小尾寒羊β2微球蛋白的表达及其二级结构预测分析

为研究小尾寒羊绵羊白细胞抗原Ⅰ（Ovis aries leukocyte antigen classⅠ，OLAⅠ）轻链四聚体前体链β₂微球蛋白（β₂-microglobulin，β₂m）的结构和功能，根据GenBank中公布的绵羊β₂m基因设计特异性引物，提取小尾寒羊全血中的RNA并运用RT-PCR方法扩增绵羊β₂m基因，将扩增的绵羊β₂m基因克隆到pMD18-T载体，筛选出阳性克隆菌pMD18T-OLAⅠ-β₂m，经双酶切后与表达载体pET-28a（+）连接，再转化大肠杆菌BL21（DE3）感受态细胞中构建pET-28a（+）-OLAⅠ-β₂m重组表达菌，经IPTG诱导表达，SDS-PAGE检测目的蛋白大小及表达情况；运用SOMPA在线软件预测OLAⅠ-β₂m蛋白的二级结构。PCR扩增结果显示，目的基因大小为357 bp，与理论值相符，扩增片段成功克隆到pMD18-T载体，经EcoR Ⅰ和Hind Ⅲ双酶切筛选及测序，成功获得阳性克隆菌株pMD18-T-OLAⅠ-β₂m，插入的目的片段大小为357 bp。阳性克隆菌株与表达载体pET-28a（+）经EcoR Ⅰ和Hind Ⅲ双酶切后连接，转化大肠杆菌BL21（DE3）感受态细胞后获得pET-28a（+）-OLAⅠ-β₂m重组表达菌，经IPTG诱导表达，Western blotting检测目的蛋白大小为17.3 ku，目的蛋白在大肠杆菌中主要以包涵体的形式表达，经洗涤、变性、纯化、初步获得SDS-PAGE纯化的OLAⅠ-β₂m蛋白；PORTER在线软件预测OLAⅠ-β₂m蛋白的二级结构元件α-螺旋（Hh）、β-折叠（Ee）和无规则卷曲（Cc）分别占22.03%、22.03%和55.93%。本研究成功构建了小尾寒羊β₂m基因的pET-28a（+）重组表达体系，运用SOMPA在线软件预测OLAⅠ-β₂m的二级结构，为下一步绵羊OLAⅠ类分子四聚体的构建奠定基础。

Expression and Secondary Structure Analysis of β2-microglobulin in Small-tailed Han Sheep

In order to study the structure and function of Small-tailed Han sheep leukocyte antigen (OLA) class Ⅰ tetramer light chain β₂-microglobulin (β₂m).A pair of specific primers based on the published sequence of this gene was designed and the cDNA of full coding region for β₂m precursor was obtained by RT-PCR from the Small-tailed Han sheep whole blood.The Small-tailed Han sheep β₂m gene was cloned into the pMD18-T vector and the positive clones of pMD18T-OLAⅠ-β₂m were selected.After double digestion,the β₂m gene in positive clones was further ligated into the pET-28a(+) expression vector and transformed into E.coli BL21(DE3) to construct the recombinant strain of pET-28a(+)-OLAⅠ-β₂m.Then the targeted protein was detected by SDS-PAGE within induction of IPTG.The secondary structure of the OLAⅠ-β₂m protein was predicted and analyzed by SOMPA software.The PCR results showed that the length of OLAⅠ-β₂m was about 357 bp which was consistent with the theoretical value.The amplified fragment was successfully cloned into pMD18-T vector,and the positive clones were verified by EcoR Ⅰ and Hind Ⅲ digestion and DNA sequencing analysis.The cut β₂m gene from pMD18T-OLAⅠ-β₂m was inserted into pET-28a(+) and transformed into E.coli BL21(DE3) successfully.After IPTG induction,the expressed protein was detected by western blotting and SDS-PAGE,the results showed that the target protein (17.3 ku) was expressed efficiently with inclusion body.Finally,the secondary structure α-helix,extended strand and random coil of the target protein was 22.03%,22.03% and 55.93%,respectively.In this study,the recombinant tetramer precursor of OLAⅠ-β₂m light chain was constructed into pET-28a(+) expression line successfully,and the secondary structure of the OLAⅠ-β₂m protein was predicted and analyzed by SOMPA software,which would lay a solid foundation for constructing the OLAⅠtetramer of Small-tailed Han sheep.