沙眼衣原体Omp2的表达、纯化及其单克隆抗体的研制与鉴定
发布时间：2019-06-28 12:01
【摘要】：目的:用构建含沙眼衣原体(Chlamydia trachomatis, Ct)血清型D型外膜蛋白2(outer membrane protein 2, Omp2)167～434位氨基酸编码基因(omp2’)的重组表达载体,在大肠杆菌E. coli BL21(DE3)中表达Omp2重组蛋白;免疫BALB/c雌鼠,研制并鉴定其单克隆抗体,为Ct感染诊断试剂盒研制与其致病机制的研究提供实验依据。 方法:将含重组质粒pET28b(+)/omp2’在原核细胞中用IPTG诱导表达,SDS-PAGE、Western blot分析和鉴定表达的蛋白;采用强变性尿素溶解包涵体,经过亲和层析法对重组蛋白进行纯化;Bradford法测定纯化的重组蛋白的浓度。采用分步稀释,逐渐降低变性剂的浓度,并在复性缓冲溶液中加入一定量氧化、还原型谷胱苷肽和甘氨酸等,进行复性;用纯化的Omp2重组蛋白(rOmp2’)皮下多点免疫BALB/c雌鼠;无菌操作取其脾细胞,以聚乙二醇作为融合剂,与骨髓瘤细胞融合;有限稀释法进行单克隆化;间接ELISE法筛选阳性克隆;以琼脂糖免疫双扩散法鉴定单抗亚类;间接ELISE法测定腹水效价;细胞涂片染色镜检杂交瘤细胞株染色体数目;流式细胞仪分析杂交瘤细胞DNA含量;Western blot分析其特异性。 结果:SDS-PAGE分析显示,在IPTG诱导下,基因工程菌表达一相对分子质量(Mr)约为35KDa的目的蛋白,且主要以包涵体形式存在;经亲和层析后可获得纯度在96%左右的重组蛋白。纯化的rOmp2’免疫BALB/c小鼠,经细胞融
[Abstract]:Aim: to construct a recombinant expression vector containing Chlamydia trachomatis (Chlamydia trachomatis, Ct) serotype D outer membrane protein 2 (outer membrane protein 2, Omp2) 167 / 434 amino acid coding gene (omp2') and express Omp2 recombinant protein in E. coli BL21 (DE3), and to develop and identify its monoclonal antibody against BALB/c female mice, so as to provide experimental basis for the development of Ct infection diagnostic kit and the study of its pathogenic mechanism. Methods: the recombinant plasmid pET28b () / omp2' was induced and expressed in prokaryotic cells by IPTG, the expressed protein was analyzed and identified by SDS-PAGE,Western blot, the inclusion body was dissolved by strong denatured urea, and the recombinant protein was purified by affinity chromatography, and the concentration of the purified recombinant protein was determined by Bradford method. The concentration of denaturant was gradually decreased by step dilution, and a certain amount of oxidation, reduced glutathione and glycine were added to the renaturation buffer solution for renaturation. BALB/c female mice were immunized with purified Omp2 recombinant protein (rOmp2') subcutaneally. the spleen cells were obtained by aseptic operation and fused with myeloma cells with polyethylene glycol as fusion agent. The positive clones were screened by indirect ELISE. The monoclonal antibody subclasses were identified by agarose immunodouble diffusion method, the ascitic titer was determined by indirect ELISE method, the chromosome number of hybridoma cell line was detected by cell smear staining microscope, and the specificity of hybridoma cell DNA content; Western blot was analyzed by flow cytometry. Results: SDS-PAGE analysis showed that under the induction of IPTG, the relative molecular weight (Mr) was about the target protein of 35KDa, and mainly in the form of inclusion body, and the recombinant protein with purity of about 96% could be obtained by affinity chromatography. BALB/c mice were immunized with purified rOmp2' and melted by cells.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R374
本文编号：2507286
[Abstract]:Aim: to construct a recombinant expression vector containing Chlamydia trachomatis (Chlamydia trachomatis, Ct) serotype D outer membrane protein 2 (outer membrane protein 2, Omp2) 167 / 434 amino acid coding gene (omp2') and express Omp2 recombinant protein in E. coli BL21 (DE3), and to develop and identify its monoclonal antibody against BALB/c female mice, so as to provide experimental basis for the development of Ct infection diagnostic kit and the study of its pathogenic mechanism. Methods: the recombinant plasmid pET28b () / omp2' was induced and expressed in prokaryotic cells by IPTG, the expressed protein was analyzed and identified by SDS-PAGE,Western blot, the inclusion body was dissolved by strong denatured urea, and the recombinant protein was purified by affinity chromatography, and the concentration of the purified recombinant protein was determined by Bradford method. The concentration of denaturant was gradually decreased by step dilution, and a certain amount of oxidation, reduced glutathione and glycine were added to the renaturation buffer solution for renaturation. BALB/c female mice were immunized with purified Omp2 recombinant protein (rOmp2') subcutaneally. the spleen cells were obtained by aseptic operation and fused with myeloma cells with polyethylene glycol as fusion agent. The positive clones were screened by indirect ELISE. The monoclonal antibody subclasses were identified by agarose immunodouble diffusion method, the ascitic titer was determined by indirect ELISE method, the chromosome number of hybridoma cell line was detected by cell smear staining microscope, and the specificity of hybridoma cell DNA content; Western blot was analyzed by flow cytometry. Results: SDS-PAGE analysis showed that under the induction of IPTG, the relative molecular weight (Mr) was about the target protein of 35KDa, and mainly in the form of inclusion body, and the recombinant protein with purity of about 96% could be obtained by affinity chromatography. BALB/c mice were immunized with purified rOmp2' and melted by cells.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R374
【参考文献】
相关期刊论文 前2条
1 谭文庆;单链抗体的研究进展[J];国外医学(放射医学核医学分册);2001年02期
2 曲东明;抗体工程发展进程[J];河北医科大学学报;2003年01期
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