斑马鱼Tph1a蛋白表达、纯化及对脂代谢基因的影响

Expression,purification and effect of Tph1aon lipid metabolism-related genes in zebrafish(Danio rerio)

对斑马鱼色氨酸羟化酶1a (Tph1a)进行原核表达、纯化,探究其在斑马鱼脂代谢中的作用。根据大肠埃希菌的偏爱密码序列,优化斑马鱼tph1a编码区,插入原核表达载体pET-28a,构建融合蛋白表达质粒,并诱导表达、纯化和复性后,腹腔注射斑马鱼,检测其对脂代谢基因表达的影响。结果表明:重组蛋白Tph1a表达形式为包涵体;纯化和复性后的蛋白质具有57 ku的特异性条带;腹腔注射复性蛋白后,高脂组斑马鱼acc、pgc1α基因等表达上调,lpl基因表达下调,且pgc1α基因的表达丰度远高于其他脂代谢基因。这一研究成功构建了原核表达载体pET-28a-tph1a,获得体外高效表达;该蛋白质不仅诱导肥胖斑马鱼的长链脂肪酸合成阻滞,更促进脂肪酸氧化与分解,为探究Tph1a在鱼类脂代谢调控中的功能奠定了基础。

This study was aimed to express zebrafish tryptophan hydroxylase 1 a(Tph1 a) through prokaryotic expression system and explore its role in lipid metabolism in zebrafish using the purified Tph1 a. The optimization of tph1 a coding region was performed based on the preferred genetic codes of E.coli, after which the prokaryotic expression vector of pET-28 a-tph1 a was constructed and transformed into Rosetta competent cells for IPTG-induced expression. The recombinant Tph1 a with activity was obtained after purification and renaturation. The expressions of lipid metabolism-related genes in zebrafish after intraperitoneal injection of Tph1 a were evaluated by quantitative real-time PCR. SDS-PAGE showed that the expressed protein was present in inclusion bodies. The target protein was purified by affinity chromatography and imidazole elution and then renaturated, which showed a single band at 57 ku analyzed by Western blot. After treated with the recombinant Tph1 a, the expression levels of acc, pgc1α and pparγ increased significantly in zebrafish fed with high-fat diet(HFD), whereas lpl expression decreased in the same group of individuals. Moreover, pgc1α mRNA exhibited higher transcript level than the expression of other related genes. In summary, the optimized expression vector pET-28 a-tph1 awas successfully constructed.The recombinant Tph1 awas highly expressed and showed the biological activity after purification and renaturation.The recombinant Tph1 acould lead to the blocking of long-chain fatty acid synthesis in obese zebrafish,and more importantly it may play a major role in enhancing activity of fatty acids oxidation and catabolism.The data in this study may provide the theoretical foundation for investigating the detailed regulatory effect of Tph1 aon lipid metabolism in teleost fish.