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CYP2J酶主要参与机体内源性物质代谢,尤其是对花生四烯酸和亚油酸代谢发挥着重要作用。本试验通过克隆CYP2J基因,构建其原核表达载体,为蛋白层面深入研究双峰驼CYP2J酶提供特异性抗体奠定基础。从双峰驼肾组织提取RNA,反转录后PCR扩增出双峰驼CYP2J基因,胶回收纯化后进行TA克隆,连接pMD18-T质粒并转化大肠杆菌DH5α的感受态细胞中,提取质粒进行HindⅢ和KpnⅠ双酶切和PCR鉴定,并借助上述内切酶双酶切重组质粒pMD18-T-CYP2J后,连接于经同样双酶切处理的原核表达载体pET-32a,转化于大肠杆菌DH5α细胞中,并经HindⅢ和KpnⅠ双酶切、PCR鉴定及测序鉴定重组质粒。结果表明,在重组质粒pMD18-T-CYP2J和pET-32a-CYP2J双酶切电泳中均能得到约1500bp的特异性目的条带及两种质粒的特异性条带。以2种重组质粒为模板进行PCR扩增后电泳均能得到约1500bp的CYP2J特异性条带,测序结果与GenBank中公布的双峰驼CYP2J预测序列的相似度为99%。由此可见,本试验成功构建了双峰驼CYP2J基因原核表达载体pET-32a-CYP2J,为后续该基因的高效原核表达和蛋白层面的研究奠定了基础。
CYP2J enzyme is mainly involved in the metabolism of endogenous substances in the body, especially plays an important role in the metabolism of arachidonic acid and linoleic acid. In this study, CYP2J gene was cloned and its prokaryotic expression vector was constructed to lay a foundation for the further study of CYP2J enzyme in bactrian camels to provide specific antibodies. RNA was extracted from the kidney of Bactrian Camel, and the baculovirus CYP2J gene was amplified by PCR after reverse transcription. The cDNA was purified by TA cloning and ligated into plasmid pMD18-T and transformed into competent cells of E. coli DH5α. Plasmids were extracted Hind Ⅲ and Kpn Ⅰ double digestion and PCR identification, and with the help of the restriction endonuclease digested recombinant plasmid pMD18-T-CYP2J, connected to the same double digestion of prokaryotic expression vector pET-32a, was transformed into E. coli DH5α cells , And digested with Hind Ⅲ and Kpn Ⅰ, PCR identification and sequencing identified recombinant plasmid. The results showed that a specific band of about 1500 bp and a specific band of two plasmids could be obtained in the double digestion electrophoresis of pMD18-T-CYP2J and pET-32a-CYP2J. The PCR products of the two recombinant plasmids were used as templates for PCR amplification. CYP2J specific bands of about 1500bp were obtained. The similarity between the sequencing results and the predicted CYP2J locus in Bactrian camels was 99%. Thus, we successfully constructed the prokaryotic expression vector pET-32a-CYP2J of Bactrian camel CYP2J gene, which lays the foundation for the subsequent high-efficiency prokaryotic expression and protein profiling of this gene.