【摘要】 目的全基因合成并原核表达免疫球蛋白M的重链(IgMμ链)及其各肽段,与天然的IgM、IgMμ链和IgG进行免疫原性的分析比较,进一步了解IgMμ链恒定区的结构与功能。方法根据GenBank数据库IgMμ链恒定区(CH1-CH4)的cDNA序列并进行必要的大肠杆菌密码子优化,利用重叠延伸PCR(OE-PCR)体外基因合成人IgMμ链恒定区全长(CH1-CH4)及其截断片段CH1-CH2和CH3-CH4。原核表达并纯化相应的3种融合蛋白PET32a-rMμCH1-CH4、PET32a-rMμCH1-CH2和PET32a-rMμCH3-CH4,并分别免疫BALB/c小鼠。同时设天然人IgM、IgMμ链和IgG免疫组为对照。再用ELISA法检测6种血清,分析比较其抗原性及免疫原性。结果利用OE-PCR方法体外成功合成1 359 bp的IgMμ链(CH1-CH4)全长基因、651 bp的IgMμ链CH1CH2和708 bp的IgMμ链CH3CH4,并构建相应原核表达质粒PET32a-rMμ1-4、PET32a-rMμ1-2和PET32a-rMμ3-4,血清ELISA检测结果显示:①IgG免疫原性>IgM>IgMμ链>rMμ1-4>rMμ1-2>rMμ3-4;②IgM免疫的血清与IgG有较强的交叉反应性,IgG免疫的血清与IgM也存在较强的交叉反应性;IgMμ链和重组μ链的各肽段免疫的血清与IgG均无明显的交叉反应性。结论重组的IgMμ链及其各肽段与天然IgMμ链有着相似的免疫原性和特异性,为基因工程得到抗人IgMμ链单克隆抗体奠定了研究基础。更多还原

【Abstract】 Objective Gene synthesis in vitro and prokaryotic expression of constant region of human IgMμ chain,then make immunogenicity and antigenicity analysis with other natrural IgM,IgMμ chain and IgG.The results made people to learn more about the structure and function of constant region of human IgMμ chain furthermore and lay the foundation for the study of human monoclonal antibody.Methods The cDNA sequence of human IgMμ chain constant region(CH1-CH4) was obtained from GenBank and necessary coden optimization was made due to the special preferance of E.coli,then the nucleotide sequence and its truncated sequences CH1-CH2 and CH3-CH4 were synthesized by OE-PCR.The three recombinant proteins PET32a-rMμ1-4,PET32a-rMμ1-2 and PET32arMμ3-4 were purified by affinity chromatography,and were used to immunize BALB / c mice.In the meanwhile the natural human IgM,IgMμ chain and IgG were also included as controls.The six kinds of mouse sera were tested by ELISA and analyze their immunogenicity and antigenicity.Results In the study,the gene encoding IgMμ chain constant region protein was synthesized by OE-PCR and three prokaryotic expression plasmids,PET32a-rMμ1-4a,PET32a-rMμ1-2 and PET32a-rMμ3-4 were constructed successfully.The results of ELISA showed that:① Immunogenicity analysis IgG > IgM > IgMμ chain > rMμ1-4 > rMμ1-2 > rMμ3-4;② Specificity analysis,intensity cross reaction between immune sera of IgM and immune sera of IgG.There is no obviously cross reaction between immune sera of IgMμ chain / recombinant each peptide constant of human IgMμ chain and immune sera of IgG.Conclusion Recombinant each peptide constant of human IgMμ chain have affinis immunogenicity and specificity with native IgMμ chain,which provides a valuable potential in further research on gene engineering obtained human IgMμ monoclonal antibodies.更多还原