【摘要】 试验利用Trizol法从鸡肠道组织提取总RNA,采用特异性引物通过RT-PCR扩增出α-2,3-唾液酸转移酶Ⅰ(α-2,3-sialyltransferaseⅠ,ST3GALⅠ)基因的cDNA片段,并将其克隆至pGEM-T easy载体,获得阳性重组质粒,再以阳性重组质粒为模板亚克隆ST3GALⅠ基因的完整ORF区,定向插入到真核表达载体pcDNA3.1(+)上,进行PCR、限制性酶切和DNA序列分析鉴定。结果表明,ST3GALⅠ基因全长1029bp,测序结果同GenBank数据库收录的序列一致,无任何密码子缺失与突变,插入到真核表达载体pcDNA3.1(+)上的目的基因大小方向均正确。本研究成功构建了pcDNA3.1(+)-ST3GALⅠ真核表达载体,为下一步的真核表达及对ST3GALⅠ基因的功能研究奠定了基础。更多还原

【Abstract】 Total RNA was extracted from intestinal of chicken by the method of Trizol.The α-2,3-sialyltransferase Ⅰ(ST3GAL Ⅰ) gene’s cDNA was amplified by reverse transcription polymerase chain reaction with specific primer.The amplified fragment was cloned into pGEM-T easy vector,then got the recombinant plasmid.Sub-cloning the ORF of ST3GAL Ⅰ gene of pGEM-ST3GAL Ⅰ and inserted the eukaryotic expression vector pcDNA3.1(+).The target segment in recombinant plasmid was confirmed by PCR,restriction enzyme digestion and sequencing methods.The results indicated that ST3GAL Ⅰ gene was 1029 bp in length,the result of sequence was consistent with the sequence which included in GenBank,and without any mutation and deletion of codon.The length and directions of gene which inserted to eukaryotic expression vector pcDNA3.1(+) were all correct.The recombinant eukaryotic expression vector pcDNA3.1(+)-ST3GAL Ⅰ had been constructed successfully,which laid the foundation for further eukaryotic expressing and studying of functional of ST3GAL Ⅰ gene.更多还原