青虾血蓝蛋白基因的克隆、表达分析及多克隆抗体的制备  

Molecular cloning and expression analysis of the hemocyanin gene from oriental river prawn(Macrobrachium nipponense)

作　　者：孙盛明[1] 戈贤平[1,2] 傅洪拓[1,2] 朱健[1] 张世勇[2] 乔慧[1] 

机构地区：[1]中国水产科学研究院淡水渔业研究中心,农业部淡水渔业与种质资源利用重点实验室,江苏无锡214081 [2]南京农业大学无锡渔业学院,江苏无锡214081

出　　处：《中国水产科学》2014年第2期235-243,共9页Journal of Fishery Sciences of China

基　　金：国家科技支撑计划项目(2012BAD26B04;2012BAD25B07)

摘　　要：应用RACE技术克隆了青虾(Macrobrachium nipponense)的Hc基因全长cDNA序列,并对该基因序列特征进行了分析。青虾Hc基因cDNA全长2 235 bp,包括10 bp的5′末端非翻译区(UTR),2 074 bp的开放阅读框(ORF),151 bp的3′UTR,开放阅读框编码688个氨基酸。蛋白相似度比对显示,青虾血蓝蛋白含有典型的6个保守的铜离子结合位点。系统进化树分析表明,青虾Hc与脊尾白虾(Exopalaemon carinicauda)Hc聚在一起,具有最近的亲缘关系。荧光定量PCR检测显示,Hc基因在青虾不同组织中均有表达,其表达量在肝胰腺中最高;使用荧光定量PCR检测青虾Hc基因在低氧胁迫和复氧条件下在肝胰腺中的mRNA时空表达情况,结果显示,经低氧和复氧刺激后,与对照组相比,Hc在肝胰腺中的表达量分别在低氧胁迫12 h、24 h和复氧6 h出现了3次明显上调,由此推测Hc基因参与低氧应激分子过程。此外,本研究通过血蓝蛋白的分离纯化技术制备了多克隆抗体,本研究结果可为更进一步的了解青虾血蓝蛋白的生理功能提供理论支撑。Hemocyanin is a copper-binding protein that plays a crucial role in the physiological processes of crustacean. In this study, the cDNA-encoding hemocyanin subunit （MnHc） from the oriental river prawn, Macrobrachium nippo-nense, was cloned using expressed sequence tag （EST） analysis and a rapid amplification of cDNA ends （RACE） ap-proach. The full-length cDNA of MnHc was 2 235 bp, consisting of a 5′untranslated region of 10 bp, a 3′untranslated region of 151 bp, and an open reading frame of 2 064 bp. The deduced protein had 688 amino acid residues with a mo-lecular mass of 79.16 kD. Based on a protein similarity comparison, the two copper-binding sites and six histidine resi-dues necessary for stabilization of oxygen-binding were highly conserved. Phylogenetic tree analysis indicated that M. nipponense has the closest relationship with Exopalaemon carinicauda. Quantitative real-time RT-PCR analysis showed that the MnHc gene was expressed in hemocytes, the hepatopancreas, muscles, testis and intestines, with the highest level of expression in the hepatopancreas and the lowest in muscles. After environmental hypoxia and reoxygenation challenge, the relative expression level of MnHc in the hepatopancreas was significantly higher compared with the con-trol group at 12 h, 24 h post-hypoxia and 6 h reoxygenation, followed by a gradual recovery at 24 h reoxygenation. The highest titer of antiserum was up to 1︰120 000, as revealed by ELISA assay. The specificity of antibodies against hemocyanin was verified by westernblotting. The study indicates that the expression levels of MnHc in the hepatopan-creasmay be affected by environmental dissolve oxygen, and hemocyanin may potentially be involved in the hypoxia stress response of M. nipponense.

关 键 词：青虾 血蓝蛋白 基因克隆 低氧胁迫 复氧刺激 多克隆抗体 

分 类 号：S941[农业科学—水产养殖]