尼罗罗非鱼P2X4R基因克隆及原核表达分析

摘要

[目的]克隆尼罗罗非鱼P2X4R基因,并构建原核表达载体进行诱导表达,为深入研究P2X4R在鱼类中的生物学功能打下基础.[方法]利用PCR克隆尼罗罗非鱼P2X4R基因的3个片段(G1、G2和G3),拼接获得目的基因后连接pCold II载体构建pCold II-P2X4R重组质粒,再转化大肠杆菌BL21(DE3)感受态细胞,以IPTG进行诱导表达.分别采用SDS-PAGE和Western blotting检测分析重组蛋白P2X4R的表达情况,并运用生物信息学在线分析软件对其理化性质、糖基化位点、跨膜区域、亚细胞定位及信号肽等进行预测分析.[结果]克隆获得的尼罗罗非鱼P2X4R基因大小为1108 bp,与pCold II载体重组后转化BL21(DE3)感受态细胞获得的原核表达载体经IPTG诱导可表达获得目的蛋白,在IPTG 1.0 mmol/L、37℃诱导4 h的条件下重组蛋白表达量高于在IPTG 0.1 mmol/L、16℃过夜诱导的表达量.重组蛋白P2X4R的分子量约43.0 kD,其氨基酸数量为354个,理论等电点(pI)为6.78,不稳定指数为37.62,属于稳定蛋白,脂肪族指数为74.35;重组蛋白P2X4R具有3个N-糖基化位点和1个O-糖基位点;该蛋白未见跨膜区,其蛋白几乎100%位于细胞膜内,不含信号肽.[结论]诱导表达获得的尼罗罗非鱼P2X4R蛋白具有3个N-糖基化位点和1个O-糖基化位点,推测其存在糖基化现象,可制备相应抗体用于揭示罗非鱼巨噬细胞的抗原呈递作用机制.%[Objective]Gene P2X4R of Nile tilapia(Oreochromis niloticus)was cloned and its prokaryotic expression vector was constructed to induce its expression in order to lay a foundation for studying biological function of P2X4R in fish.[Method]Three fragments(G1,G2 and G3)of gene P2X4R of Nile tilapia were cloned by PCR,then assembled into the target gene.The target gene linked pCold II vector to construct recombinant plasmid of pCold II-P2X4R.Bacillus coli BL21(DE3)competent cells were transformed to be induced by IPTG. The expression of recombinant protein P2X4R were detected by SDS-PAGE and Western blotting.Bio-informatics was used to analyze the physicochemical properties of protein P2X4R,glycosylation site,transmembrane region,subcellular localization and signal peptide.[Result]Gene P2X4R of Nile tilapia was cloned and its length was 1108 bp.The pCold II-P2X4R expression vector,which was constructed re-combinatly with pCold II vector,could obtain target protein after being induced by IPTG.The expression of recombinat protein induced by 1.0 mmol/L IPTG at 37℃for 4 h was higher than that induced by 0.1 mmol/L IPTG at 16℃overnight. Recombinat protein P2X4R contained 43.0 kD molecular weight and 354 amino acids.Its theoretical isoelectric point(pI) was 6.78,instability index 37.62 and aliphatic index 74.35,which meant it was stable protein.The recombinant protein P2X4R has three N-glycosylation sites and one O-glycosylation site.There was no transmembrane region in the protein, its protein sequence was almost 100% within the membrane. No signal peptide was observed.[Conclusion]Nile tilapia protein is obtained by induction.It contains three N-glycosylation sites and one O-glycosylation sites.It is inferred that gly-cosylation phenomenon exists in it.Therefore,it could be used for preparation of relevant antibodies which can reveal an-tigen presentation of Nile tilapia macrophage.