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 .
出处
《华南农业大学学报》
CAS
CSCD
北大核心
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)
关键词
抗菌肽
表达
纯化
生物活性
antimicrobial peptide
expression
purification
biological activity
