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目的构建携带CDCP1胞外段基因的原核表达载体。方法以A549的mRNA为模板,应用所设计的引物通过RT-PCR法扩增CDCP1胞外段基因;将PCR产物与pGEX-KG载体连接,获得重组质粒pGEX-KG/CDCP1;经双酶切、PCR及DNA序列测定进行鉴定;IPTG诱导表达。结果①RT-PCR扩增得到约270bp大小的CDCP1胞外段基因目的片段;②目的片段正确插入到pGEX-KG中;③经IPTG诱导表达,可见在相对分子质量约35kD处出现明显的诱导蛋白条带,与预期一致。结论成功构建了携带CDCP1胞外段基因的原核表达载体,在大肠杆菌中获得大量表达。
Objective To construct a prokaryotic expression vector carrying CDCP1 extracellular domain gene. Methods A549 mRNA was used as a template to amplify the extracellular domain of CDCP1 by RT-PCR using the designed primers. The PCR product was ligated into pGEX-KG vector to obtain the recombinant plasmid pGEX-KG / CDCP1. PCR and DNA sequencing to identify; IPTG induced expression. Results ①The target fragment of extracellular domain of CDCP1 with about 270bp was amplified by RT-PCR. ②The inserted fragment was inserted into pGEX-KG correctly. ③The expression of induced protein band was induced by IPTG. At the relative molecular mass of about 35kD, Belt, as expected. Conclusion The prokaryotic expression vector carrying CDCP1 extracellular domain gene was successfully constructed and expressed in E. coli.