牛乳溶菌酶在毕赤酵母中的分泌表达及活性分析  

Secreted expression of Bovine lysozyme-1 in Pichia pastoris

作　　者：付世新[1] 齐长学[1] 罗春海[1] 张丽[1] 王瑶[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《中国预防兽医学报》2010年第6期428-431,454,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省青年科学技术专项资金项目(QC06C001)

摘　　要：为真核表达牛乳溶菌酶,本研究在通过酵母偏爱密码子改造并人工合成LYZ1基因的基础上,将LYZ1基因经克隆构建了高效表达具有生物活性牛乳溶菌酶的分泌型表达载体pPICZα-A-LYZ1,将其经SacⅠ酶切线性化后电转化毕赤酵母菌株GS115中,通过Zeocin筛选和PCR鉴定后的阳性重组菌用甲醇诱导60h后,进行SDS-PAGE和western blot鉴定,用溶壁微球菌对其进行活性检测,并对其进行体外抑菌效果检测分析。结果表明:分泌表达的重组目的蛋白约16ku,而且抗血清具有良好的反应原性。活性检测表明,培养液中重组溶菌酶活性达到2842u/mL。体外抑菌试验结果表明,重组牛乳溶菌酶对标准葡萄球菌及大肠杆菌菌株具有较好的抗菌作用,而对无乳链球菌、停乳链球菌、乳房链球菌作用较弱。To obtain efficient expression of Bovine lysozyme-1 （LYZ1）, LYZ1 gene was designed and synthesized using the preference codons of Pichia pastoris, and inserted into the Pichia pastoris expression vector pPICZ a -A. The recombinant plasmid pPICZ-LYZ1 was linearized and transformed into Pichia pastoris strain GS 115 by electroporation. Recombinant Pichia pastoris was screened on YPDS plates containing Zeocin and identified by PCR. Expression of LYZ1 was detected by SDS-PAGE and confirmed by western blot analysis. Lysozyme activity was detected by Micrococcus lysodeikticus （ML）. Result showed that recombinant protein was expressed in soluble form with a molecular weight approximately 16 ku. The biological activity were shown up to 2 842 u/mL was detected in the supernatant by ML assay. The recombinant LYZ1 had better antibacterial activity on staphylococci and E.coli, but was inferior against Streptococcus mastitidis, Streptococcus dysgalactiae and Streptococcus uberis.

关 键 词：牛乳溶菌酶 毕赤酵母 分泌表达 抗菌活性 

分 类 号：S852.42[农业科学—基础兽医学] S858.31[农业科学—兽医学]