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目的 获得鼠抗人精浆蛋白 m Ab重链 Fd段基因 ,并将其转染到 He L a细胞进行表达 .方法 从分泌抗人精浆蛋白 m Ab的杂交瘤细胞系 E4 B7中提取总 RNA,用 RT- PCR法获取重链 Fd段 c DNA.将其克隆入真核表达载体 pc D-NA3,用脂质体转染法转染 He L a细胞 ,并用免疫荧光染色方法检测重链 Fd段的表达 .结果 从分泌抗人精浆蛋白的鼠m Ab杂交瘤细胞系 E4 B7中克隆出了重链 Fd段基因 ,序列测定表明 ,VH 与鼠免疫球蛋白 MUSIGHAEI同源性最高 .将含重链 Fd段基因的真核表达载体 pc DNA3- E4 B7Fd中转染He L a细胞后 ,在 He L a细胞中检测到了重链 Fd段的表达 .结论 获得了序列正确的重链 Fd段基因 ,它在 He L a细胞中可以成功地表达 ,为进一步构建和表达 Fab奠定基础 .
OBJECTIVE: To obtain the Fd fragment of murine anti-human seminal plasma m Ab heavy chain and to transfect it into He L a cells for expression.Methods Total RNA was extracted from the hybridoma cell line E4 B7 secreting anti-human seminal plasma protein m Ab The cDNA of heavy chain Fd was obtained by RT-PCR. The recombinant plasmid was cloned into eukaryotic expression vector pc D-NA3 and transfected into HeLa cells by lipofection. The immunofluorescence staining was used to detect the heavy chain Fd .Results The heavy chain Fd gene was cloned from mouse mAb hybridoma cell line E4 B7 secreting anti-human seminal plasma protein.Its sequence showed that VH had the highest homology with murine immunoglobulin MUSIGHAEI, The heavy chain Fd fragment was detected in HeLa cells transfected with pcDNA3-E4 B7Fd in HeLa cells.Conclusion The Fd gene with the correct heavy chain sequence , Which can be successfully expressed in HeLa cells and lay the foundation for further construction and expression of Fab.