抗脂肪细胞40000特异膜蛋白scFv-Fc融合抗体毕赤酵母表达载体的构建  

Construction of Pichia pastoris expression vector for production of scFv-Fc fusion antibody against 40 000 adipocyte-specific plasma membrane protein

作　　者：高士争[1] 尹革芬[2] 张玉静[3] 汪磊[4] 张曦[1] 

机构地区：[1]云南农业大学云南省动物营养与饲料重点实验室,云南昆明650201 [2]云南农业大学动物科技学院,云南昆明650201 [3]吉林大学畜牧兽医学院,吉林长春130062 [4]华东师范大学生命科学系,上海200062

出　　处：《中国兽医学报》2007年第3期376-381,386,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30260079);云南省自然科学重点基金项目(2000C005Z)

摘　　要：采用RT-PCR方法从人外周血淋巴细胞克隆IgG1Fc片段基因,经序列分析表明,该Fc片段基因长699bp,具有完整的铰链区、CH2和CH3结构区,与GenBank中人IgG1重链Fc片段基因序列完全相同。将Fc片段基因与pPICZαA酵母载体连接先构建pPIgG1质粒,然后将抗脂肪细胞40000特异膜蛋白的噬菌体scFv抗体基因插入到pPIgG1质粒,构建pPIgG1-scFv酵母表达载体。序列分析表明,scFv基因与Fc片段基因拼接正确,阅读框正确无误,表明成功构建了抗脂肪细胞40000特异膜蛋白scFv-Fc融合抗体毕赤酵母表达载体。本试验为下一步在毕赤酵母中大量表达与IgG结构相似、具有较高亲和力和生物活性的scFv-Fc融合抗体分子奠定了基础。The Fc fragment DNA sequence of the immune gamma globulins （IgG1） was amplified from the human peripheral blood lymphocyte by reverse transcriptase-polymerase chain reaction （RT-PCR）. The nucleotide sequence analysis revealed that this 699 bp DNA fragment possessed the complete hinge area, CH2 and CH3 structural region, and showed 100% identity to the Fc fragment DNA sequence of human IgG1 heavy chain which had been deposited in the GenBank database. Subsequently this Fc fragment DNA was inserted into pPICZaA vector of Pichia pastoris to construct the pPIgG1 plasmid. Next, scFv antibody gene of Pichia pastoris whose protein prod- uct was against the 40 000 adipocyte-specific plasma membrane protein was inserted into the pPIgG1 plasmid and constructed the pPIgGl-scFv Pichia pastoris expression vector. Validation analysis showed that ligation direction of the scFv gene of Pichia pastoris and the Fc fragment DNA was correct and was in agreement with the correct reading frame of scFv-Fc. These results suggested that the scFv-Fc Pichia pastoris expression vector had been suc- cessfully constructed and this established the primary foundation to the express a great quantity of scFv-Fc fusion antibody protein which had the similar structure to IgG and also possessed the desirable affinity and bioactivity through this vector.

关 键 词：脂肪细胞 单链抗体 毕赤酵母 表达载体 

分 类 号：Q78[生物学—分子生物学]