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目的克隆Ang-1基因,分析其结构特征,构建pPIC9K/Ang-1毕氏酵母表达载体,为进一步研究Ang-1防治糖尿病视网膜病变的血管渗漏奠定物质基础。方法提取人胎盘组织总RNA,RT-PCR扩增出Ang-1基因片段,并将其克隆到PGEM-T Easy载体中进行序列分析,再亚克隆到毕氏酵母表达载体pPIC9K中。结果从胎盘组织总RNA中,RT-PCR扩增出1.5kb的cDNA片段,成功构建了PGEM-T/Ang-1载体,B last序列分析与GenBank中AY121504一致。两者成熟区cDNA的核苷酸和氨基酸同源性分别大于99%和98%,成功构建pPIC9K/Ang-1毕氏酵母表达载体。结论从人的胎盘中克隆Ang-1基因无突变,成功构建pPIC9K/Ang-1毕氏酵母表达载体,满足表达蛋白质的需要。
Objective To clone Ang-1 gene and analyze its structural characteristics, and to construct pPIC9K / Ang-1 Pichia pastoris expression vector, which will lay a material foundation for further study of Ang-1 in prevention and treatment of vascular leakage of diabetic retinopathy. Methods The total RNA of human placenta was extracted and the Ang-1 gene fragment was amplified by RT-PCR. The fragment was cloned into PGEM-T Easy vector and sequenced, then subcloned into pichia pastoris expression vector pPIC9K. Results A 1.5 kb cDNA fragment was amplified by RT-PCR from total placental total RNA. PGEM-T / Ang-1 vector was successfully constructed. The B last sequence analysis was consistent with that of AY121504 in GenBank. The nucleotide and amino acid homology of cDNA in mature region of the two strains were greater than 99% and 98%, respectively. The pPIC9K / Ang-1 Pichia pastoris expression vector was successfully constructed. Conclusion Cloning of Ang-1 gene from human placenta has no mutation, and pPIC9K / Ang-1 Pichia pastoris expression vector was successfully constructed to meet the needs of protein expression.