P-糖蛋白基因疫苗的构建与鉴定

目的　制备P 糖蛋白基因疫苗。方法　利用PCR方法扩增编码人类P 糖蛋白多药耐药基因序列胞外区约 1kb片段 ,与真核表达载体pcDNA3进行定向重组 ,限制性内切酶BamHI和XhoI双酶切反应及测序鉴定该重组基因疫苗 ,磷酸钙共沉淀法转染人类K5 62红白血病细胞 ,经G418筛选后 ,免疫组化分析该重组基因疫苗表达产物的抗原特异性。结果　构建了pcDNA3 MDR1重组基因疫苗。限制性内切酶BamHI和XhoI双酶切分别显示 5 .4kb的载体片段及约 1kb的插入序列 ,测序 948个碱基中除 2个碱基突变外均与原序列排列相符。免疫组化方法证实细胞膜表面MDR1约 1kb片段的表达产物可被抗Pgp抗体识别。结论　构建的pcDNA3 MDR1重组基因疫苗可被市售的Pgp抗体特异性识别 ,具有Pgp抗原特异性

Construction and Characterization of P-glycoprotein DNA Vaccine

Objective To prepare recombinant P glycoprotein DNA vaccine.Methods A 1kb fragment of the extracellular region of multiple drug resistant(MDR1) gene encoding human P glycoprotein was amplified by PCR,inserted into eucaryotic expression vector pcDNA3,digested with BamHI and Xho I,and tranfected to human K562 cells by calcium phosphate co precipitation.The expressed product was screened by G418 and analyzed for antigen specificity by immunohistochemical method.Results A recombinant DNA vaccine pcDNA3 MDR1 was constructed.The digestion with BamHI and Xho I showed a 5.4kb vector fragment and a 1kb inserted sequence respectively.Sequencing showed the mutation of only 2 bases.Immunohistochemical method proved that the expressed product could be recognized by antibody to Pgp.Conclusion The constructed pcDNA3 MDR1 vaccine showed Pgp antigen specificity.