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①采用RT PCR技术 ,以HL 60细胞mRNA为模板扩增获得编码BPI氨基端 1 93个氨基酸 (rBPI2 1)的基因片段 (BPI60 0 ) ;EcoRI/BamHI酶切扩增产物获得BPI 2 0 0bp和BPI 4 0 0bp 2个基因片段。②成功构建PUC1 8 BPI2 0 0和PUC1 8 BPI4 0 0重组克隆载体 ,DNA测序分析结果与文献报道一致。③成功构建pBV2 2 0 BPI60 0重组表达载体 ;转化E .coliDH5α感受态细胞 ,诱导目的重组蛋白表达 ;经SDS PAGE电泳和Western blot鉴定 ,证实表达的重组蛋白确为外源基因 (BPI 60 0bp)编码的产物BPI2 1重组蛋白。
(1) RT-PCR was used to amplify the gene fragment of BPI60 with the amino acid sequence of BPI60 of BPI by using HL60 cell mRNA as a template; BPI60 0 was obtained by digesting the amplified product with EcoRI / BamHI to obtain BPI of 200 bp and BPI 4 0 0bp 2 gene fragments. ② The PUC1 8 BPI2 0 0 and PUC1 8 BPI4 0 0 recombinant cloning vectors were successfully constructed, and the results of DNA sequencing analysis were consistent with those reported in the literature. (3) The recombinant plasmid pBV220 BPI60 0 was successfully constructed; transformed E. coli DH5α competent cells to induce the expression of the recombinant protein of interest; SDS-PAGE electrophoresis and Western blot analysis confirmed that the expressed recombinant protein was a foreign gene (BPI 60 0 bp) The encoded product BPI2 1 recombinant protein.