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目的: 通过整合,用巴氏毕赤酵母表达抗人肝癌mAbHAb18的cFab的嵌合轻链.方法: 将抗人肝癌mAbcFab/HAb18原核表达载体pET32a/cFab中的cL亚克隆到酵母表达载体pPIC9K,构建成重组质粒pPIC9K/cL测序鉴定.重组质粒pPIC9K/cL整合到酵母菌GS115的染色体上,经G418筛选得到高拷贝转化子及Mut表型鉴定后,用含5mL/L甲醇的培养基诱导表达.结果: 该系统成功表达了抗人肝癌mAbHAb18cFab的嵌合轻链,表达水平为22mg/L;WesternBlotting证实,表达产物具有良好的HAb18GE结合活性和特异性.结论: 嵌合轻链/HAb18在巴氏毕赤酵母获得成功表达,为高效分泌表达cFab抗体奠定了基础.
Objective: To express the chimeric light chain of cFab against human hepatoma mAb HAb18 by using Pichia pastoris.Methods: The cL of anti-human hepatoma mAbcFab / HAb18 prokaryotic expression vector pET32a / cFab was subcloned into yeast expression vector pPIC9K, The recombinant plasmid pPIC9K / cL was cloned into the chromosome of yeast GS115. After high-copy transformants were identified by G418 and mut phenotype was identified, the recombinant plasmid pPIC9K / cL was induced to express in medium containing 5 mL / L methanol Results: The system successfully expressed the chimeric light chain of anti-human hepatoma mAb HAb18cFab with a level of 22 mg / L. Western Blotting confirmed that the expressed product had good HAb18GE binding activity and specificity.Conclusion: Pichia pastoris was successfully expressed, which laid the foundation for the efficient secretion and expression of cFab antibody.