凡纳滨对虾溶菌酶基因在毕赤酵母中的分泌表达和活性检测  

SECRETED EXPRESSION OF LITOPENAEUS VANNAMEI LYSOZYME GENE IN PICHIA PASTORIS AND ITS BIOACTIVITY DETECTION

作　　者：卞曙光[1,2] 陈华新[2] 姜鹏[2] 张海波[3] 刘兆普[1] 秦松[2] 

机构地区：[1]南京农业大学海洋生物学江苏省重点实验室,南京210095 [2]中国科学院海洋研究所,青岛266071 [3]上海海洋大学水产与生命学院,上海200090

出　　处：《水生生物学报》2010年第6期1091-1096,共6页Acta Hydrobiologica Sinica

基　　金：国家863重点项目计划"抗病功能蛋白渔药研制"(编号:2006AA100311)资助

摘　　要：原核重组表达的凡纳滨对虾(Litopenaeus vannamei)溶菌酶蛋白主要以包涵体形式存在,经变性和复性处理后活性仍较差。研究将凡纳滨对虾溶菌酶基因(Lvlyz基因)克隆至毕赤酵母分泌型表达载体pPIC9K中,电击转化毕赤酵母GS115细胞,经组氨酸营养缺陷培养基筛选和PCR检测获得转化子。对其进行连续甲醇诱导表达,利用SDS-PAGE和C端携带的6×His标签,对发酵液上清进行Western blot检测,结果表明19.3 kD左右的条带即是重组表达的溶菌酶蛋白。用溶壁微球菌平板抑菌法鉴定表达产物具有较强的抑菌能力。研究首次利用毕赤酵母真核表达系统实现对虾溶菌酶基因的可溶性表达,并且表达产物的活性良好。Litopenaeus vannamei,commonly known as White shrimp,belong to Arthropoda,is a widely eurythermal and euraline tropical shrimp.Since the late 80s of last century,L.vannamei has become the major species of cultured shrimp in China.However,during the last decade,the outbreak of infectious diseases has caused significant losses of farmed prawns.The latest research shows that,immunological system of L.vannamei are powerful and useful to in-crease its resistance and evaluate the health state.The immunologies of shrimp mainly consist of cellular immunity and humoral immunity and the existing studies have shown that,shrimp lysozyme,as an important factor for non-specific immune,plays an important role in the process of body’s humoral immunity.Up to present,a variety of shrimp lysozyme gene has been cloned and the recombinant lysozymes have been expressed.However,all the obtained recombi-nant lysozymes were using the prokaryotic expression system to achieve the expression of lysozyme genes.The resulting problem is that： when expressed in procaryotic expression system,the L.vannamei recombinant lysozyme protein was mainly insoluble inclusion bodies with low bioactivity.Therefore,it is necessary to test the other expression system,such as Pichia pastoris eukaryotic expression system,which is one of the most perfect expression system in eukaryotic gene expression.The object of this study was thus to construct a L.vannamei lysozyme gene（lvlyz gene） expression vectors of Pichia pastoris and express a new recombinant lysozyme protein with high bioactivity.A DNA fragment coding lvlyz gene was obtained from the plasmid pMD18-T/Lvlyz and cloned into the Not Ⅰ site of the P.pastoris expression vector pPIC9K.The constructed plasmid,pPIC9K/Lvlyz,was transformed into the P.pastoris strain GS115 by electroporation.The positive transformants,screened out from histidine-deficient medium plates,were confirmed by polymerase chain reaction amplification.The recombinant strains were induced continuously by methanol and the induced

关 键 词：凡纳滨对虾 溶菌酶 毕赤酵母 分泌表达 抑菌活性 

分 类 号：Q786[生物学—分子生物学]