合浦珠母贝nacrein基因真核表达载体构建及生物信息学分析  

Bioinformatics analysis and construction of eukaryotic expression vector of nacrein gene from Pinctada fucata

作　　者：刘鹏 兰太进 杨宇华 杨大航 焦扬 李论 黎睿 林江 LIU Peng;LAN Taijin;YANG Yuhua;YANG Dahang;JIAO Yang;LI Lun;LI Rui;LIN Jiang(School of Basic Medical, Guangxi University of Chinese Medicine, Nanning 530200, China)

机构地区：[1]广西中医药大学基础医学院,广西南宁530200

出　　处：《邵阳学院学报（自然科学版）》2021年第5期8-17,共10页Journal of Shaoyang University：Natural Science Edition

基　　金：广西自然科学基金青年科学基金(2018GXNSFBA281100);广西中医药大学校级自然科研项目(2018QN001);广西壮族自治区大学生创新创业训练计划项目(202010600102)。

摘　　要：目的克隆合浦珠母贝(Pinctada fucata)水溶性基质蛋白Nacrein的基因,构建真核表达载体,并对Nacrein蛋白进行生物信息学分析。方法根据GenBank公布的Pinctada fucata nacrein基因CDS序列(D83523.1),设计合成特异引物,以合浦珠母贝肉cDNA为模板,克隆获得nacrein基因,构建到真核表达载体pPICZαA上,PCR筛选阳性克隆子,并对其进行测序鉴定和生物信息学分析。结果合浦珠母贝nacrein基因全长1344 bp,编码447个氨基酸,分子量约为50.11 kD,为一稳定的亲水性蛋白质,无跨膜结构域,为分泌性胞外蛋白质。具有2个碳酸酐酶结构域和1个Gly-Xaa-Asn重复结构域,存在多个蛋白酶酶切位点,其降解片段可能具有抑菌活性,且含有多个磷酸化位点,Nacrein蛋白糖基化程度低。Nacrein蛋白二级结构主要为无规卷曲,其次为α-螺旋和延伸链及β-折叠,其三维结构与所预测的Nacrein蛋白二级结构一致。结论成功克隆获得合浦珠母贝nacrein基因及构建真核表达载体,并进行了生物信息学分析,为后续深入开发Nacrein蛋白生物功能提供了一定的理论和实验数据。Objective To clone the CDS sequence of water-soluble matrix protein Nacrein gene of Pinctada fucata,construction of eukaryotic expression vector and bioinformatics analysis of Nacrein protein.Methods According to the CDS sequence of Pinctada fucata nacrein gene(D83523.1)published by GenBank,the specific primer was designed and synthesized.The nacrein gene was cloned and constructed into the eukaryotic expression vector pPICZαA.The positive clones were screened by PCR and then sequenced,the Nacrein protein was further analyzed by bioinformatics.Results The nacrein gene of Pinctada fucata was 1344 bp,encoding a protein of 447 amino acids with a calculated molecular weight of about 50.11 kDa.It was predicted to be a stable hydrophilic protein without transmembrane domain,but with a signal peptide.There were two carbonic anhydrase domains and one Gly-Xaa-Asn repeat domain,and had a series of protease cleavage sites.The degradation fragment of Nacrein protein had potential antibacterial activity and contained many potential phosphorylation sites,and Nacrein protein with low level of glycosylation.The secondary structure of Nacrein was mainly random curl,followed byα-helix,extended chain andβ-fold,the three-dimensional structure was consistent with the predicted secondary structure.Conclusion The nacrein gene of Pinctada fucata is cloned and constructed its eukaryotic expression vector,and the bioinformatics analysis is carried out,which provides some theoretical and experimental data for further development of Nacreen protein biological function.

关 键 词：合浦珠母贝 nacrein基因 真核表达载体 生物信息学 

分 类 号：S917.4[农业科学—水产科学]