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目的克隆并原核表达 α4亚基片段。方法从 IL- 6 0细胞系中提取总 RNA,以 RT- PCR方法分两段扩增人整合素分子 α4亚基片段 2 .0 kb c DNA,将两片段连接 ,并将其中的 1.7kb基因片段亚克隆至表达载体 PGEX- KG中 ,IPTG诱导表达。结果 RT- PCR扩增并克隆了α4的两段 c DNA,序列分析表明 :与文献报道的α4c DNA序列基本一致。两片段成功连接后 ,将其中的 1.7kb亚克隆至表达载体 PGEX- KG中 ,经诱导在大肠杆菌中得到高效表达。结论获得了α4亚基 2 .0 kbc DNA片段 ,及其中 1.7kb的原核表达产物对于研究α4整合素的功能有重要意义
Objective cloning and prokaryotic expression of α4 subunit fragment. Methods Total RNA was extracted from IL-60 cell line, and the 2.0-kb cDNA of human integrin α4 subunit fragment was amplified by RT-PCR in two steps. The two fragments were ligated and the 1.7 kb The fragment was subcloned into the expression vector PGEX-KG and IPTG induced expression. Results The two cDNA fragments of α4 were amplified by RT-PCR and sequenced. The sequence analysis showed that the sequence of α4c DNA was consistent with that reported in the literature. After the two fragments were successfully ligated, 1.7 kb of them were subcloned into the expression vector PGEX-KG and induced to be highly expressed in E. coli. Conclusion Obtaining the 2.0 kb DNA fragment of α4 subunit and the prokaryotic expression product of 1.7 kb is of great significance for studying the function of α4 integrin