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目的构建人源靶向补体抑制物CR2-CD59,并筛选中国仓鼠卵巢细胞(Chinese hamster ovary cell,CHO)高效表达细胞株。方法运用FuGENE6转染试剂,将含有人CR2-CD59的重组PEE14.1质粒转入CHO细胞,蛋氨酸亚氨基代砜(MSX)筛选出阳性克隆,并利用无血清培养基对CHO细胞表达株进行培养获得重组蛋白,以ELISA、SDS-PAGE和Western blot对表达蛋白进行鉴定。结果成功构建PEE14.1-CR2-CD59重组质粒,获得CHO细胞稳定表达株。SDS-PAGE结果表明,重组蛋白CR2-CD59的相对分子质量同预期结果一致。ELISA和Western blot鉴定重组蛋白CR2-CD59可与CR2、CD59多克隆抗体特异性结合。且与含血清培养基相比,无血清培养基能明显提高CHO细胞的蛋白表达量(P<0.05)。结论在CHO细胞中成功表达人源靶向补体抑制物CR2-CD59。
Objective To construct human targeted complement inhibitor CR2-CD59 and screen the Chinese hamster ovary cell (CHO) highly expressing cell line. Methods Recombinant PEE14.1 plasmids containing human CR2-CD59 were transfected into CHO cells using FuGENE6 transfection reagent. The positive clones were screened by methionine sulfoximine (MSX) and the CHO cells were cultured in serum-free medium The recombinant protein was obtained and the expressed protein was identified by ELISA, SDS-PAGE and Western blot. Results The recombinant plasmid PEE14.1-CR2-CD59 was successfully constructed and stable CHO cell lines were obtained. SDS-PAGE results showed that the relative molecular mass of recombinant protein CR2-CD59 was consistent with the expected results. The recombinant protein CR2-CD59 was identified by ELISA and Western blot, which could specifically bind with CR2 and CD59 polyclonal antibody. Compared with serum-containing medium, serum-free medium significantly increased the protein expression of CHO cells (P <0.05). Conclusion The human-targeted complement inhibitor CR2-CD59 was successfully expressed in CHO cells.