人血管生成素-1的克隆和毕氏酵母表达载体pPIC9K/Ang-1的构建  

Cloning of Human Angiopoietin-1 Gene and Construction of its Pichia Pastoris Expression Vector pPIC9K/Ang-1

作　　者：马腾[1] 刘丽波[2] 黄波[1] 刘学政[1] 刘学[1] 

机构地区：[1]锦州医学院解剖教研室,辽宁锦州121001 [2]中国医科大学神经生物学教研室,辽宁沈阳110000

出　　处：《解剖科学进展》2005年第3期194-196,200,共4页Progress of Anatomical Sciences

基　　金：辽宁省自然基金项目(No.20022152);辽宁省科技计划资助项目(No.2003225007)

摘　　要：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 human angiopoietin-1 cDNA, analyse its structure and construct its Pichia Pastoris expression vector for making future base for the defence of blood vessel seepage of diabetic retinopathy（DR）. Methods Total RNA was extracted from human placenta by TRIZOL reagents. The angiopoietin-1 cDNA fragment was amplified by RT-PCR, cloned into PGEM-T Easy vector and then sequenced, furthermore subeloned into Pichia Pastoris expression vector pPIC9K. Results High-quality total RNA was extracted from human placenta. A fragment of human angiopoietin-1 gene, 1.5kb, was amplified by RT-PCR. PGEM-T/Ang-1 vector was constructed successfully, and the result of Blast sequence analysis is in accordance with that of AY121504 in GenBank. The results also showed that the homology of nucleotides and amino acids in mature fragment between cDNA of chinese Ang-1 and the published data was more than 99% and 98% respectively. The pPIC9K/Ang-1 yeast expression vector was constructed successfully. Conclusion A fragment of human angiopoietin-1 gene did not mutate. The pPICgK/Ang-1 yeast expression vector was constructed successfully for the need of protein expressing.

关 键 词：血管生成素-1 逆转录聚合酶链式反应 克隆 序列分析 

分 类 号：R331[医药卫生—人体生理学]