[1]刘海峰,刘平,李楠楠,等.家蚕抗菌肽CM4和人可溶性BAFF融合基因的克隆、表达及活性鉴定[J].南京师大学报(自然科学版),2008,31(02):87-91.
　Liu Haifeng,Liu Ping,Li Nannan,et al.Cloning Expression and Characterization of a Bi-functional Antibacterial Peptides CM4 and hsBAFF Fusion Protein in Escherichia Coli[J].Journal of Nanjing Normal University(Natural Science Edition),2008,31(02):87-91.
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家蚕抗菌肽CM4和人可溶性BAFF融合基因的克隆、表达及活性鉴定()
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《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

卷:
第31卷
期数:
2008年02期
页码:
87-91
栏目:
生命科学
出版日期:
2008-06-30

文章信息/Info

Title:
Cloning Expression and Characterization of a Bi-functional Antibacterial Peptides CM4 and hsBAFF Fusion Protein in Escherichia Coli
作者:
刘海峰;刘平;李楠楠;张双全;
江苏省分子医学生物技术重点实验室, 南京师范大学生命科学学院, 江苏南京210046
Author(s):
Liu Haifeng; Liu Ping; Li Nannan; Zhang Shuangquan
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology,School of Life Science,Nanjing Normal University,Nanjing 210097,China
关键词:
抗菌肽CM4;  人的可溶性BAFF;  抗菌活性;  融合基因;  大肠杆菌;  表达
Keywords:
Antibacter ia l peptide CM4;  hsBAFF;  fusion gene;  E. co li;  expression
分类号:
R346
摘要:
采用PCR法从含有人可溶性BAFF基因的pET30a( + )扩增得到人sBAFF基因, 通过rPCR 将抗菌肽 Cecrop in CM 4基因的2条单核苷酸链进行扩增得到CM 4基因, 再采用ov er-lap PCR法通过linke r将hsBAFF与 Cecrop in CM 4融合基因相连接. 经纯化和鉴定后, 定向插入到原核表达载体pET30a ( + )中, 然后转化E. co li BL21( DE3), 通过实验确定了表达该融合基因的最佳诱导条件: IPTG 终浓度为1.0 mm o l/L, 诱导时间为5 h, 温度为30 ℃ , 其表达量占全菌蛋白的40%. 表达产物经SDS- PAGE 分析, 得到相对分子质量约为22 000的重组蛋白并且存在超声裂解后的上清中. 重组蛋白经W estern b lo t检测, 结果显示重组蛋白可被鼠抗人可溶性BAFF的抗体识别. 采用分子筛Sephadex G - 75对重组融合蛋白进行纯化, 并经SDS - PAGE对其鉴定. 通过对其生物学功能的检测得知, 纯化后的重组融合蛋白对大肠杆菌K12D31和真菌有明显的抑菌能力.
Abstract:
The cDNA o f hum an so lub le B lymphocyte stim ulato r ( hsBAFF) w as am plified by PCR from pET30a ( + ) and the gene o f an tibac terial peptide CM 4 by rPCR. The fusion gene o f hsBAFF and CM4 w as am plified by us ing over- lap PCR. The prokaryotic expression plasm id pET30a ( + ) /hsBAFF - CM 4 w as constructed w ith recomb inant DNA techniques after pur ify ing and iden tify ing the DNA fragm ent. Then the plasm id pET30a( + ) /hsBAFF- CM4 w as trans form ed in to BL21( DE3) cells and the expression w as optim ized w ith proper induc ing cond itions of 1. 0mm o l/L IPTG, 5 h and 30 ℃ induc tion. The expression leve l o f the target fusion pro te in reached 40% of the to tal bac terial pro tein. The resu lts o f SDS- PAGE indicated tha tm o lecularw e ight of the expressed prote in was abou t 22 000 and the expressed pro te in m a inly ex isted in the supe rnatan t after sonication. W estern b lo t ana lysis proved tha t the recom b inant pro tein has good reactive ability aga instm ouse anti-hum an so lub le BAFF IgG. The expression product w as purified by Sephadex G- 75. The pur ified recom bina tion prote in displayed antim icrob ial activ ity obviously.

参考文献/References:

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备注/Memo

备注/Memo:
基金项目: 国家自然科学基金( 30271093) , 江苏省自然科学基金( BK2005140 )资助项目.
通讯联系人: 刘 平, 博士, 副教授, 研究方向: 生化与分子生物学的教学与研究. E-mail:l-ping0805@ yahoo. com. cn
更新日期/Last Update: 2013-05-05