美丽硬仆骨舌鱼Raf激酶基因的克隆、组织表达及原核表达分析

Molecular Cloning,Tissue Expression and Prokaryotic Expression of raf Gene from Scleropages formosus

【目的】Raf激酶是调控细胞增殖、分泌和运动的重要蛋白激酶,参与多种生命活动。研究Raf激酶基因(raf)的结构特征和表达模式,以探究其在美丽硬仆骨舌鱼(Scleropagesformosus)中的功能。【方法】研究通过RACE技术克隆了美丽硬仆骨舌鱼raf基因cDNA序列(GenBank登录号MW679580)并进行生物信息学分析;用荧光定量PCR检测raf在精巢等8个组织内的表达模式;通过构建原核表达载体诱导美丽硬仆骨舌鱼raf融合蛋白。【结果】美丽硬仆骨舌鱼raf序列全长3747 bp,开放阅读框(ORF)为1827 bp,编码609个氨基酸。生物信息学分析显示,Raf蛋白的分子量为68.8 ku,等电点PI为9.45,含有1个Ras结合域,1个蛋白激酶C保守区(C1)结构域,1个低合成复杂度区域和1个丝氨酸/苏氨酸蛋白激酶催化结构域,是亲水性跨膜蛋白。序列同源性分析显示,美丽硬仆骨舌鱼raf编码蛋白序列与雀鳝相似度最高,为84.28%。美丽硬仆骨舌鱼与雀鳝都是较早分化出的物种,序列同源性分析与两者进化地位相符。用荧光定量PCR检测raf在8个组织内的表达量,结果表明,raf基因在美丽硬仆骨舌鱼多种组织中均有表达,其中在精巢和肝中的表达量最高,其次是脾和头肾。精巢和肝中的表达量显著高于卵巢、鳃、心脏和脑组织(P<0.05)。构建pET-B2m-raf原核表达载体,通过0.1 mmol/L IPTG诱导,成功获得融合蛋白,经SDS-PAGE检测,其分子量符合预期。【结论】raf在美丽硬仆骨舌鱼精巢和肝脏中的表达量较高,推测其可能在精巢发育和免疫机能调控中发挥作用,构建原核表达载体并成功诱导出重组蛋白,为进一步研究raf在美丽硬仆骨舌鱼体内的生物功能奠定基础。

[Objective]Raf kinase is an important protein kinase that regulates cell proliferation,secretion and movement,which is important for maintaining body vital activities.The structural feature and expression pattern of Raf kinase gene(raf)were studied so as to explore the function of raf in Scleropages formosus.[Method]The full-length cDNA sequence of raf was cloned by rapid-amplification of cDNA ends(RACE)(GenBank No.MW679580)and the bioinformatics analysis was performed.The expression pattern of vtg was evaluated using real-time fluorescent quantitative PCR.The expression of fusion protein was induced by constructing prokaryotic expression vectors.[Result]The full length of the sequence was 3747 bp,and the open reading frame(ORF)was 1827 bp,which encoding 609 amino acids.Bioinformatics analysis showed that the molecular weight of raf encoded protein was 68.8 ku,and the isoelectric point was 9.45.The results of domain analysis showed that there were 4 conserved domains in ORF,RAS binding domain,protein kinase C conserved domain(C1),low synthesis complexity domain and serine/threonine protein kinase catalytic domain.It was a hydrophilic transmembrane protein.The results of homologous analysis of Raf kinase showed that the similarity compared with Lepisosteus oculatu was the highest of 84.28%.Both S.formosus and Lepisosteus oculatus were early-differentiated species,and the result of homologous analysis was in accordance with their evolutionary status.The expression pattern of raf in eight tissues was evaluated by real-time fluorescent quantitative PCR,and the result showed that raf gene was expressed in various tissues of S.formosus.The expression of raf was the highest in the testis and liver,and the next was the spleen and the head kidney.The expression levels in testis and liver were significantly higher than those in ovary,gill,heart and brain tissues(P<0.05).The prokaryotic expression vector of pET-B2m-raf was successfully constructed,and transferred to expression competent cells.The fusion protein was obtained by induction with 0.1 mmol/L IPTG.The molecular weight of the fusion protein was consistent with the expected results.[Conclusion]The expression of raf was high in testis and liver,and it was speculated that raf might play a role in testis development and the immune regulation in S.formosus.The prokaryotic expression vector was constructed and the recombinant protein was successfully induced,which laid a foundation for the further study of the biological function of raf in S.formosus.