脑钠肽前体及氨基末端脑钠肽前体基因的克隆与表达

目的在大肠杆菌中表达脑钠肽前体(proBNP)及氨基末端前体蛋白(NT-proBNP)。方法采用分子生物学技术构建重组质粒PGEX-20T-proBNP和PGEX-20T-NT-proBNP,分别对其进行PCR、双酶切和测序鉴定,然后将已测序鉴定的包含两种重组质粒的工程菌,转化至大肠杆菌BL21菌中表达脑钠肽前体及氨基末端前体蛋白,并用Western-blot鉴定纯化的重组蛋白。结果在大肠杆菌中成功的表达脑钠肽前体及氨基末端前体蛋白,两种蛋白均以可溶性形式表达,更有利于保存其生物学活性。结论两种蛋白的成功表达将有利于后续的单抗制备及检测试剂盒的开发奠定基础。

Brain natriuretic peptide precursor and N-terminal Brain natriuretic peptide precursor genes cloning and expression

Objective Brain natriuretic peptide precursor and N-terminal Brain natriuretic peptide precursor proteins were expressed in E. coll. Methods Recombinant plasmid PGEX-20T proBNP and PGEX-20T-NT proBNP were con structed by molecular biological methods, which were identified by PCR, restriction enzyme digestion and sequence anal ysis. Those two recombinant plasmids,identified by sequencing , were transformed into E. coli BL21 and expressed proBNP and NT-proBNP proteins. Those two recombinant proteins which were identified by Western-blot test. Results Brain natriuretic peptide precursor and N-terminal Brain natriuretic peptide precursor proteins were expressed successfully in E. coli. Both proteins which were expressed in dissolubility pattern conduced to conserve biologic activity. Conclusion Those two recombinant proteins which were expressed successfully conduced to preparation on mAb and foundation for detection kits development in next step.