重组抗菌肽Cecropin P1在毕赤酵母X-33中的表达以及纯化后的生物活性分析  

Expressions of Recombinant Antimicrobial Peptide Cecropin P1 in Pichia pastoris X-33 and Biological Activity Analysis After Purification

作　　者：唐丽云[1] 郭春和[2] 郑红玉[1] 项林盛[1] 刘德辉 黄毓茂[1] 焦茂兴[4] 

机构地区：[1]华南农业大学兽医学院,广东广州510642 [2]中山大学生命科学学院,广东广州510006 [3]广州沃德生物技术有限公司,广东广州510623 [4]宜春学院,江西宜春336000

出　　处：《华南农业大学学报》2013年第4期569-573,共5页Journal of South China Agricultural University

基　　金：宜春市科技创新"六个一"工程项目;广东省现代农业生猪产业技术体系(F10021);江西省教育厅科技项目(2002年)

摘　　要：为了在毕赤酵母Pichia pastoris表达系统中表达猪源Cecropin P1并分析其生物活性,根据Cecropin P1的氨基酸序列和毕赤酵母的密码子偏嗜性设计引物,通过SOEing法合成全基因片段载入到PpicZαA质粒中,酶切线性化重组质粒PpicZαA-cecropin P1后电穿孔导入毕赤酵母X-33中进行整合,经Zection筛选和PCR检测获得高拷贝的转化子.发酵表达后将表达产物经加热预处理和Sephadex G-25层析及冻干浓缩后,Tricine-SDS-PAGE显示明亮单一条带.采用琼脂扩散法对纯化后的抗菌肽的热稳定性、酸碱稳定性、耐胰酶和胃蛋白酶稳定性进行了生物活性分析,结果表明Cecropin P1具有较强的热稳定性、酸稳定性及胃蛋白酶稳定性,但是在碱性环境下和胰酶作用下抑菌活性明显下降.To express the porcine antibacterial peptide Cecropin P 1 in Pichia pastoris and analyze the bio-logical activity of the expressed product , Cecropin P1 gene was designed and synthesized to include the partiality codons of P.pastoris based on the gene sequences encoding swine antibacterial Cecropin P 1.The linearized recombinant was shocked into P.pastoris X-33 by electroporation , and the high copy transfor-mants were screened with Zection and PCR .After being pured , Tricine -SDS-PAGE showed a single bright band .Agar diffusion method was used to analyze the biological activity of the purified antimicrobial peptides under the thermal , different pH , trypsin and pepsin .The results indicated that the cecropin P 1 had a strong thermal stability , acid stability and pepsin stability .However , in alkaline environment and pancreatin , the antibacterial activity of cecropin P 1 decreased significantly .

关 键 词：抗菌肽 表达 纯化 生物活性 

分 类 号：S188[农业科学—农业基础科学]