摘要
目的克隆人软骨调节素I(CHM-I)cDNA,并在原核表达系统中表达和纯化。方法利用RT-PCR技术从人软骨组织中扩增出CHM-I基因片段,并将其克隆到原核表达载体pET28a中,转化至大肠杆菌BL21(DE3),IPTG进行诱导表达;对在N末端融合了6×His纯化标签的表达产物进行Westernblot分析和用Ni2+2NTA离子交换树脂进行纯化;纯化蛋白进行SDS-PAGE纯度分析。结果获得了人软骨调节素cDNA,并成功构建了高效原核表达质粒pET(CHM-I),能有效表达CHM-I蛋白,且CHM-I蛋白的纯度高于90%。结论原核表达系统可有效克隆较高纯度的CHM-I基因。
Objective To obtain the gene of human Chondromodulin-I（CHM-I） gene and express and purify it in procaryotic expression system. Methods A fragment of human Chondromodulin-I gene was amplified from tissue by RT-PCR, and cloned into procaryotic expression vector pET28a（ ＋ ）. The recombinant plasmid pET（CHM-I）was transformed into E. coli B121 （DE3） and expressed under the induction of IPTG. The expression product fused with 6 × His at N-terminal was analyzed by Western blotting, and purified by using Ni2 ＋ 2NTA ion exchange resin. The purity of CHM-I protein was analyzed by SDS-PAGE. Results Human CHM-I cDNA was obtained, and the plasmid pET（CHM-I）was constructed successfully. The human CHM-I was expressed with the purity higher than 90%. Conclusion The more effective CHM-I clone with high purity could be expressed by procaryotic expression system.
出处
《华中医学杂志》
2006年第5期394-396,共3页
Central China Medical Journal
