肝靶向穿膜肽与家蝇天蚕素基因的融合及其分子特征分析

本研究利用改进SOE-PCR技术构建肝靶向穿膜肽(HTPP)与家蝇天蚕素(MDC)融合基因并对其分子特征进行了预测和分析。结果表明:成功融合了HTPP与MDC,并构建了HTPP-MDC融合基因的克隆重组质粒HTPP-MDC/pMD20-T。PCR和KpnⅠ/HindⅢ双酶切结果显示获得与预期大小一致的基因片段,测序结果显示获得的基因序列没有发生突变,与预期完全一致。分子特征分析表明,该融合基因编码60个氨基酸,分子量为6516.2Da,理论等电点为9.31,二级结构主要由α-螺旋、无规则卷曲、延伸链和β-转角组成。研究结果为HTPP-MDC后续的功能研究奠定了基础,同时也为应用SOE-PCR技术构建融合基因提供了有益借鉴。

Construction and Molecular Characteristics of Fusion Gene HTPP-MDC

A no vel fusion gene combining hepatocyte-targeted cell penetrating peptide (HTPP) with Musca domestica cecropin (MDC) was constructed by improved SOE-PCR. The molecular characteristics of HTPP-MDC were analyzed with bioinformatics analysis software. The results show that the fusion gene HTPP-MDC and recombinant plasmid HTPP-MDC/pMD20-T were constructed successfully. The results of PCR amplification and restriction endonuclease KpnⅠ/Hind Ⅲ digesting show that the acquired gene fragment is as long as what have been expected. DNA sequencing confirms that the sequence of HTPP-MDC is identified with the sequences of HTPP and MDC reported in Genbank. The analysis of molecular characteristics indicates that HTPP-MDC is cationic molecule containing 60 amino acids withthe theoretical PI of 9.31 and the molecular weight of 6 516.2 Da. The second structure of HTPP-MDC contains α -helix, random coil, extended strand and β-turn. This research could provide foundation for further study on the biological activity of HTPP-MDC, and give useful reference for constructing fusion gene with SOE-PCR.