coli was chosen to express the mature Ch-penaeidin (mChp) as a foreign protein. The DNA fragment encoding mature Ch-penaeidin obtained by PCR amplification with the following primers designed from the mChp sequence we cloned by forwarding primer 5′GCGC GAA TTC AAG GGT GGT TAC ACA CGC 3′ containing an EcoR Ⅰ site (underlined) and reversing primer 5′ GCGC CTC GAG ACT TAC ATC CCA CAT GCA C3′ that contains an Xho Ⅰ site (underlined). The amplified fragment was digested with EcoR Ⅰ and Xho Ⅰ and subcloned into the EcoR Ⅰ and Xho Ⅰ sites of pGEX-4T-1 expression vector. The plasmid constructed was denoted as mChp-pGEX-4T-1 and transformed into competent cells of E. coli BL21 for fusion expression. First, small-scale expression (3ml) was tested and the mChp-pGEX recombinants were screened for fusion protein expression on 12.5% SDS-PAGE. Then large-scale expression and purification were performed according to the following steps to obtain mCHP. A single colony of BL21 containing mChp-pGEX plasmid was grown overnight at 37℃ in 5ml of LB medium containing 100μg/ml ampicillin. Cultures were then diluted (1∶100) in fresh 2YT medium and grown at 37℃ for about 4h until the OD 600 reached approximately 0.8—1.0. The expression of GST-mCHP was initiated by adding isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 1mmol/L and the cultures grown for additional 4h. Cells were pelleted by centrifugation at 7000r/min for 10min and resuspended in 5%

关 键 词：中国对虾 重组表达 融合蛋白 大肠杆菌表达 原核表达载体 E.coli 特异性抗体 特异性引物 外源蛋白 基因设计 层析纯化 抑制方法 抑菌活性 血细胞 表达量 CHP 抗菌肽 成熟肽 GST 酶裂解 分子量 克隆 菌株 凝血 测序 

分 类 号：Q959.223.5[生物学—动物学] Q55