摘要
目的:制备昆虫抗冻蛋白MpAFP149特异性抗血清,以便从细胞水平阐明昆虫抗冻蛋白在转基因烟草中的功能基础。方法:将具有完整读码框的MpAFP149基因连接到真核表达载体pcDNA3上,用做DNA免疫质粒。另外,将编码成熟肽的抗冻蛋白基因片段MpsAFP149插入原核表达载体pGEX-4T-1中,形成的重组质粒pGEX-4T-1-MpsAFP149转化大肠杆菌BL21(DE3),获得融合蛋白GST-sAFP149,用于蛋白免疫。采用"DNA初免-蛋白质加强"的策略免疫小鼠,制备抗血清,Western blot检测抗血清的特异性。制备野生型和转有MpAFP149转基因烟草叶片的超薄切片,利用免疫胶体金技术检测异源抗冻蛋白的表达及亚细胞水平的表达定位。结果:构建了昆虫抗冻蛋白基因MpAFP149的原核和真核表达载体;获得了预期的相对分子质量(Mr)约为36 000的融合蛋白。Western blot和转基因烟草的免疫定位结果表明此免疫策略可使小鼠产生特异性的昆虫抗冻蛋白抗血清。抗冻蛋白MpAFP149在转基因烟草中得到了表达并主要分布植物细胞质外体中的细胞壁上。结论:含有抗冻蛋白基因MpAFP149的真核表达质粒pcDNA3-MpAFP149和融合蛋白GST-sAFP149,通过"DNA初免-蛋白质加强"的免疫策略,可使小鼠产生专一性的抗血清。该抗血清可有效地用于抗冻蛋白基因MpAFP149转基因烟草的免疫定位研究。
AIM： To prepare the polyclonal antibody against insect antifreeze protein MpAFP149 and use it to identify the expression of the heterologous antifreeze protein in transgenic tobacco.METHODS： Eukaryotic expression vector pcDNA3-MpAFP149 was constructed as a DNA vaccine by inserting MpAFP149 gene into pcDNA3 vector.The gene fragment encoding MpAFP149 mature peptide was cloned into pGEX-4T-1 vector to yield recombinant pGEX-4T-1-MpsAFP149.The pGEX-4T-1MpsAFP149 was transformed into E.coli BL21（DE3）.GST-sAFP149P fusion protein was obtained after IPTG induction,which was used as a protein vaccine.The polyclonal antibody was generated by the DNA prime-protein boost vaccination strategy and its speciality was analyzed by Western blot.Ultra-thin sections for leaves of transgenic tobacco were assayed for the expression and distribution of heterologous protein MpAFP149 by immunogold particle technique.RESULTS： The prokaryotic and eukaryotic expression vectors carrying MpAFP149 were constructed.The recombinant protein GST-MpAFP149 was expressed with the expected molecular weight at 36 kDa.The results of Western blot and immunogold localization confirmed that mouse polyclonal antibody against MpAFP149 was obtained.CONCLUSION： The recombinant expression vectors carrying MpAFP149 were successfully constructed and the polyclonal antibody against MpAFP149 was obtained.The immunogold localization by TEM（transmission electron microscope） showed that the heterologous MpAFP149 protein was mainly rocalized in the cell wall in apoplast of the transgenic tobacco plant.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2010年第11期1101-1104,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
农业部转基因生物新品种培育重大专项(2009ZX08005-022B)
