CAJ | 학술논문

本研究通过RT-PCR从正常鸡外周血淋巴细胞总RNA中扩增chβ2m 基因全长片段，然后将其克隆至原核表达载体pET-32a（+），转化大肠杆菌BL21（DE3），用IPTG对其进行诱导表达，利用HiTrap亲和柱对表达产物进行纯化。结果显示，采用RT-PCR扩增出chβ2m基因全长约360 bp，编码120个氨基酸，与基因库公布的相一致，同源性为100%；经0.8 mM IPTG诱导表达后，SDS-PAGE分析发现chβ2m融合蛋白主要以可溶性形式存在于细菌裂解上清；利用His标签纯化该蛋白， SDS-PAGE分析仅见约34 kDa大小的chβ2m融合蛋白条带；Western-blot分析表明，纯化后的chβ2m融合蛋白与特异单克隆抗体反应呈现特异性的条带。以上结果证实，本研究成功表达并获得了纯化的可溶性chβ2m融合蛋白，为进一步对chβ2m结构及其功能研究奠定基础。
본연구통과RT-PCR종정상계외주혈림파세포총RNA중확증chβ2m 기인전장편단，연후장기극륭지원핵표체재체pET-32a（+），전화대장간균BL21（DE3），용IPTG대기진행유도표체，이용HiTrap친화주대표체산물진행순화。결과현시，채용RT-PCR확증출chβ2m기인전장약360 bp，편마120개안기산，여기인고공포적상일치，동원성위100%；경0.8 mM IPTG유도표체후，SDS-PAGE분석발현chβ2m융합단백주요이가용성형식존재우세균렬해상청；이용His표첨순화해단백， SDS-PAGE분석부견약34 kDa대소적chβ2m융합단백조대；Western-blot분석표명，순화후적chβ2m융합단백여특이단극륭항체반응정현특이성적조대。이상결과증실，본연구성공표체병획득료순화적가용성chβ2m융합단백，위진일보대chβ2m결구급기공능연구전정기출。
chβ2m gene was amplified from normal chicken peripheral blood lymphocytes by RT-PCR. It was cloned into pET-32a(+) vector and induced by IPTG, then purified with HiTrap affinity column. The results showed that the sequence of chβ2m gene amplified from chickens by RT-PCR was the same as that previously reported (M84767, AY989898, Z48921) and consists of 360 bp encoding 120 amino acids (aa). Most of the soluble recombinant chβ2m proteins existed in the bacterial supernatant induced with 0.8 M IPTG. Only one band of molecular weight of 34 kDa was purified by ion-exchange chromatography. Western blotling assay demonstrated that anti-His-tag monoclonal antibody could specifically recognize the recombinant protein. This work would provid us the basis materials for further study of the structure and functions of chβ2m.