牛源HSP70基因点突变、分子进化和pET32a-c(+)原核表达

Point mutation, molecular evolution and prokaryotic expression in E.coli DE3 of bovine HSP70 gene

设计引物PCR方法克隆了奶牛1926bp的HSP70基因,连接到pGEMR○-T Easy Vector上测序,与NCBI上的序列比对,发现开放阅读框内的点突变(941位点,T→C),致使314位L(亮氨酸)→P(脯氨酸)。DNAstar Protean分析点突变对蛋白的原核表达一、二级结构、等电点进行了分析,结果显示螺旋转角区数目增多,不规则卷曲发生轻微变化,但其它指标均不变。此外,通过DNAstar MegAlign用Clustal V方法分析HSP70家族蛋白的同源性、氨基酸残基替换频率并构建了分子进化树,结果发现碱性、酸性氨基酸(R和K)、分支的氨基酸(V和L)、极性氨基酸(S和T)等在生物HSP70分子进化中氨基酸发生替换的频率最高,HSP70可以作为研究生物系统演化的一个重要的工具。在上述基础上,在HSP70基因上、下游引物分别加上EcoRⅠ和HindⅢ酶切位点,构建了pET-32a-c(+)-bHSP70原核表达质粒,IPTG诱导成功得到重组牛源HSP70蛋白,293细胞和Hela细胞的体外实验表明原核表达的蛋白有抗凋亡生物活性[动物学报51(6)1080-1090,2005]。

The 1 926 bp bHSP70 gene was successfully cloned and linked with pGEM-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 [Acta Zoologica Sinica 51 （6）： 1080 - 1090, 2005].