论文部分内容阅读
目的:利用噬菌体展示技术构建抗脐带间充质干细胞表面分子噬菌体ScFv抗体库。方法:收集P3代培养的UC-MSCs免疫BALB/c小鼠,提取其脾细胞总RNA,RT-PCR扩增全套VH和VL基因片段,将其先后克隆入噬菌粒pSEX81中,构建成完整的噬菌体ScFv抗体库。结果:构建的噬菌体ScFv抗体库的库容为2×107cfu,ScFv插入重组率为93%,BstN1酶切图谱呈不同多样性。ScFv抗体库经3轮初步筛选后插入重组率达100%,3个克隆出现了相同的酶切图谱,并且随着筛选次数的增加,输出/输入比明显提高,这说明抗体库得到了特异性富集。结论:成功地构建了抗脐带间充质干细胞表面分子噬菌体ScFv抗体库,这为将来筛选特异性抗体和进一步用于间充质干细胞表面特异性分子研究奠定了坚实的基础。
OBJECTIVE: To construct an anti-ScFv library against phage display anti-umbilical cord mesenchymal stem cells using phage display technology. METHODS: BALB / c mice were immunized with P3-cultured UC-MSCs and the total RNA was extracted from the splenocytes. The full-length VH and VL gene fragments were amplified by RT-PCR and cloned into the phagemid pSEX81 to construct a complete Phage ScFv antibody library. Results: The constructed phage ScFv library had a capacity of 2 × 107cfu and the inserted and recombined rate of ScFv was 93%. The diversity of BstN1 digestion maps was different. After 3 rounds of primary screening, the recombinant plasmid of ScFv was inserted into the recombination rate of 100%. The same digestion pattern appeared in the three clones, and the output / input ratio increased significantly with the increase of the number of screening, indicating that the antibody library was specific Enriched. CONCLUSION: The anti-ubiquitin-derived scFv library against umbilical cord mesenchymal stem cells has been successfully constructed, which lays a solid foundation for the future screening of specific antibodies and further study on surface-specific molecules of mesenchymal stem cells.