摘要
目的:在大肠杆菌中分别重组表达拟南芥甘露糖苷酶Ⅰ(ATMDSI)和人N-乙酰葡萄糖胺转移酶I(HsGnTI),制备其多克隆抗体,为基因表达鉴定提供检测抗体。方法:用PCR方法克隆ATMDSI、HsGnTI基因片段,连接至pBV220表达载体后转化大肠杆菌DH5α,获得表达菌株,通过42℃升温诱导表达,制备纯化ATMDSI和HsGnTI;纯化的蛋白以80μg/kg的剂量免疫大耳白兔,经3次免疫后,采集血清制备其相应的多克隆抗体;采用Western印迹检测多克隆抗体的特异性。结果:获得ATMDSI和HsGnTI基因片段,并构建了其相应的原核表达载体pBV220-ATMDSI、pBV220-HsGnTI,在大肠杆菌中表达了重组ATMDSI和HsGnTI,SDS-PAGE分析显示其相对分子质量分别为45.3×103和46.9×103,与理论值一致;用纯化的蛋白免疫大耳白兔后制备了抗ATMDSI、HsGnTI多克隆抗体,Western印迹结果证明该抗体具有较高的特异性。结论:获得了特异性较高的抗ATMDSI、HsGnTI多克隆抗体血清,为甘露糖苷酶Ⅰ和N-乙酰葡萄糖胺转移酶Ⅰ的研究提供了检测抗体。
Objective： Arabidopsis thaliana mannosidaseⅠ （ATMDSI） and Homo sapiens N-acetylglucosaminyl trans- ferase Ⅰ （HsGnTI） were expressed in Escherichia coli DHSα for producing polyclonal antibodies respectively. Methods： The ATMDSI and HsGnTI genes were amplified and subcloned into prokaryotic expression vector pBV220 and transformed to E.coli DHSα to express. Rabbits were immunized with the purified ATMDS1 and HsGnTI proteins for developing the polyclonal antibodies against ATMDSI and HsGnTI, then their specificities were detected by Western blot. Results： The ATMDSI and HsGnTI genes were cloned and expressed in E.coli successfully. The recombinant proteins were expressed as the predicated molecular mass by SDS-PAGE. The result of Western blot demonstrated that the polyclonal antibodies were of high specificity. Conclusion： The polyclonal antibodies against ATMDSI and HsGnTI were prepared, which provides an experimental basis for the detecting expression and local- ization of mannosidase and N-acetylglucosaminyl transferase.
出处
《生物技术通讯》
CAS
2012年第6期817-820,共4页
Letters in Biotechnology
基金
国家自然科学基金(31200082)
北京市自然科学基金(5102037)
