鲤鱼g型溶菌酶基因的cDNA克隆及其在毕赤酵母中的表达

cDNA cloning of g-type lysozyme gene from common carp and its expression in Pichia pastoris

从鲤鱼鳃组织中分离克隆到一种新的g型溶菌酶同工型基因(CLYG).该基因的全长cDNA为558 bp,除去终止密码子,编码185个氨基酸,推断的氨基酸序列没有信号肽,含3个催化位点(谷氨酸73、脯氨酸86和天冬氨酸97),具有鱼类g型溶菌酶的基本特征.通过PCR方法在CLYG基因的5'和3'端分别引入XhoⅠ和XbaⅠ酶切位点.扩增到的目的片段与表达载体pPICZαA连接构建重组表达载体pPICZαA-CLYG后,电转至毕赤酵母X-33,筛选得到的阳性转化子在1.0%甲醇、29℃、pH 6.0的条件下诱导表达72 h,获得重组体CLYG.SDS-PAGE和蛋白质印迹分析显示,在毕赤酵母中成功表达了重组体CLYG.

G-type lysozyme plays an important role in fishes immune response against microbial invasion. In the present study, a new g-type lysozyme isofor^n gene （ CLYG） w as separated from common carp （ Cyprinus ca rp io ） gill. T h e full-length c D N A of CLYG is 558 bp, which encodes 185 amino acid residues. The amino acid sequence deduced from the cDNA of CLYG contains 3 catalytic res-idues （Glu73, Pro86 and Asp97） without any signal peptide, which is similar to those of other teleost species. Subsequently, Xho I and Xba I restriction sites were added to the 5r and 3r ends of CLYG gene by P C R , respectively. After that, the expected fragment was ligated with expression vector pPICZaA, and followed by being transfor^med into competent P ich ia pastoris X - 3 3 cells. Then recombinant CLYG was induced in 1.0% methanol under pH 6.0 at 29 ^ for 72 h. SDS-PAGE analysis and Western blot anal-ysis demonstrated that the recombinant CLYG was successfully expressed.