牛ATG10基因的克隆、原核表达、纯化和生物信息学分析

Cloning,prokaryotic expression,purification and bioinformatics analysis of bovine ATG10 gene

为了获得牛自噬相关基因10(ATG10)蛋白,试验参照GenBank中牛ATG10基因设计引物,PCR扩增ATG10基因,与pET-N-His-TEV载体连接,测序鉴定正确后用IPTG诱导ATG10蛋白表达,利用Ni-TED琼脂糖树脂进行蛋白纯化,Western-blot鉴定ATG10蛋白表达情况,通过生物信息学在线软件对ATG10蛋白进行分析,获得其相应的理化性质及参数。结果表明:克隆得到的ATG10基因序列长度约为705 bp,测序鉴定正确后得到pET-N-His-TEV-ATG10原核表达载体,IPTG成功诱导表达了ATG10蛋白,分子量为28 ku;ATG10蛋白与兔抗ATG10抗体特异性结合;ATG10蛋白是一种较为稳定的、含38个潜在的磷酸化位点、无跨膜区及信号肽的亲水性蛋白,二级结构中无规则卷曲占比44.44%,与ATG5、ATG12、ATG16L1、ATG3等多个自噬相关蛋白相互作用。说明试验成功构建出pET-N-His-TEV-ATG10原核表达载体,并获得ATG10蛋白。

In order to obtain the bovine autophagy related gene 10(ATG10)protein,in this experiment,primers were designed with reference to ATG10 gene in GenBank,and PCR amplified ATG10 gene,which was connected to pET-N-His-TEV vector.After correct sequencing,IPTG was used to induce ATG10 protein expression.The protein was purified by Ni-TED agarose resin,and the expression of ATG10 protein was identified by Western-blot.The physicochemical properties and parameters of ATG10 protein were obtained by bioinformatics online software.The results showed that the cloned ATG10 sequence was about 705 bp in length,and the pET-N-His-TEV-bovine ATG10 plasmid was obtained after correct sequencing and identification.IPTG successfully induced the expression of ATG10 protein with a molecular mass of 28 ku.ATG10 protein was binded specifically to ATG10 antibody.ATG10 protein was a relatively stable hydrophilic protein with 38 phosphorylated modification sites,no transmembrane region and signal peptide were contained.Irregular curling accounted for 44.44% of the secondary structure,and it interacted with many autophagy related proteins such as ATG5,ATG12,ATG16L1 and ATG3.The results indicated that the recombinant pET-N-His-TEV-ATG10 prokaryotic expression vector was successfully constructed and ATG10 protein was obtained.