人β防御素4在大肠杆菌中的融合表达及其多克隆抗体的制备和鉴定  

Fusion expression of human β defensin 4 in Escherichia coli and preparation of its polyclonal antibody

作　　者：曹玉红[1] 张光运[2] 张国成[1] 

机构地区：[1]第四军医大学西京医院儿科,陕西西安710032 [2]第四军医大学西京医院神经内科,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2008年第2期150-152,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：第四军医大学西京医院科技创新基金(XJCX06M21)

摘　　要：目的:构建人β防御素4(human β defensin4,HBD4)基因的原核融合表达载体PGEX-4T-2/mHBD4,诱导GST-HBD4融合蛋白在大肠杆菌中表达,并制备其多克隆抗体。方法:从重组克隆载体PMD18-T/HBD4中扩增HBD4成熟肽编码基因,并克隆入PGEX-4T-2中,在IPTG诱导下,表达GST-HBD4融合蛋白。以表达的融合蛋白GST-HBD4作为免疫原免疫家兔制备抗GST-HBD4的抗血清,抗体效价及特异性分别用ELISA和Western blot法鉴定。结果:在大肠杆菌中成功表达Mr约为32000的融合蛋白GST-HBD4。ELISA法检测抗血清的效价可达到1∶128000,Western blot分析抗血清可与原核表达的融合蛋白GST-HBD4特异结合。结论:在大肠杆菌中成功表达了GST-HBD4融合蛋白,并制备了GST-HBD4的抗血清,为进一步研究HBD4蛋白的结构和功能奠定了基础。AIM： To construct the prokaryotic fusion expression vector of human β defensin 4 （ HBD4）, and to express GST-HBD4 fusion protein in Escherichia coil （ E. coil ） and prepare polyclonal antibody of GST-HBD4. METHODS： The gene encoding mature peptide of HBD4 （mHBD4） was amplified by PCR from cloning vector PMD18-T/HBD4 which contained the full-length HBD4 cDNA and then cloned into prokaryotic expression vector PGEX-4T-2 to construct PGEX-4T-2/mHBD4. GST-HBD4 expression was induced by IPTG. The antiserum was prepared by immunizing rabbit with GST-HBD4. The titer and specificity of the antibody were detected by ELISA and Western blot, respectively. RESULTS： The recombinant expression vector PGEX-4T-2/ mHBD4 was successfully constructed. After being induced by IPTG, The fusion protein with relative molecular mass of 32 000 was successfully expressed in E. coil and partly expressed in the soluble form in supematant. The rabbit anti- body against GST-HBD4 was obtained. The ELISA titer of antiserum against GST-HBD4 was about 1： 128 000. Westem blot analysis showed that the antiserum could bind to the expressed GST-HBD4 specifically. CONCLUSION： The rabbit antibody against GST-HBD4 has been successfully prepared, which lays the foundation for further studying the structure and function of HBD4.

关 键 词：人β防御素4 基因克隆 融合表达 

分 类 号：Q786[生物学—分子生物学]