粉尘螨Ⅰ类变应原瞬时表达载体的构建及其在烟草中的表达

Construction on transgenic recombinant vector of group Ⅰ allergen from Dermatophagoides farinae and its expression in tobacco

目的构建粉尘螨I类变应原瞬时表达载体TRBO-Der f1并观察其在烟草中的表达。方法以粉尘螨总RNA为模板,采用RT-PCR方法扩增Der f1基因并定向克隆到瞬时表达载体TRBO中,转化根癌农杆菌(Agrobacterium tumefa-ciens)GV1301株后,抽提质粒进行酶切及PCR鉴定。将含TRBO-Der f1重组质粒的GV1301注射本氏烟叶片,SDS-PAGE电泳检测注射3、4、5、6d后Der f 1的表达,并进行Western blot鉴定。结果电泳及测序表明Der f1基因克隆成功,大小为627bp。经PCR及酶切结果证实重组质粒TRBO-Der f1成功转入根癌农杆菌。SDS-PAGE电泳检测表明,该蛋白于第5和6d在烟草叶片中高表达,并通过Western blot得到验证。结论粉尘螨I类变应原Der f 1成功表达于烟草叶片,为进一步研究源于植物的粉尘螨疫苗奠定了基础。

To construct the transgenic recombinant vector of TRBO-Der f1and conduct its expression in tobacco,and the total RNA was extracted from D.farinae,the Der f1gene was amplified using total RNA as template by reverse transcription polymerase chain reaction(RT-PCR)and inserted into the transient expression vector TRBO for constructing the recombinant plasmid TRBO-Der f 1.The recombinant plasmid was identified using digestion and PCR after transforming to Agrobacterium tumefaciens(A.tumefaciens)line GV1301.Tobacco leaves were harvested at 3,4,5,and 6day respectively,following by infiltration with GV1301containing recombinant plasmid TRBO-Der f1,and Der f1proteins were identified by SDS-PAGE electrophoresis.Electrophoretic analysis and sequencing showed that the Der f 1gene,with 627bp,was cloned successfully and the recombinant plasmid TRBO-Der f1was transformed to A.tumefaciens line GV1301as expected.Der f 1proteins were expressed highly in tobacco leaves at 5and 6day after infiltration.The transgenic vector of recombinant plasmid TRBO-Der f1was constructed,and the Der f 1proteins were expressed in tobacco leaves successfully.Results in the present investigation would provide a basis for the further study on the transgenic plant vaccine.