粉尘螨Ⅱ类抗原cDNA原核表达质粒的构建与表达

CONSTRUCTION AND EXPRESSION OF PROKARYOTIC EXPRESSION PLASMID OF cDNA CODING FOR GROUP Ⅱ ALLERGEN OF DERMATOPHAGOIDES FARINAE

目的 构建粉尘螨Ⅱ类抗原cDNA基因的重组表达质粒 ,并在大肠埃希菌中表达。 方法 采用亚克隆技术 ,用SacⅠ和NotⅠ从重组质粒pMD 18T Derf 2上切下Derf 2cDNA片段 ,插入表达载体 pET 3 2a(+ )质粒 ,转化大肠埃希菌BL2 1,在含氨苄青霉素的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 pET 3 2a(+ ) Derf2转化大肠埃希菌 ,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 ,获得 45 5bp大小的目的基因片段 ,与预期结果相符 ,证明已成功构建携带Derf 2基因的重组原核表达质粒pET 3 2a(+ ) Derf 2。核酸序列测定及同源性分析证实 ,所构建的原核表达质粒pET 3 2a(+ ) Derf 2中所含的Derf 2cDNA片段与GenBank中的Derf 2序列同源性达到 10 0 %。Derf 2cDNA在大肠埃希菌诱导表达后获得分子质量单位为 3 4ku的蛋白 ,蛋白含量占菌体蛋白含量的 16%。 结论 成功构建了粉尘螨Ⅱ类抗原cDNA基因的重组表达质粒pET 3 2a(+ ) Derf 2 ,并在大肠埃希菌中获得高效表达 ,为获得重组纯化Derf 2变应原并用于尘螨变应性疾病的诊治奠定基础。

Objective To construct and express prokaryotic expression plasmid of NA coding for group Ⅱ allergen of Dermatophagoides farinae (Der f 2). W Methods The NA of Der f 2 was digested with restriction endonuclease from recombinant plasmid pMD 18T Der f 2 and was inserted into expression vector pET 32a(+) by subclone technique, then the recombinants were transferred into Escherichia coli DL21 and identified by restriction endonuclease digestion and PCR. After that, the genetically engineered bacteria which including pET 32a(+) Der f 2 plasmids were induced by IPTG, the expression product was analyzed by SDS PAGE and densitometric scanning. W Results The positive recombinant plasmids pET32a(+) Der f 2 were identified by restriction endonuclease digestion and PCR, the objective gene fragment with the size of 455 bp were acquired, in accordance with the expected results. Sequence determination analysis showed that the gene homology with Der f 2 reported in GenBank was 100%. Plasmid pET 32a(+) Der f 2 could express a specific 34 ku protein in E. coli DL21, the protein accounted for 16% of total protein of recombinant bacteria. W Conclusion The prokaryotic expression plasmids, which contain Der f 2 gene fragment of D. farinae have been successfully constructed. Plasmid pET 32a(+) Der f 2 can express specific protein in E. coli DL21.