Point mutation, molecular evolution and prokaryotic expression in E.coli DE3 of bovine HSP70 gene
Point mutation, molecular evolution and prokaryotic expression in E.coli DE3 of bovine HSP70 gene
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作者：
Cai Ya
成功克隆1 926 bp的bHSP70基因，并与pGEMD○R-T Easy Vector连接。使用ABI PRISM TM 377进行双向测序。在HSP70 ORF中发现突变（941，T→C），导致HSP70蛋白序列发生变化（314，L→P）。用DNAstar 2.0 Megalign软件将其蛋白序列与NCBI中HSP70蛋白的另一序列进行比对。还用 DNAstar 2.0 Protean 探索了一级结构、二级结构和滴定曲线。结果表明:蛋白质序列发生突变(314,L→P)时,转角区数量增多,线圈区变化不大。分子进化证据表明HSP70基因足以作为进化研究的完美研究工具。我们设计并构建了pET-32a-c(+)-bHSP70载体，并在大肠杆菌DE3中表达bHSP70蛋白。 IPTG成功诱导rbHSP70蛋白。纯化重组蛋白并用于检测其活性。本研究包括两种体外细胞系（293 细胞和 Hela 细胞）。在我们的重组蛋白中发现了主要的抗凋亡活性。
The 1 926 bp bHSP70 gene was successfully cloned and linked with pGEMD○R-T Easy Vector. It was sequenced in both directions using an ABI PRISM TM 377. A mutation (941,T→C) was found in HSP70 ORF, leading to the change(314, L→P) of the sequence of HSP70 protein. Its protein sequence was aligned with the other sequence of HSP70 protein in NCBI with software DNAstar 2.0 Megalign. The primary structure, secondary structure and titration curve were also explored with DNAstar 2.0 Protean. The results were shown as follows:the number of the turn-region increased and little change was found in coil-region with the mutation(314, L→P) in protein sequence. The molecular evolutionary evidence indicated that HSP70 gene was sufficient for acting as a perfect research tool for evolutionary research. We designed and reconstructed a pET-32a-c(+)-bHSP70 vector and made bHSP70 protein be expressed in E.coli DE3. rbHSP70 protein was successfully induced by IPTG. The recombinant protein were purified and used to detection of its activity. Two cell lines (293 cells and Hela cells) in vitro were included in this study. The primary anti-apoptosis activity was found in our recombinant protein.