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目的 构建pBV BPI60 0 Fcγ170 0 重组表达载体 ,转化E .coliDH5α诱导表达BPI2 3 Fcγ1抗菌重组蛋白。方法 采用RT PCR技术 ,从HL 6 0细胞和正常人白细胞mRNA中扩增编码BPI2 3 和IgG1Fc(Fcγ1)的基因 ;通过连接反应构建重组克隆载体和重组表达载体 ;转化感受态E .coliDH5α细胞 ,通过温控诱导表达BPI2 3 Fcγ1重组蛋白。结果 (1)RT PCR获得预期的扩增产物BPI60 0bp和Fcγ170 0bp基因片段 ;(2 )成功构建pUC18 BPI180 、pUC18 BPI4 2 0 、pUC18 Fcγ170 0 重组克隆载体 ,DNA测序结果与文献报道一致 ;(3)成功构建pBV BPI60 0 Fcγ170 0 重组表达载体 ,酶切图谱分析与预期结果相符 ;(4 )转化感受态E .coliDH5α细胞 ,通过温控诱导表达BPI2 3 Fcγ1重组蛋白 ,表达量约占菌体蛋白总量的 2 0 %～ 30 % ;(5 )复性后重组蛋白具有抗菌活性和结合蛋白A的作用。结论 pBV BPI60 0 Fcγ170 0 重组表达载体构建成功 ;在E .coli中得到表达 ,获得具有抗菌活性的BPI2 3 Fcγ1重组蛋白
Objective To construct pBV BPI60 0 Fcγ170 0 recombinant expression vector and transform E.coli DH5α to induce the expression of BPI2 3 Fcγ1 antimicrobial recombinant protein. Methods RT-PCR was used to amplify the genes coding for BPI2 3 and IgG1 Fc (Fcγ1) from HL 60 cells and normal human leukocyte mRNA. The recombinant cloning vector and recombinant expression vector were constructed by ligation reaction. The competent E. coli DH5α cells were transformed, The BPI2 3 Fcγ1 recombinant protein was expressed by temperature-control. Results (1) The expected amplification products BPI60 0bp and Fcγ170 0bp gene fragments were obtained by RT PCR. (2) The recombinant cloning vector pUC18 BPI180, pUC18 BPI4 2 0, pUC18 Fcγ170 0 was successfully constructed. The sequencing results of DNA were consistent with those reported in the literature (3) ) Successfully constructed pBV BPI60 0 Fcγ170 0 recombinant expression vector, digestion map consistent with the expected results; (4) transformed E.coli DH5α cells by temperature-induced expression of BPI2 3 Fcγ1 recombinant protein, the expression of about bacterial protein The total amount of 20% to 30%; (5) renaturation of the recombinant protein has antibacterial activity and binding protein A role. Conclusion The recombinant plasmid pBV BPI60 0 Fcγ170 0 was constructed successfully and expressed in E.coli. The recombinant protein BPI2 3 Fcγ1 with antibacterial activity