VT2-B亚单位的重组表达及单克隆抗体的制备

Prokaryotic Expression of Vero Toxin Ⅱ B Subunit from Escherichia coli O157 and Preparation of Monoclonal Antibodies

摘要:

以大肠杆菌O157 DNA为模板扩增VT2-B亚单位，纯化后经EcoRⅠ和XhoⅠ酶切，连入表达载体pGEX_4T-2，构建重组表达质粒pGEX_4T-2-VT2-B。转化到大肠杆菌BL21中，经IPTG诱导，实现了VT2-B-GST融合蛋白的可溶性表达，表达量约占菌体总蛋白的35％。重组蛋白纯化后，免疫BALB/C小鼠制备单克隆抗体，得到2株融合细胞，制备了腹水单抗，测得单抗的ELISA效价为10⁴。Western blot表明，该单抗能与重组蛋白特异性的结合。特异性单抗的获得为VT2毒素检测方法的建立奠定了基础。

Abstract:

The segment of VT2-B subunit was amplified by PCR，then purified and digested with the endonucleases EcoRⅠand XhoⅠ.The digested segment was inserted into the double-cleavaged expression vector pGEX_4T-2.The recombinant expression vector was named as pGEX_4T-2-VT2-B.The expression vector was transformed into E.coli BL21 compotent cells,and highly expressed a VT2B-GST fusion protein after IPTG inducing.The target fusion protein occupied 35% of total cell protein and in soluble form.The high-purified fusion protein was obtained by GSH-Sepharose Resin.The BALB/C mice were immunized with the purified recombinant protein.Two strains of monoclonal antibody 4B×F10×D4 and 4B×8G×11F were obtained.Ascitic monoclonal antibodies were generated by using cells 4B×8G×11F.Western blot analysis showed that the recombinant protein had a specific affinity for monoclonal antibody,while not for vector protein.The results showed that the McAb generated was against the VT2-B subunit,not against the GST.