Objective To try making huZP3a^22-176 and huZP3b^177-348 polypeptides （representing an intact huZP^322-348 protein without its N-terminal signal peptide and C-terminal transmembrane domain ） express in E. coli at a ...Objective To try making huZP3a^22-176 and huZP3b^177-348 polypeptides （representing an intact huZP^322-348 protein without its N-terminal signal peptide and C-terminal transmembrane domain ） express in E. coli at a higher level Methods The cDNAs encoding huZP3a and huZP3b were obtained with PCR method. The pBV221 plasmid was used to construct thermo-inducible recombinant expression vector. Purification of two target expression products employed an improved method of preparative gel polyacrylamide gel electrophoresis. Results Two polypeptides of recombinant huZP3a （rhuZP3a） and recombinant huZP3b （rhuZP3b） were all expressed respectively in an E. coli BL21（DE3）pLysS strain at a higher level, which were recognized by two specific polyclonal antisera in Western blotting test which recognize a linear B cell epitope present in rhuZP3a or rhuZP3b respectively. Using the shake-flask method, approximately 5 mg of rhuZP3a and rhuZP3b with more than 95% relative homogeneity were harvested from 1 L culture respectively. Conclusion The availability of two rhuZP3 polypeptides will help in detecting the immunogenicities of rhuZP3a and rhuZP3b through animal experiments and confirming the function domain of non-glycosylated huZP3 to induce acrosome reaction in vitro.展开更多
目的:提高草原兔尾鼠(L agurus lagurus)卵透明带3(LZP 3)基因在酵母细胞中表达的水平.方法:利用重叠PCR技术,定点突变LZP 3基因上6个稀有的密码子簇,将LZP 3基因中11个稀有的密码子更换成毕赤酵母(P ich ia Pastoris)最常用的相应密码...目的:提高草原兔尾鼠(L agurus lagurus)卵透明带3(LZP 3)基因在酵母细胞中表达的水平.方法:利用重叠PCR技术,定点突变LZP 3基因上6个稀有的密码子簇,将LZP 3基因中11个稀有的密码子更换成毕赤酵母(P ich ia Pastoris)最常用的相应密码子.将获得的LZP 3突变基因(LZP 3m)插入pGAPZαA中,构建穿梭表达载体.以重组体转化P ich ia Pastoris SM D 1168菌株进行表达.结果:LZP 3m基因的表达量比野生型LZP 3基因明显提高.结论:通过密码子优化,能显著提高LZP 3基因在酵母细胞中的表达水平.展开更多
目的:构建草原兔尾鼠卵透明带3 DNA疫苗pVAX1-sig-LTB-lZP3-C3d3进行小鼠的黏膜免疫,增强该疫苗的免疫不育效果。方法:将两种佐剂分子大肠杆菌不耐热肠毒素B亚基和C3d基因通过酶切鉴定,构建重组质粒pVAX1-sig-LTB-lZP3-C3d3,采用RT-PCR...目的:构建草原兔尾鼠卵透明带3 DNA疫苗pVAX1-sig-LTB-lZP3-C3d3进行小鼠的黏膜免疫,增强该疫苗的免疫不育效果。方法:将两种佐剂分子大肠杆菌不耐热肠毒素B亚基和C3d基因通过酶切鉴定,构建重组质粒pVAX1-sig-LTB-lZP3-C3d3,采用RT-PCR和W estern b lot技术,检测其在mRNA和蛋白水平的表达,并通过肌肉注射、滴鼻和灌服3种途径免疫雌性C57BL/6小鼠,通过ELISA检测抗体水平及分型。结果:酶切鉴定,RT-PCR和W estern b lot结果表明重组质粒构建正确并可在mRNA和蛋白水平的表达,ELISA结果表明以重组质粒pVAX1-sig-LTB-lZP3-C3d3免疫诱导的特异性IgG、IgA的水平明显高于对照组(P<0.01)。抗生育实验表明该疫苗免疫的小鼠平均生仔数与对照组相比差异极显著(P<0.01)。结论:构建的草原兔尾鼠卵透明带3DNA疫苗重组质粒pVAX1-sig-LTB-lZP3-C3d3可高效地激发小鼠特异性免疫应答,提高抗生育的效果。展开更多
基金This work was supported by grant (No. 03JG05014) from the Population Family Planning Commission ofShanghai. China and the Medical and Health Science Research Foundation of Zhejiang Province(No. 2004A002)
文摘Objective To try making huZP3a^22-176 and huZP3b^177-348 polypeptides （representing an intact huZP^322-348 protein without its N-terminal signal peptide and C-terminal transmembrane domain ） express in E. coli at a higher level Methods The cDNAs encoding huZP3a and huZP3b were obtained with PCR method. The pBV221 plasmid was used to construct thermo-inducible recombinant expression vector. Purification of two target expression products employed an improved method of preparative gel polyacrylamide gel electrophoresis. Results Two polypeptides of recombinant huZP3a （rhuZP3a） and recombinant huZP3b （rhuZP3b） were all expressed respectively in an E. coli BL21（DE3）pLysS strain at a higher level, which were recognized by two specific polyclonal antisera in Western blotting test which recognize a linear B cell epitope present in rhuZP3a or rhuZP3b respectively. Using the shake-flask method, approximately 5 mg of rhuZP3a and rhuZP3b with more than 95% relative homogeneity were harvested from 1 L culture respectively. Conclusion The availability of two rhuZP3 polypeptides will help in detecting the immunogenicities of rhuZP3a and rhuZP3b through animal experiments and confirming the function domain of non-glycosylated huZP3 to induce acrosome reaction in vitro.
文摘目的:提高草原兔尾鼠(L agurus lagurus)卵透明带3(LZP 3)基因在酵母细胞中表达的水平.方法:利用重叠PCR技术,定点突变LZP 3基因上6个稀有的密码子簇,将LZP 3基因中11个稀有的密码子更换成毕赤酵母(P ich ia Pastoris)最常用的相应密码子.将获得的LZP 3突变基因(LZP 3m)插入pGAPZαA中,构建穿梭表达载体.以重组体转化P ich ia Pastoris SM D 1168菌株进行表达.结果:LZP 3m基因的表达量比野生型LZP 3基因明显提高.结论:通过密码子优化,能显著提高LZP 3基因在酵母细胞中的表达水平.
文摘目的:构建草原兔尾鼠卵透明带3 DNA疫苗pVAX1-sig-LTB-lZP3-C3d3进行小鼠的黏膜免疫,增强该疫苗的免疫不育效果。方法:将两种佐剂分子大肠杆菌不耐热肠毒素B亚基和C3d基因通过酶切鉴定,构建重组质粒pVAX1-sig-LTB-lZP3-C3d3,采用RT-PCR和W estern b lot技术,检测其在mRNA和蛋白水平的表达,并通过肌肉注射、滴鼻和灌服3种途径免疫雌性C57BL/6小鼠,通过ELISA检测抗体水平及分型。结果:酶切鉴定,RT-PCR和W estern b lot结果表明重组质粒构建正确并可在mRNA和蛋白水平的表达,ELISA结果表明以重组质粒pVAX1-sig-LTB-lZP3-C3d3免疫诱导的特异性IgG、IgA的水平明显高于对照组(P<0.01)。抗生育实验表明该疫苗免疫的小鼠平均生仔数与对照组相比差异极显著(P<0.01)。结论:构建的草原兔尾鼠卵透明带3DNA疫苗重组质粒pVAX1-sig-LTB-lZP3-C3d3可高效地激发小鼠特异性免疫应答,提高抗生育的效果。
