CAJ | 학술논문

目的构建人脂蛋白相关磷脂酶A2(Lp-PLA2)的表达载体,表达Lp-PLA2重组蛋白,并制备其单克隆抗体(mAb).方法合成Lp-PLA2基因并插入融合表达载体pBV220的多克隆位点,构建重组表达质粒pBV220/Lp-PLA2.以重组质粒转化大肠杆菌DH5α,筛选阳性重组子诱导目的蛋白的表达,表达产物的免疫学活性用Western blot进行鉴定.以基因重组的Lp-PLA2蛋白为抗原,常规方法免疫BALB/c小鼠,取其脾细胞与NS-1细胞融合,获得稳定分泌Lp-PLA2 mAb的杂交瘤细胞株,ELISA检测mAb的效价;Western blot检测mAb的特异性.结果酶切鉴定和DNA测序显示Lp-PLA2重组表达载体中含有人Lp-PLA2全长编码序列.将该重组载体转化入大肠杆菌DH5α中表达所得蛋白经Western blot验证为目的蛋白,筛选出2株能稳定分泌特异性抗Lp-PLA2的mAb杂交瘤细胞株.结论该研究成功地构建了Lp-PLA2表达载体、表达出Lp-PLA2重组蛋白,制备出抗Lp-PLA2 mAb,为进一步用于Lp-PLA2的体外免疫学检测奠定了基础.
목적구건인지단백상관린지매A2(Lp-PLA2)적표체재체,표체Lp-PLA2중조단백,병제비기단극륭항체(mAb).방법합성Lp-PLA2기인병삽입융합표체재체pBV220적다극륭위점,구건중조표체질립pBV220/Lp-PLA2.이중조질립전화대장간균DH5α,사선양성중조자유도목적단백적표체,표체산물적면역학활성용Western blot진행감정.이기인중조적Lp-PLA2단백위항원,상규방법면역BALB/c소서,취기비세포여NS-1세포융합,획득은정분비Lp-PLA2 mAb적잡교류세포주,ELISA검측mAb적효개;Western blot검측mAb적특이성.결과매절감정화DNA측서현시Lp-PLA2중조표체재체중함유인Lp-PLA2전장편마서렬.장해중조재체전화입대장간균DH5α중표체소득단백경Western blot험증위목적단백,사선출2주능은정분비특이성항Lp-PLA2적mAb잡교류세포주.결론해연구성공지구건료Lp-PLA2표체재체、표체출Lp-PLA2중조단백,제비출항Lp-PLA2 mAb,위진일보용우Lp-PLA2적체외면역학검측전정료기출.
Objective To prepare monoclonal antibody(mAb) against recombinant human lipoprotein-associated phospholipase A2(Lp-PLA2).Methods The full length gene encoding Lp-PLA2 was synthesized and inserted into expression plasmid pBV220 to construct recombinant plasmid pBV220/Lp-PLA2.The recombinant plasmid was transformed into E.coli DH5α which then expressed Lp-PLA2.The immunological activity of the expressed Lp-PLA2 was analyzed by Western blot.Recombinant human Lp-PLA2 protein was used as antigen to immunize BALB/c mice.Monoclonal anti-bodies against Lp-PLA2 were prepared by normal hybridoma technology.The antibody titer of mAbs was determined by ELISA.Specificity of mAbs was analyzed by Western blot.Results Human Lp-PLA2 gene was synthesized and confirmed by DNA sequencing.Positive recombinant clones were identified by restriction enzyme digestion analysis and DNA sequencing.Westem blot analysis showed that the Lp-PLA2 protein could be recognized by an anti-Lp-PLA2 antibody.Two hybridmas producing antibodies against Lp-PLA2 were obtained.Conclusion The recombinant expression plasmid of Lp-PLA2 was constructed successfully and expressed in E.coli.The method of ELISA established to test serum Lp-PLA2 is useful.The Lp-PLA2 mAb using Lp-PLA2 as antigen prepared in this paper can be used for immunoassay in vitro.