鸭MHCⅠ轻链cDNA克隆及其蛋白的二级结构分析  

cDNA Cloning and Protein Structure Analysis of Duck MHC ClassⅠ Light Chain (Anpl-β2m )

作　　者：阮小飞[1] 蒋一男[1] 高凤山[1] 陈卫红[1] 郝慧芳[1] 夏春[1] 

机构地区：[1]中国农业大学动物医学院,北京100094

出　　处：《农业生物技术学报》2007年第1期41-45,共5页Journal of Agricultural Biotechnology

摘　　要：为了阐明鸭主要组织相容性复合体I（MHCⅠ）的结构并推进其功能研究，采用Full RACE PCR法从鸭淋巴细胞cDNA文库中克隆了鸭MHCⅠ轻链（Anp1-β2m）cDNA，GenBank登陆号为AB246408。随后，采用pMAL-p2X／E．coli TB1原核表达系对Anp1-β2m成熟肽基因进行了可溶性表达，表达的融合蛋白命名为MBP-β2m。融合蛋白经过Amylose树脂亲和层析纯化、Western blot分析和蛋白酶切割后，用圆二色谱测定了其二级结构，并同源模建了Anp1-β2m蛋白的三级结构（3D）。cDNA克隆结果显示Anp1-β2m cDNA长792bp，包含18bp5’端和414bp3’端非翻译区。其ORF为119个氨基酸（aa），包含20aa信号肽和998aa成熟肽。成熟肽序列的第25位和80位为半胱氨酸（C），第80位半胱氨酸附近的“YTCRVDH”序列与免疫球蛋白超基因家族的特征基序“YxCxVxH”相符。氨基酸序列与鸡、鱼、蛙、人和鼠等的β2m基因比较，同源性30．3％-65．9％之间。圆二色谱测定结果显示Anp1-β2加蛋白呈典型的β折叠结构，α螺旋、β折叠、转角和随机卷曲含量分别为0、50、23和26。同源模建显示Anp1-β2m蛋白三级结构（3D）与人和小鼠β2m3D结构类似，由7个β片层组成，不含α螺旋。In order to clarify the structure of duck major histocompatibility complex MHC class Ⅰ （MHC Ⅰ ）, duck MHC class Ⅰ light chain cDNA （Anp1-β2m, GenBank accession No.AB246408） was cloned from a duck eDNA library by Full RACE PCR. Subsequently, the mature protein of β2m gene was expressed solubility in pMAL-p2X/E, coli TB 1 system, and the fusion protein was named MBP-β2m. Then the fusion protein was purified, analyzed by Western blot and cleaved by the Factor Xa protease. At last, the purified MBP-β2m protein was estimated by circular dichroism （CD） spectrum, and the three-dimentional （3D） structure of the Anp1-β2m protein was analyzed by homology modeling. As result, the sequence of Anp1-β2m eDNA was 792 bp length, containing 18 bp 5＇-UT and 414 bp 3＇-UT. the Anp1-β2m gene encoded 119 amino acids, including 20 aa of signal peptide and 99 aa of mature protein. According to the sequence analysis, two cysteines were located in the 25 and 80 sites of the Anp1-β2m mature protein, and the ＂YTCRVDH＂arround the cysteine at the 80 site was similar to the YxCxVxH Ig-motif character. The Anp1-β2m gene shared a 30.3% -65.9% aa homology with the chicken, fish, flog, human and mouse＇s β2m. Analysis by CD spectrum revealed that the Anp1-β2m protein displayed typical β-sheet structure, and the contents of α-helix, β-sheet, turn, and random coil were 0, 50, 23 and 26, respectively. The 3D structure of the Anp1-β2m protein by homology modeling was similar as the 3D structure of human and mouse β2m, consisting of seven b-sheets without the α-helix.

关 键 词：鸭 Β2微球蛋白 二级结构 同源模建 

分 类 号：S188[农业科学—农业基础科学]