| 金鱼HSC70和HSP40基因克隆及其原核表达 |
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| 引用本文： | 金新萍,谢倩,吕斌,曹诣斌.金鱼HSC70和HSP40基因克隆及其原核表达[J].南方农业学报,2018,49(2):367-374. |
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| 作者姓名： | 金新萍 谢倩 吕斌 曹诣斌 |
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| 作者单位： | 浙江师范大学 化学与生命科学学院,浙江 金华,321004 |
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| 摘 要： | 目的]克隆金鱼热激同源蛋白70(HSC70)和热休克蛋白(HSP40)基因,构建原核表达载体并通过IPTG诱导表达获得目的蛋白,明确HSC70和HSP40蛋白的生物学功能,为揭示金鱼的高温应答机制打下基础.方法]提取金鱼鳃组织总RNA,利用RT-PCR扩增金鱼HSC70和HSP40基因,将目的基因片段克隆至线性空表达载体pET-32a上构建原核表达载体pET-32a-HSC70和pET-32a-HSP40,转化大肠杆菌Rosetta表达菌株后用IPTG进行诱导表达,并以SDS-PAGE和Western blotting检测分析表达获得的融合蛋白.结果]金鱼HSC70基因CDS序列全长1950 bp,编码650个氨基酸;金鱼HSP40基因CDS序列全长996 bp,编码332个氨基酸.金鱼HSC70氨基酸序列与斑马鱼、人类、家鼠、金线鲃和草鱼的相似度分别为96.28%、95.50%、95.35%、98.15%和98.61%;HSP40氨基酸序列与斑马鱼的完全一致(相似度100.00%),与普通鲤鱼、金线鲃、人类和家鼠的相似度均为94.12%.原核诱导表达获得的融合蛋白Trx-HSC70和Trx-HSP40为可溶性表达,其大小约91.5和55.0 kD,均能与Anti-Trx抗体发生特异性反应.结论]HSC70和HSP40蛋白在鱼类及哺乳动物中高度保守,经原核诱导表达获得的金鱼融合蛋白Trx-HSC70和Trx-HSP40为可溶性表达,且携带有Trx标签,能与Anti-Trx抗体发生特异性反应,可作为互作蛋白用于RNA pull down试验.
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| 关 键 词： | 金鱼 HSC70基因 HSP40基因 克隆 原核表达载体 |
Cloning and prokaryotic expression of genes HSC70 and HSP40 in goldfish |
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| JIN Xin-ping,XIE Qian,LYU Bin,CAO Yi-bin.Cloning and prokaryotic expression of genes HSC70 and HSP40 in goldfish[J].Journal of Southern Agriculture,2018,49(2):367-374. |
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| Authors: | JIN Xin-ping XIE Qian LYU Bin CAO Yi-bin |
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| Abstract: | Objective]Heat shock cognate protein 70 gene(HSC70)and heat shock protein 40 gene(HSP40)of gold-fish were cloned,their prokaryotic expression vectors were constructed and induced by IPTG to obtain target proteins,and the biological functions of proteins HSC70 and HSP40 were surveyed to laid the foundation for revealing the mechanism of high-temperature response of goldfish.Method]Genes HSC70 and HSP40 were amplified from total RNA isolated from goldfish liver tissues using RT-PCR.The target gene fragments were cloned to linear empty expression vector pET-32a, then prokaryotic expression vectors pET-32a-HSC70 and pET-32a-HSP40 were constructed on it.Transformed Escherichia coli Rosetta expression strains were induced by IPTG,and the obtained fusion protein was analyzed by SDS-PAGE and Western blotting.Result]CDS sequence of goldfish gene HSC70 was 1950 bp in length,encoding 650 amino acids;CDS sequence of goldfish gene HSP40 was 996 bp in length,encoding 332 amino acids. Amino acid sequence of HSC70 shared 96.28%,95.50%,95.35%,98.15% and 98.61% similarity with zebrafish,human,house mouse,golden-line bar-bell and grass carp.Amino acid sequence of HSP40 was completely the same with that of zebrafish(similarity 100.00%), and it shared 94.12% similarity with common carp,golden-line barbell,human and house mouse.The fusion proteins Trx-HSC70 and Trx-HSP40 obtained by protokaryon induction were in soluble expression,and they were 91.5 and 55.0 kD re-spectively.They could both specifically react to antibody Anti-Trx.Conclusion]Proteins HSC70 and HSP40 are modera-tely or highly conservative in fish and mammals.The goldfish fusion proteins obtained by protokaryon induction are in soluble expression,and have Trx tag.They can both specifically react to antibody Anti-Trx,and can be used in RNA pull down experiment as interacting proteins. |
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