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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
Objective To construct a recombinant plasmid expressing cDNA of Dermatophagoides pteronyssinidae antigen (Derf1) and express it in E.coli. Methods The Derf1 gene was cut out from the recombinant plasmid pMD18T Derf 1 using BamHⅠ and SacⅠ and inserted into the expression vector pET32a (+). The recombinant plasmid was transformed into E. coli BL21. The positive recombinant was screened on ampicillin-positive LB plates and double- Cut and PCR amplification identification. The recombinant plasmid pET32a (+) Derf 1 was transformed into Escherichia coli. After IPTG induction, it was analyzed by SDS PAGE electrophoresis and thin layer gel scanning. Results The recombinant plasmids were digested with restriction endonucleases and identified by PCR. The results showed that the recombinant plasmid pET32a (+) Derf 1 carrying Derf1 gene was successfully constructed. Nucleotide sequencing and homology analysis confirmed that the Derf1 gene contained in the constructed prokaryotic expression plasmid pET32a (+) Derf1 has a homology of 99.5% with the Derf1 sequence in GenBank. Derf 1 gene was expressed in E. coli induced Mr about 45000 protein, protein content accounted for 15% of the whole body protein content. Conclusion The recombinant plasmid pET32a (+) Derf 1 was successfully constructed. The recombinant plasmid pET32a (+) Derf 1 was successfully expressed in Escherichia coli. In order to obtain the recombinant Derf 1 allergen, it was used in the diagnosis and treatment of allergic diseases of dust mites Lay the foundation