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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。
Objective: To prepare insect antithrombotic protein MpAFP149 specific antiserum to clarify the functional basis of insect antifreeze protein in transgenic tobacco at the cellular level. Methods: MpAFP149 gene with complete reading frame was ligated to eukaryotic expression vector pcDNA3 and used as DNA immunization plasmid. In addition, MSPAFP149was inserted into the prokaryotic expression vector pGEX-4T-1 and the recombinant plasmid pGEX-4T-1-MpsAFP149 was transformed into E.coli BL21 (DE3) to obtain the fusion protein GST-sAFP149, For protein immunization. The mice were immunized with the strategy of “DNA prime-protein boost” to prepare antiserum and Western blot to detect the specificity of antiserum. Ultrathin sections of wild-type and transgenic MpAFP149 transgenic tobacco leaves were prepared. The expression of heterologous anti-freeze protein and expression of sub-cellular level were detected by immunogold assay. Results: The prokaryotic and eukaryotic expression vectors of insect antifreeze protein MpAFP149 were constructed. The expected fusion protein with a relative molecular mass (Mr) of 36,000 was obtained. The immunolocalization results of Western blot and transgenic tobacco showed that this immunization strategy can produce specific antifreeze antisera in mice. The antifreeze protein MpAFP149 was expressed in transgenic tobacco and mainly distributed on the cell wall in plant cytoplasm. CONCLUSION: The eukaryotic expression plasmid pcDNA3-MpAFP149 containing the anti-freeze protein gene MpAFP149 and the fusion protein GST-sAFP149 can produce specific antisera to the mice through the immunization strategy of “DNA prime-protein boost”. The antiserum can be effectively used for immunolocalization of anti-freeze protein gene MpAFP149 transgenic tobacco.