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目的:研究前蛋白转化酶枯草溶菌素6(proprotein convertase subtilisin/kextin type 6,PCSK6)对可溶性corin的活化作用,以及PCSK6作用后的可溶性corin是否具有生物学活性。方法:以野生型corin质粒为模板,通过PCR扩增出不含跨膜区的corin基因片段,并将目的基因克隆至真核表达载体p SEC得到重组质粒p SECsol Corin,测序验证。分别将可溶性corin、PCSK6及心钠肽前体(pro-atrial natriuretic peptide,pro-ANP)的表达载体转染人胚胎肾(human embryonic kidney,HEK)293细胞,收取上清液。然后将可溶性corin上清液与不同体积的PCSK6上清液共同孵育,免疫印迹(Western blot)方法检测corin蛋白的活化情况。再将PCSK6作用后的可溶性corin与pro-ANP上清液共孵育,检测可溶性corin的生物学活性。结果:构建了可溶性corin的表达质粒,并在HEK293细胞成功表达可溶性corin蛋白。可溶性corin的上清液与pcsk6上清液共同孵育后可检测到活化条带,并可使pro-ANP活化为ANP。结论:PCSK6同样可以剪切活化可溶性corin蛋白,活化后的corin具有生物学活性。为可溶性corin用于心衰的临床治疗提供了重要基础。
OBJECTIVE: To investigate the activation of soluble corin by proprotein convertase subtilisin / kextin type 6 (PCSK6) and the biological activity of soluble corin after PCSK6 treatment. Methods: The corin gene fragment containing no transmembrane region was amplified by PCR using the wild-type corin plasmid as a template. The recombinant plasmid p SECsol Corin was cloned into the eukaryotic expression vector p SEC and sequenced. The expression vectors of soluble corin, PCSK6 and pro-atrial natriuretic peptide (pro-ANP) were respectively transfected into human embryonic kidney (HEK) 293 cells and the supernatant was collected. The soluble corin supernatant was then incubated with different volumes of PCSK6 supernatants, and the activation of corin protein was detected by Western blot. The soluble corin after PCSK6 treatment was co-incubated with pro-ANP supernatant to detect the biological activity of soluble corin. Results: The soluble corin expression plasmid was constructed and the soluble corin protein was successfully expressed in HEK293 cells. The soluble corin supernatant co-incubated with pcsk6 supernatants detected activation bands, and pro-ANP can be activated to ANP. Conclusion: PCSK6 can also cleave and activate soluble corin protein, and the activated corin has biological activity. It provides an important basis for the clinical use of soluble corin for heart failure.