基于酵母双杂交技术的绒山羊波形蛋白互作蛋白鉴定

Identification of vimentin interaction protein of cashmere goats based on the yeast two-hybrid technology

为进一步探索波形蛋白(vimentin)调控绒山羊绒毛生长的作用机制,以内蒙古遗传种子资源阿尔巴斯白绒山羊皮肤中的毛囊组织为材料,利用PCR技术和酵母双杂交技术对根据GenBank中山羊的vimentin蛋白序列,运用蛋白质互作在线软件预测和液相色谱质谱联用采集免疫共沉淀数据筛选出肌动蛋白(ACTB)和vimentin进行基因引物设计、克隆、酵母表达载体构建和蛋白互作鉴定。结果显示:成功克隆出绒山羊vimentin基因CDS区序列的长度为1 401 bp, ACTB基因CDS区序列的长度为1 128 bp;构建了酵母表达载体;试验组共转化pGBKT7-vimentin与pGADT7-ACTB的Y2HGold菌株在二缺板SD/-Trp/-Leu/X-α-GaL/AbA培养基和四缺板SD/-Trp/-Leu/-His/-Ade/X-α-GaL/AbA培养基上,均能生长出淡蓝色菌落,而在对照组共转化pGBKT7空质粒与pGADT7-ACTB重组质粒的二缺板SD/-Trp/-Leu/X-α-GaL/AbA培养基上长出白色菌落,表明共转化的pGBKT7-vimentin与pGADT-ACTB重组质粒的下游报告基因Lac被激活,共转化pGBKT7空质粒与pGADT7-ACTB重组质粒的下游报告基因Lac未被激活。提示:vimentin与ACTB两种蛋白间存在相互作用。

To further explore the mechanism of vimentin regulating the growth of goat cashmere, the hair follicle tissues in the skin of Arbas white cashmere goats, a genetic seed resource in Inner Mongolia, were used as the material, and ACTB was predicted and vimentin was screened to be the interaction protein related to the development and growth of cashmere goat hair follicles. Based on the goat vimentin sequence in GenBank, using the protein interaction online software and co-immunoprecipitation combined with liquid chromatography mass spectrometry data, the primers of vimentin and ACTB genes were designed to clone the genes, to construct yeast expression vector and to identify the protein interactions using PCR and the yeast two-hybrid technology. The results showed that the sequence length of the CDS region of the vimentin gene and ACTB gene of cashmere goats were 1 401 and 1 128 bp, respectively, and the yeast expression vector was constructed successfully. Y2 HGold strains co-transformed with pGBKT7-vimentin and pGADT7-ACTB grew into light blue colonies on the two-deficiency plate SD/-Trp/-Leu/X-α-GaL/AbA medium and the four-deficiency plate SD/-Trp/-Leu/-His/-Ade/X-α-GaL/AbA medium in the experimental group, while Y2 HGold strains co-transformed by pGBKT7 empty plasmid and pGADT7-ACTB recombinant plasmid grew into white colonies on the two-deficiency plate SD/-Trp/-Leu/X-α-GaL/AbA medium in the control group. It suggested that the downstream reporter gene Lac co-transformed by the pGBKT7-vimentin and the pGADT-ACTB recombinant plasmid was activated, but the downstream reporter gene Lac co-transformed by the pGBKT7 empty plasmid and the pGADT7-ACTB recombinant plasmid was not activated. These results revealed the interaction relations between vimentin and ACTB.