鲤鱼g型溶菌酶基因的cDNA克隆及其在毕赤酵母中的表达

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.

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

G-type lysozyme plays an important role in fish's immune response against microbial invasion. In the present study, a new g-type lysozyme isoform gene (CLYG) was separated from common carp (Cyprinus carpio) gill. The full-length cDNA 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, XhoⅠ and XbaⅠ restriction sites were added to the 5′ and 3′ ends of CLYG gene by PCR, respectively. After that, the expected fragment was ligated with expression vector pPICZαA, and followed by being transformed into competent Pichia pastoris X-33 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.