衔接蛋白beta亚单位不同剪切体的克隆与鉴定
发布时间：2018-10-25 07:57
【摘要】：衔接蛋白2(adaptin protein 2,Ap2)是披网格蛋白小泡(clathrin-coated vesicles,CCVS)的重要组成部分,在蛋白质通过细胞膜进入胞质的转运过程中发挥着重要作用。我们通过人类睾丸cDNA芯片的方法将成人和6月龄的胚胎睾丸进行杂交比较,克隆了一个衔接蛋白2β(Ap2β)亚单位的不同剪接体-Ap2β-NY。芯片杂交结果显示,Ap2β-NY在成人睾丸中mRNA的表达量是胚胎睾丸的3.62倍。对Ap2β-NY基因进行测序,获得全长序列并输入GenBank,登录号为AY341427。生物学软件分析表明,全长为3457bp的Ap2β-NY基因编码了一个具有880个氨基酸的蛋白质,预测此蛋白质的分子量为98.12KDa,等电点PI为5.01,定位于人17号染色体上的q11.2-q12。对预测蛋白进行生物信息学分析发现,Ap2β-NY具有Ap2β的全部三个结构域,即具有一个Ap2β氮末端结构域,一个低度重复序列和一个Ap2β炭末端结构域。但是Ap2β-NY的氮末端结构域比Ap2β短了57个氨基酸残基,并且这57个氨基酸残基是疏水性的,很有可能位于蛋白质的表面,是蛋白质相互作用的结合位点。用反转录PCR(reverse transcription PCR,RT-PCR)的方法检测成人和胚胎睾丸Ap2β-NY的表达状况,证实了芯片杂交结果。同时精子RT-PCR表明Ap2β-NY在精子中也高表达。用Southern杂交的方法检测Ap2β-NY在16种正常人体组织中的表达情况,发现Ap2β-NY高表达于脾脏、胸腺、前列腺、卵
[Abstract]:Binding protein 2 (adaptin protein 2 / Ap2 is an important component of drape grid protein vesicles (clathrin-coated vesicles,CCVS), which plays an important role in the transport of proteins into the cytoplasm through the cell membrane. Using human testis cDNA microarray, we compared adult testis with 6-month-old embryonic testis and cloned Ap2 尾 NY., a splice of different splicing units of Ap2 尾. The results of microarray hybridization showed that the expression of Ap2 尾-NY in adult testis was 3.62 times higher than that in embryonic testis. The Ap2 尾-NY gene was sequenced, the full-length sequence was obtained and the GenBank, login number was entered as AY341427.. The analysis of biological software shows that the Ap2 尾-NY gene with full length of 3457bp encodes a protein with 880 amino acids. The predicted molecular weight of the protein is 98.12 KDaand the isoelectric point PI is 5.01, which is located at q11.2-q12 on human chromosome 17. Bioinformatics analysis showed that Ap2 尾-NY had all three domains of Ap2 尾, namely, one Ap2 尾 N-terminal domain, one low-degree repeat sequence and one Ap2 尾 C-terminal domain. However, the nitrogen terminal domain of Ap2 尾-NY is 57 amino acid residues shorter than Ap2 尾, and the 57 amino acid residues are hydrophobic and probably located on the surface of protein and are binding sites for protein interaction. Reverse transcription PCR (reverse transcription PCR,RT-PCR (RT PCR (reverse transcription PCR,RT-PCR) was used to detect the expression of Ap2 尾-NY in adult and embryonic testis, which confirmed the results of microarray hybridization. At the same time, sperm RT-PCR showed that Ap2 尾-NY was also highly expressed in spermatozoa. The expression of Ap2 尾-NY in 16 normal human tissues was detected by Southern hybridization. It was found that Ap2 尾-NY was highly expressed in spleen, thymus, prostate and egg.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R341
本文编号：2293098
[Abstract]:Binding protein 2 (adaptin protein 2 / Ap2 is an important component of drape grid protein vesicles (clathrin-coated vesicles,CCVS), which plays an important role in the transport of proteins into the cytoplasm through the cell membrane. Using human testis cDNA microarray, we compared adult testis with 6-month-old embryonic testis and cloned Ap2 尾 NY., a splice of different splicing units of Ap2 尾. The results of microarray hybridization showed that the expression of Ap2 尾-NY in adult testis was 3.62 times higher than that in embryonic testis. The Ap2 尾-NY gene was sequenced, the full-length sequence was obtained and the GenBank, login number was entered as AY341427.. The analysis of biological software shows that the Ap2 尾-NY gene with full length of 3457bp encodes a protein with 880 amino acids. The predicted molecular weight of the protein is 98.12 KDaand the isoelectric point PI is 5.01, which is located at q11.2-q12 on human chromosome 17. Bioinformatics analysis showed that Ap2 尾-NY had all three domains of Ap2 尾, namely, one Ap2 尾 N-terminal domain, one low-degree repeat sequence and one Ap2 尾 C-terminal domain. However, the nitrogen terminal domain of Ap2 尾-NY is 57 amino acid residues shorter than Ap2 尾, and the 57 amino acid residues are hydrophobic and probably located on the surface of protein and are binding sites for protein interaction. Reverse transcription PCR (reverse transcription PCR,RT-PCR (RT PCR (reverse transcription PCR,RT-PCR) was used to detect the expression of Ap2 尾-NY in adult and embryonic testis, which confirmed the results of microarray hybridization. At the same time, sperm RT-PCR showed that Ap2 尾-NY was also highly expressed in spermatozoa. The expression of Ap2 尾-NY in 16 normal human tissues was detected by Southern hybridization. It was found that Ap2 尾-NY was highly expressed in spleen, thymus, prostate and egg.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R341
【参考文献】
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1 席晔斌,王保国,陈广洁,包士三,李伟毅;大鼠Leydig细胞分泌的IL-1和TGF-β_1在感染时的免疫调节作用[J];生殖与避孕;2002年05期
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