人β2微球蛋白基因在大肠杆菌中的稳定、高效表达

Efficient and constant expression of cloned human β2-microglobulin gene in E.coli

目的应用基因工程技术表达人β2微球蛋白基因(β2m)。方法采用逆转录PCR技术,从Raji细胞株获得β2m的基因序列;将其与改建后的质粒pBV220连接,转染大肠杆菌BL21,经42℃诱导后,以SDS-PAGE鉴定阳性表达克隆,产物经过柱纯化。结果获得了编码成熟β2m的全长cDNA和高效、稳定表达人β2m的大肠杆菌克隆。结论成功地在大肠杆菌中获得了β2m基因的高效表达,为制备主要组织相容性复合体(MHC)-四聚复合物系统奠定了基础。

Objective To o btain human a2-microglobulin (a2m) gene that is to be efficiently expressed in E.coli. Methodsa2m cDNA including only the coding region for the protein was a mplified from Raji cell line by reverse transcriptase-PCR via the primers 5'-GGT GGTCATATGGCTATCCAGCGTACTCCA-3' and 3'-GGTGGTTGCTCTTCCGACATGTCTCGATCC-3'. The pr oduct was subsequently cloned into a modified pBV220 vector after digestion with NdeⅠ/SapⅠ. The recombinant plasmid pBV220-a2m was transformed into E.coli B L21 after sequenc analysis, and the fusion protein was then expressed via induct ion at 42 ℃for 5 h at D 600 of 0.55-0.60, followed by purification throu gh chitin beads. Results The a 2m cDNA was iden-tical with those published in Genbank. The expressed fusion pro tein was identified in the form of inclusion body at the ratio more than 45% of the E.coli proteins, and was denatured with 8 mol/L urea, followed by refold an d purification to a high puri-ty, displaying a relative molecular mass of 12 000 on 10% SDS-PAGE gel. Conclusion The human a2m gene was cloned successfully and expressed efficiently and constantly in E.coli BL21,which lays the ground f or engineering MHC-tetramers.