山地乌骨鸡β防御素Gal-2基因的克隆及酵母表达载体的构建

Cloning of β-defensin Gal-2 gene in Sichuan Mountain dark-bone chickens and construction of its yeast expression vector

为了构建山地乌骨鸡β防御素Gal-2基因酵母表达载体,试验根据GenBank中鸡β防御素Gal-2基因序列设计引物,采用RT-PCR技术从山地乌骨鸡骨髓细胞中扩增出Gal-2基因,经PCR和酶切鉴定后测序,并采用DNAStar软件进行生物信息学分析;再根据毕赤酵母偏爱密码子和山地乌骨鸡β-防御素Gal-2成熟肽编码基因序列设计并合成1对引物,采用PCR技术从重组质粒pMD18-T-Gal-2扩增出其成熟肽编码基因,并将其插入载体pPICZaA,构建重组质粒pPICZaA-Gal-2,将pPICZaA-Gal-2线性化,通过电击转入毕赤酵母菌株GS115,用1%甲醇诱导表达120 h,表达产物纯化后进行Tricine-SDS-PAGE电泳鉴定。结果表明:山地乌骨鸡β防御素Gal-2基因克隆成功;山地乌骨鸡β-防御素Gal-2成熟肽编码基因重组真核表达载体构建成功;山地乌骨鸡Gal-2基因在酵母中得到表达。

To construct the yeast expression vector for β defensin Gal - 2 gene in the Mountain dark - bone chickens, the primers were designed according to the gene sequence of β defensin Gal - 2 published in GenBank. RT - PCR was used to amplify the Gal - 2 genes of the bone marrow cells from the Mountain dark - bone chickens. The PCR products were cloned into pMD18 - T and transformed into E. coli. Subsequently, the positive plasmids were identified by PCR, restriction enzyme analysis, and sequencing. Bioinformatics analysis was also carried out by using DNAStar software. According to the sequence which encodes beta - defensin Gal - 2 of chicken, a pair of primer was designed. The coding sequence of mature peptide was cloned from plasmid pMD18 - T - Gal - 2 by PCR and inserted downstream to signal peptide to construct recombinant plasmid pPICZa - A - Gal - 2. The plasmid was transformed into E. coli, and the positive clones were selected and sequenced. After linearization, the plasmid pPICZa- A -Gal -2 was electro -transformed into Pichia pastoris GS115 strains. 1% methanol was used to induce Gal -2 gene. At 120 h of expression, the products were purified preliminarily and identified by Tricine - SDS - PAGE electrophoresis. The resuits showed that ： Gal - 2 genes which contains complete cds were cloned successfully. The recombinant eukaryntic expression vector containing chicken Gal - 2 gene was constructed successfully. It indicates that the Gal - 2 gene of Mountain dark - bone chickens was expressed in yeast.