粉尘螨Ⅰ类抗原cDNA的克隆表达和初步鉴定

目的　构建粉尘螨Ⅰ类抗原 (Derf1)cDNA基因的重组表达质粒 ,并于E .coli表达。方法　用BamHⅠ和SacⅠ从重组质粒pMD 18T Derf 1上切下Derf1基因 ,插入表达载体pET32a( )质粒 ,转化大肠杆菌BL2 1,在氨苄青霉素阳性的LB平板上筛选阳性重组子 ,并经双酶切及PCR扩增鉴定。重组质粒pET32a( ) Derf 1转化大肠杆菌 ,IPTG诱导表达后进行SDS PAGE电泳和薄层凝胶扫描定量分析。结果　对重组质粒进行酶切和PCR鉴定 ,与预期结果相符 ,证明已成功构建携带Derf1基因的重组原核表达质粒pET32a( ) Derf 1。核酸序列测定及同源性分析证实所构建的原核表达质粒pET32a( ) Derf1中所含的Derf1基因与GenBank中的Derf1序列同源性达到 99.5 %。Derf 1基因在大肠杆菌诱导表达后获得Mr 约4 5 0 0 0的蛋白 ,蛋白含量占全菌体蛋白含量的 15 %。结论　成功构建了粉尘螨Ⅰ类抗原cDNA基因的重组表达质粒pET32a( ) Derf 1,并在大肠杆菌中获得高效表达 ,为获得重组纯化Derf 1变应原并用于尘螨变应性疾病的诊治奠定基础

Construction and expression of prokaryotic expression plasmid of Der f 1 cDNA of Dermatophagoides farinae

Objective To construct and express prokaryotic expression plasmid of Der f 1 cDNA of Dermatophagoides farinae. Me- thods The Der f 1 gene was digested from recombinant plasmid pMD-18T-Der f 1 by restriction endonuclease, and then inserted into expression vector pET-32a ( ) by subclone technique. The recombinants were transferred into E.coli DL21 and identified by restriction endonuc- lease digestion and PCR. The expression of the recombinant plasmid pET-32a ( )-Der f 1 in the genetically engineered bacteria was induced by IPTG, and the expression products were analyzed by SDS-PAGE and densitometric scanning. Results The identified results of the positive recombinant plasmids pET32a( )-Der f 1 by restriction endonuclease digestion and PCR were in accordance with the expected results. Sequence determination analysis showed that the gene homology with the Der f 1 reported in GenBank was 99.5%. The plasmid pET-32a ( )-Der f 1 could express a specific M r 45 000 protein in E.coli BL21, 15% of total protein of recombinant bacterial. Conclusion The prokaryo-tic expression plasmids, which contain Der f 1 cDNA of Dermatophagoides farinae have been constructed successfully. Plasmid pET-32a( )-Der f 1 can express specific protein in E.coli DL21.