Cloning and molecular characterization of heat shock cognate 70 from tiger shrimp (Penaeus monodon)
Cloning and molecular characterization of heat shock cognate 70 from tiger shrimp (Penaeus monodon)
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DOI：
10.1379/csc-47r.1
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发表时间：
2004-12-01
影响因子：
3.8
通讯作者：
Song, YL
中科院分区：
文献类型：
--
作者：
Lo, WY;Liu, KF;Song, YL
克隆了斑节对虾热休克同源基因70（hsc 70）的互补脱氧核糖核酸（cDNA）。该基因全长2207 bp，包括1959 bp的编码区，5'端40 bp的侧翼区，3'端208 bp的侧翼区。推导的，652个氨基酸的序列具有71 481 Da的分子量和估计的等电点（pI）为5.2。基于系统发育分析，该基因与脊椎动物和无脊椎动物的hsc 70蛋白聚在一起。在天然凝胶电泳中，在大肠杆菌系统中表达的重组P monodon hsc 70与羧甲基化α-乳白蛋白（CMLA）紧密相关，这表明hsc 70可能起伴侣作用。在体外腺苷三磷酸酶测定中，重组hsc 70将腺苷三磷酸水解为腺苷-5 '-二磷酸，并通过与未折叠肽CMLA结合来增加水解活性。利用反义核酸探针进行原位杂交，结果显示hsc 70基因在非应激状态下的大部分组织中都有活性。在热休克后的第1小时和恢复0.5小时，血细胞中hsc 70信使核糖核酸（mRNA）的表达增加了2- 3倍。Hsc 70 mRNA逐渐下降至背景水平。克隆和鉴定单齿对虾hsc 70基因是研究热休克蛋白与对虾应激或免疫反应关系的第一步，也是关键的一步。
We cloned the complementary deoxyribonucleic acid (cDNA) of the heat shock cognate 70 (hsc70) gene of tiger shrimp (Penaeus monodon). It was 2207 bp long and included a 1959-bp coding region, a 40-bp flanking region at the 5' end, and a 208-bp flanking region at the 3' end. The deduced, 652-amino acid sequence had a molecular mass of 71 481 Da and an estimated isoelectric point (pI) of 5.2. Based on phylogenetic analysis, the gene is clustered with the hsc70 proteins of invertebrates and vertebrates. In native gel electrophoresis, recombinant P monodon hsc70 expressed in an Escherichia coli system is tightly associated with carboxymethylated alpha-lactalbumin (CMLA), which indicates that hsc70 probably functions as a chaperone. In an in vitro adenosine triphosphatase assay, recombinant hsc70 hydrolyzed adenosine triphosphate to adenosine-5'-diphosphate and increased hydrolysis activity by binding to unfolded peptide, CMLA. In situ hybridization using an antisense riboprobe revealed that the hsc70 gene was active in most tissues of unstressed shrimp. The expression of hsc70 messenger ribonucleic acid (mRNA) in hemocytes increased 2- to 3-fold at the first hour after shrimp experienced heat shock and 0.5-hour recovery. Hsc70 mRNA decreased gradually to the background level. Cloning and characterizing the P monodon hsc70 gene is the first, crucial step in studying the relationship of heat shock proteins with the stress or immune responses of shrimp.