在烟草中瞬时表达人角质细胞生长因子1

Transient Expression of Human Keratinocyte Growth Factor 1 in Tobacco

目的在烟草中瞬时表达人角质细胞生长因子1(Keratinocyte growth factor 1,KGF1),旨在寻求一种低成本﹑方便﹑大量生产rhKGF1的方法。方法以重组质粒pET3c-hKGF1为模板,通过PCR法扩增hKGF1基因片段,插入经改造的马铃薯病毒X双元表达载体pGR107中,冻融法转化感受态农杆菌GV3101,以获得的GV3101-pGR107-rhKGF1侵染烟草。以绿色荧光蛋白基因(smGFP)为报告基因,建立马铃薯X病毒(PVX)瞬时表达体系。用紫外灯连续20 d观察经GV3101-pGR107-smGFP侵染的烟草中smGFP的表达。经RT-PCR法分析烟草中rhKGF1基因mRNA的转录,SDS-PAGE﹑Western blot分析烟草中rhKGF1蛋白的表达。结果重组表达质粒pGR107-rhKGF1经PCR及测序证实构建正确;转化的农杆菌GV3101-pGR107-rhKGF1侵染的烟草中目的蛋白的表达量在第8天达最大。目的蛋白rhKGF1在烟草中获得成功表达。结论在烟草中瞬时表达了rhKGF1,为其单克隆抗体的制备及产业化奠定了基础。

Objective To transiently express human keratinocyte growth factor 1（KGF1） in tobacco so as to explore a cheap and simple method for large-scale production of rhKGF1.Methods The hKGF1 gene fragment was amplified by PCR using recombinant plasmid pET3c-hKGF1 as a template,and inserted into modified binary expression potato virus X（pGR107）.The constructed recombinant plasmid was transformed to competent Agrobacterium GV3101.The obtained recombinant Agrobacterium GV3101-pGR107-rhKGF1 was infected to tobacco.Potato virus X（PVX） transient expression system was established using green fluorescent protein（smGFP） as a report gene.The expression of smGFP in tobacco infected with GV3101-pGR107-smGFP was observed for 20 consecutive days.The transcription of rhKGF1 mRNA in the infected tobacco was determined by RT-PCR,while the expression of rhKGF1 protein by SDS-PAGE and Western blot.Results PCR and sequencing proved that recombinant plasmid pGR107-rhKGF1 was constructed correctly.The expression level of target protein in Agrobacterium reached a peak value 8 d after transfection with GV3101-pGR107-rhKGF1.The target protein was successfully expressed in tobacco.Conclusion The rhKGF1 was transiently expressed in tobacco,which laid a foundation of preparation and industrialization of McAb against rhKGF1.