论文部分内容阅读
目的构建Cav1.2钙通道C末端远端片段(dDCT)(2 080~2 169)质粒,表达、提取、纯化蛋白并进行生物学活性鉴定。方法将dDCT的cDNA片段插入pGEX-6p-1质粒载体并转化大肠杆菌,异丙基硫代-β-D-半乳糖苷诱导蛋白表达,超声破碎法提取蛋白。GS-4B beads纯化蛋白后,pull-down方法分析其生物学活性。结果构建的dDCT质粒经限制性内切酶和测序双重鉴定成功,经超声破碎法提取的dDCT蛋白纯度和浓度均较高,并具有能够与GST-CT1融合蛋白浓度依赖性结合的生物学活性。结论本研究成功构建了dDCT重组质粒,为深入探讨Cav1.2钙通道的自身调节机制奠定了重要的物质基础。
Objective To construct a C-terminal distal fragment (dDCT) of Cav1.2 calcium channel (2080 ~ 2 169) plasmid, express, extract and purify the protein and identify its biological activity. Methods The cDNA fragment of dDCT was inserted into pGEX-6p-1 plasmid vector and transformed into E.coli. The protein expression was induced by isopropylthio-β-D-galactoside and the protein was extracted by sonication. After GS-4B beads purified the protein, its biological activity was analyzed by pull-down method. Results The constructed dDCT plasmid was successfully identified by restriction endonuclease and sequencing. The purity and concentration of dDCT protein extracted by ultrasonic disruption method were high, and the biological activity of dDCT protein was able to bind with GST-CT1 fusion protein in a concentration-dependent manner. Conclusion The recombinant plasmid of dDCT was successfully constructed in this study, which laid an important material foundation for further exploration of the self-regulatory mechanism of Cav1.2 calcium channel.