目的构建hITF毕赤酵母分泌型表达载体,表达重组hITF,为功能研究奠定基础。方法通过PCR获得hITFcDNA片段,将目的基因插入酵母表达载体pGAPZαA分泌信号下游,得到重组载体pGAPZαA-hITF。BspHⅠ线性化后氯化锂转化进入X-33,Zeoc in筛选转...目的构建hITF毕赤酵母分泌型表达载体,表达重组hITF,为功能研究奠定基础。方法通过PCR获得hITFcDNA片段,将目的基因插入酵母表达载体pGAPZαA分泌信号下游,得到重组载体pGAPZαA-hITF。BspHⅠ线性化后氯化锂转化进入X-33,Zeoc in筛选转化酵母菌,PCR鉴定目的基因。阳性转化子经摇瓶表达,取上清TCA沉淀后做Tric ine SDS-PAGE分析及W estern b lot检测。结果经测序及PCR证实,hITFcDNA准确插入酵母表达载体pGAPZαA中,氯化锂转化后,重组载体通过同源重组整合进入酵母基因组中。Tric ine SDS-PAGE分析证明hITF的分子量约为10×103,W estern b lot分析表明,表达蛋白具有良好的抗原性和特异性。结论成功构建出酵母表达载体pGAPZαA-hITF,获得重组hITF,为深入研究hITF奠定了基础。展开更多
The glycoprotein E2 contains major antigenic determinants and involved in inducing neutralization antibodies.A truncated form of the CSFV glycoprotein E2 was expressed in baculovirus.The signal sequences of E2 gene we...The glycoprotein E2 contains major antigenic determinants and involved in inducing neutralization antibodies.A truncated form of the CSFV glycoprotein E2 was expressed in baculovirus.The signal sequences of E2 gene were replaced with the honeybee melittin signal sequence,allowing efficient entrance into the secretory pathway in insect cell.In addition,the hydrophobic transmembrane anchors at the carboxyl termini of E2 proteins were removed to enable secretion rather than maintenance in the cellular membranes.Recombinant E2 protein was purified from the supernatant of infected cells by employing Ni2+ affinity chromatography.Protein purified was recognized by at least three anti-E2 pig sera and WH211 monoclonal antibody.WH211 monoclonal antibody couldn′t recognize the protein expressed in E.coli,indicating that it retain native structure.Thus,E2 expressed in insect cells can be used as a tool for diagnostic tests as well as obtaining material that could be suitable for X-ray crystallography studies.展开更多
文摘目的构建hITF毕赤酵母分泌型表达载体,表达重组hITF,为功能研究奠定基础。方法通过PCR获得hITFcDNA片段,将目的基因插入酵母表达载体pGAPZαA分泌信号下游,得到重组载体pGAPZαA-hITF。BspHⅠ线性化后氯化锂转化进入X-33,Zeoc in筛选转化酵母菌,PCR鉴定目的基因。阳性转化子经摇瓶表达,取上清TCA沉淀后做Tric ine SDS-PAGE分析及W estern b lot检测。结果经测序及PCR证实,hITFcDNA准确插入酵母表达载体pGAPZαA中,氯化锂转化后,重组载体通过同源重组整合进入酵母基因组中。Tric ine SDS-PAGE分析证明hITF的分子量约为10×103,W estern b lot分析表明,表达蛋白具有良好的抗原性和特异性。结论成功构建出酵母表达载体pGAPZαA-hITF,获得重组hITF,为深入研究hITF奠定了基础。
文摘The glycoprotein E2 contains major antigenic determinants and involved in inducing neutralization antibodies.A truncated form of the CSFV glycoprotein E2 was expressed in baculovirus.The signal sequences of E2 gene were replaced with the honeybee melittin signal sequence,allowing efficient entrance into the secretory pathway in insect cell.In addition,the hydrophobic transmembrane anchors at the carboxyl termini of E2 proteins were removed to enable secretion rather than maintenance in the cellular membranes.Recombinant E2 protein was purified from the supernatant of infected cells by employing Ni2+ affinity chromatography.Protein purified was recognized by at least three anti-E2 pig sera and WH211 monoclonal antibody.WH211 monoclonal antibody couldn′t recognize the protein expressed in E.coli,indicating that it retain native structure.Thus,E2 expressed in insect cells can be used as a tool for diagnostic tests as well as obtaining material that could be suitable for X-ray crystallography studies.
