罗非鱼hepcidin 1-5成熟肽在毕赤酵母中的表达和活性研究  

Expression of TH1-5 Mature Peptide in Pichia pastoris and Analysis of Its Biological Activites

作　　者：梁盈[1] 梁瑞[1] 张永安[2] 祁克宗[1] 涂健[1] 彭开松[1,2] 

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036 [2]中国科学院水生生物研究所淡水生态与生物技术国家重点实验室,湖北武汉430072

出　　处：《动物医学进展》2014年第7期38-43,共6页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(30800811);国家重点基础研究发展计划项目(2014CB138600);安徽农业大学引进与稳定人才基金项目(yj2006-13)

摘　　要：为获得重组酵母分泌表达的莫桑比克罗非鱼hepcidin并进行其功能研究,将罗非鱼hepcidin 1-5(TH1-5)成熟肽cDNA插入表达载体pPIC9K,获得重组分泌表达质粒pPIC9K-TH1-5,电转化毕赤酵母GS115,阳性克隆经甲醇诱导获得表达上清;表达上清冻干品经Dot-ELISA检测,并分析了表达产物对指示菌和鸡外周血淋巴细胞的影响。结果表明,TH1-5成熟肽可通过表达质粒pPIC9K在毕赤酵母中实现分泌型表达,Dot-ELISA检测表达上清中含有TH1-5。重组表达的TH1-5对大肠埃希菌(CMCC44102)、金黄色葡萄球菌(ATCC25923)、铜绿假单胞菌(ATCC27853)、粪肠球菌(ATCC29212)具有明显的抑菌活性;含重组表达TH1-5的上清冻干品(6.25mg/mL)可协同ConA(0.125μg/mL)或LPS(0.125μg/mL)促进鸡外周血淋巴细胞的增殖。To obtain the expressing recombinant hepcidin1-5of Oreochromis mossambicus（TH1-5）by Pichia pastoris and study their function,the recombinant expression vector pPIC9K-TH1-5were constructed,and then transformed into P.pastoris GS115 by electroporation.The recombinant TH1-5was expressed in the positive clones of recombinant GS115 under the induction of methanol.The expressed supernatant was detected by Dot ELISA,and antibacterial activity and the effect of proliferation of peripheral blood lymphocyte were analysis.The results showed that the recombinant TH1-5was expressed by vector pPIC9 Kin the supernatant of Pichia pastoris GS115,and could be detected by Dot ELISA.Recombinant expressed TH1-5demonstrated obvious antibacterial activities to Escherichia coli（CMCC44102）,Stapylococcus aureus（ATCC25923）,Pseudomonas aeruginosa（ATCC27853）and Enterococcus faecalis（ATCC29212）.The lyophilized supernatant of recombinant expressed TH1-5in concentration of 6.25mg/mL can synergistically promote the proliferation of chicken peripheral blood lymphocytes with LPS（0.125μg/mL）or ConA（0.125μg/mL）.

关 键 词：罗非鱼hepcidin 酵母表达 斑点-酶联免疫吸附试验 抑菌活性 淋巴细胞增殖 

分 类 号：R979.5[医药卫生—药品]