摘要
从中国鲎血细胞RT-PCR扩增抗菌肽tachyplesins基因,经改造构建原核和真核表达载体pET-KRN、pPIC9-KRY,并分别在大肠杆菌DE3和毕赤酵母GS115中诱导表达。用Tris-Tricine SDS-PAGE检测并进一步电洗脱纯化表达产物,经体外生物活性测定表明,表达产物KRN对大肠杆菌、金黄色葡萄球菌、枯草杆菌、伤寒沙门菌的抑菌环为25、22、25、16mm;MIC值为3.57、7.14、3.57、14.28mg/L。表达产物KRY对大肠杆菌、金黄色葡萄球菌、枯草杆菌、伤寒沙门菌和李氏杆菌的抑菌环为26、22、25、18、16mm;MIC值为1.57、3.14、1.57、6.28、6.28mg/L,对水霉的抑菌效果尤其显著,抑菌环达35mm。
In order to get the gene of antimicrobial peptide from horseshoe crab(Tachypleus tridentatus),the tachyplesins was amplified with the method of RT-PCR.Then they were cloned into plasmid of pMD18-T,which were identified by PCR and sequence.The expressing vector of those genes were constructed with pET28 and pPIC9,which were induced in E.coli and Pichia yeast GS115.So the antimicrobial peptide of horseshoe crab were got with the genetic engineer method.The excellent strains were selected and cultured to prepare the antimicrobial peptide.The peptides purified with the method of cuting the gel of tris-tricine SDS-PAGE.With the method of Kirby-Baller and Microdilution.Antimicrobial activities of the purified peptides were detected to some bacteria.Against Escherichia coli,Staphylococcus aureus,Bacillus subtilis, Salmonella typhi,the result of K-B of KRN is 25,22,25,16 mm respectively,the result of MM of KRN is 3.57,7.14,3.57,14.28 mg/L respectively.Against Escherichia coli,Staphylococcus aureus,Bacillus subtili,Salmonella typhi and L.monocytogenes,the result of K-B of KRY is 26,22,25,18,16 mm respectively,the result of MM of KRY is 1.57,3.14,1.57,6.28,6.28 mg/L respectively.Especially the result of K-B of KRY to mould Saprolegnia is 35 mm.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2007年第2期211-213,217,共4页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(30340087)
关键词
中国鲎
抗菌肽
抗菌活性
Tachypleus tridentatus
antimicrobial peptide
antimicrobial activity
