人血管生成素1的克隆、序列分析和单酶切法构建毕氏酵母表达载体

Cloning and sequence analysis of human angiopoietin-1 gene and construction of Pichia Pastoris expression vector with single enzyme

目的:从人胎盘中克隆血管生成素1基因,并分析其结构特征,单酶切法构建毕氏酵母表达载体pPIC9K/Ang-1,为进一步研究血管生成素1防治糖尿病视网膜病变的血管渗漏奠定物质基础。方法:实验于2003-11/2004-06在北京大学医学部细胞系完成。提取人胎盘组织总RNA,用反转录聚合酶链式反应技术扩增出血管生成素1基因片段,并将其克隆到PGEM-TEasy载体中进行序列分析,用小牛肠碱性磷酸酶法去除线性化的pPIC9K质粒DNA两端磷酸基,以防止质粒自身环化,再亚克隆到毕氏酵母表达载体pPIC9K中。结果:①经甲醛变性胶电泳鉴定,成功从胎盘组织中提取出总RNA,RNA的完整性和纯度符合进一步的酶反应要求。②胎盘组织总RNA经oligo-dT引物反转录,用人血管生成素1特异一对引物进行聚合酶链反应扩增,10g/L琼脂糖胶电泳结果显示一条特异片段,大小为1.5kbcDNA片段。③成功构建了PGEM-T/Ang-1载体,Blast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%,国人血管生成素1基因核苷酸序列的起始密码子ATG,终止密码子为TGA,共1497个碱基对。④成功构建pPIC9K/Ang-1毕氏酵母表达载体。结论:从人的胎盘中克隆血管生成素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。

AIM： To clone human angiopoietin-1 （Ang-1） cDNA, analyze its characters of structure and construct its Pichia Pastoris expression vector Ppic9k/Ang-1, so as to make future base for the defenses of blood vessel seepage of diabetic retinopathy （DR）. METHODS： The experiment was conducted in the Department of Cells, Medical Institute of Beijing University between November 2003 and June 2004. Total RNA was abstracted from human fetus by TRI7OL reagents. The Ang-1 cDNA fragment was amplified by RT-PCR, and then cloned into PGEM-T Easy Vector for sequence analysis. The linear pPIC9K plasmid DNA both ends phosphoric acid base was removed with the law of calf intestines alkaline phosphoric acid enzyme （CIP）, so as to prevent the one＇s own ring of plasmid, and then subcloned into Pichia Pastoris Expression Vector pPIC9K. RESULTS： （1） Identification with formaldehyde gel electrophoresis showed that high-quality total RNA was successfully abstracted from Human placenta. The completeness and purity of total RNA met the enzyme reaction. （2） Total RNA underwent reverse transcription by oligo-dT primer. A fragment of human Ang-1 gene, 1.5 kb, was amplified by PCR. （3） PGEM-T/Ang-1 vector was constructed successfully, and Blast sequence analysis is in accordance with 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. Ang-1 nucleotide sequencestart have 1 497 base pairs, which start codon in ATG and terminator codon is TGA in Chinese. （4） The pPIC9K/Ang-1 yeast expression vector was constructed successfully. CONCLUSION： A fragment of human Ang-1 gene does not mutate. The pPIC9K/Ang-1 yeast expression vector is constructed successfully, which fulfills the needs of protein expressing.