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为了构建人源肽抗生素hBD 2的表达质粒并在大肠杆菌中表达 ,将化学合成的hBD 2全基因插入原核融合蛋白表达质粒 pinpointXa 3的多克隆位点构建成表达质粒 ,按常规方法转化大肠杆菌JM10 9,筛选阳性重组子并诱导融合蛋白的表达。结果经酶切鉴定获得 7个阳性克隆 ,序列分析表明其中 4个克隆的插入序列与hBD 2基因完全一致 ,另 3个克隆插入序列的 39位由C突变为T ,89位多了一个G。正确的阳性克隆子在大肠杆菌中产生了约 18kD的特异性诱导蛋白。hBD 2的成功表达为进一步研究hBD 2的抗菌活性、抗菌机理打下了一定的基础。
In order to construct the expression plasmid of human peptide antibiotic hBD 2 and express it in E. coli, the chemically synthesized hBD 2 gene was inserted into the multi-cloning site of prokaryotic fusion protein expression plasmid pinpointXa 3 to construct an expression plasmid. The recombinant plasmid was transformed into Escherichia coli JM10 9. Screen positive recombinants and induce expression of the fusion protein. Results Seven positive clones were obtained by restriction enzyme digestion. The sequence analysis showed that the inserted sequences of the four clones were identical to hBD 2 gene. The other three clones inserted 39 mutations from C to T and 89 to GG. Correct positive clones produced about 18 kD of specific inducible protein in E. coli. The successful expression of hBD 2 lay a solid foundation for further study on the antibacterial activity and antibacterial mechanism of hBD 2.