CAJ | 학술논문

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.
목적:재대장간균중분별중조표체의남개감로당감매Ⅰ(ATMDSI)화인 N-을선포도당알전이매 I(HsGnTI),제비기다극륭항체,위기인표체감정제공검측항체.방법:용 PCR 방법극륭 ATMDSI、HsGnTI 기인편단,련접지pBV220표체재체후전화대장간균 DH5α,획득표체균주,통과42℃승온유도표체,제비순화 ATMDSI 화 HsGnTI;순화적단백이80μg/kg 적제량면역대이백토,경3차면역후,채집혈청제비기상응적다극륭항체;채용 Western 인적검측다극륭항체적특이성.결과:획득 ATMDSI 화 HsGnTI 기인편단,병구건료기상응적원핵표체재체 pBV220-ATMDSI、pBV220-HsGnTI,재대장간균중표체료중조 ATMDSI 화 HsGnTI,SDS-PAGE 분석현시기상대분자질량분별위45.3×103화46.9×103,여이론치일치;용순화적단백면역대이백토후제비료항 ATMDSI、HsGnTI 다극륭항체,Western 인적결과증명해항체구유교고적특이성.결론:획득료특이성교고적항 ATMDSI、HsGnTI 다극륭항체혈청,위감로당감매Ⅰ화 N-을선포도당알전이매Ⅰ적연구제공료검측항체.
Objective: Arabidopsis thaliana mannosidaseⅠ(ATMDSI) and Homo sapiens N-acetylglucosaminyl trans?feraseⅠ(HsGnTI) were expressed in Escherichia coli DH5α for producing polyclonal antibodies respectively. Meth?ods: The ATMDSI and HsGnTI genes were amplified and subcloned into prokaryotic expression vector pBV220 and transformed to E.coli DH5α to express. Rabbits were immunized with the purified ATMDSI and HsGnTI pro?teins 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.