人β-防御素2在大肠杆菌中的高效表达和纯化

目的：研究具有生物学活性的人防御素2（HBD2）多肽在大肠杆菌中的原核高效表达的可行性及融合表达蛋白的分离纯化技术。方法：PCR扩增不合前导序列的HBD2成熟肽的编码框全长的cDNA片段（smHBD2-cDNA），利用BglⅡ和BamHⅠ同尾酶构建多拷贝串联的nsmHBD2-cDNA的克隆载体，利用NcolⅠ和HindⅢ限制性内切酶构建融合表达载体pET32-nsmHBD2-cDNA，在大肠杆菌中BL21（DE3）中诱导表达含HBD2的融合蛋白，SDS-PAGE分析产量。可溶蛋白经亲和层析、肠激酶酶切、离子交换层析等方法分离、纯化HBD2多肽。采用液体培养法，测定重组人β防御素2对大肠杆菌的抑菌活性。结果：构建了n为1、2、4或8倍的多拷贝串联nsmHBD2-cDNA表达载体，1、2和4倍smHBD2-cDNA的可溶蛋白比例分别为52％、48％和31％；而8倍HBD2-cDNA几乎不合可溶蛋白，目标蛋白以包含体形式表达，集中在不可溶部分。重组HBD2对E．coli K12 D31有较强的抑制作用，在终浓度约为0．4至0．5μg／ml时，90％细胞的生长被抑制。结论：采用融合表达、多拷贝串联表达方式，可增加产物长度以弱化蛋白酶的降解作用，也减弱了目标蛋白产物对宿主的毒性；适当的多拷贝串联基因可以提高HBD2的产量，但拷贝数过高会影响重组蛋白表达产量，且易形成不溶蛋白；在大肠杆菌中可达到具有生物学活性的HBD2多肽的原核高效表达。

Efficient expression and purification of human beta-defensin-2 in E. coli

OBJECTIVE: To demonstrate the possibility of high-level expression of bioactive human beta-defensin-2 (hBD2) in E.coli, and to purify the recombinant hBD2. METHODS: DNA fragment containing mature hBD2 coding region (smhBD2-cDNA) was amplified by PCR, multiple copies of smhBD2-cDNA were linked using Bgl II and BamH I enzymes, pET32-nsmHBD2-cDNA with 1, 2, 4, or 8 copies of smhBD2-cDNA was constructed. The soluble and insoluble hBD2 proteins were separated and analyzed by SDS-PAGE analysis. The soluble protein underwent a separation process containing affinity chromatography, enterokinase digestion and ion exchange chromatography to get the recombinant hBD2 peptide. The bioactivity of recombinant hBD2 was examined by bacteria-inhibition tests in liquid culture. RESULT: The plasmids pET32-nsmHBD2-cDNA with 1, 2, 4 copies of smhBD2-cDNA were constructed and the expressed soluble protein accounted for 52 %, 48 %, and 31 % respectively. The plasmids with 8 copies expressed mainly insoluble protein with few in soluble form. The growth of E.coli K12D31 was dramatically suppressed with a inhibition rate of 90 %, when the final concentration of recombinant hBD2 reached between 0.4 to 0.5 mug/ml. CONCLUSION: Fusion expression of human beta-defensin-2 with multiple joined genes in E.coli could increase the expression of hBD2.