南方农业学报2018，Vol.49Issue(2)：367-374,8.DOI:10.3969/j.issn.2095-1191.2018.02.25
金鱼HSC70和HSP40基因克隆及其原核表达
Cloning and prokaryotic expression of genes HSC70 and HSP40 in goldfish
摘要
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.关键词
金鱼/HSC70基因/HSP40基因/克隆/原核表达载体Key words
goldfish/gene HSC70/gene HSP40/clone/prokaryotic expression vector分类
农业科技引用本文复制引用
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):367-374,8.基金项目
国家自然科学基金项目(KJZKJY13088) （KJZKJY13088）
