新城疫病毒HBNU/LSRC/F3株HN基因的克隆及生物信息学分析  

Cloning and bioinformatic analysis of the hemagglutinin-neuraminidase gene of the HBNU/LSRC/F3 strain of the Newcastle disease virus

作　　者：李冠华[1] 马媛媛[1] 王静[1] 武彩霞[1] 刘进军[1] 刘开扬[1] 

机构地区：[1]河北北方学院生命科学研究中心,河北北方学院附属第二医院,河北张家口075000

出　　处：《中国病原生物学杂志》2016年第4期305-311,共7页Journal of Pathogen Biology

基　　金：河北省自然基金资助项目(No.H2014405044);河北北方学院创新人才培育项目(No.CXRC1318)

摘　　要：目的克隆新城疫病毒HBNU/LSRC/F3株的血凝素神经氨酸酶蛋白(HN)基因,同时应用生物信息学工具分析其编码蛋白的主要特性。方法根据HN基因开放阅读框设计引物,提取新城疫病毒总RNA,通过逆转录PCR(RT-PCR)扩增HN基因,并将其连接到克隆载体上,利用蛋白分析专家系统(ExPASy)提供的ProtParam、Protscale、TMpred、Coil、MotifyScan、SWISS-MODEL等,NCBI上的CONSERVE DOMON、SMART、SignalP4.0、Bepipred 1.0、NetCTL等以及Bcepred、ABCpred、SOPMA、SYFPEITHI等生物信息学在线分析程序,结合PSORT II Prediction、rasmol等生物信息学软件,分析、预测其编码HN蛋白的主要特性及T/B细胞抗原表位等。结果 HN基因RT-PCR扩增产物与预期大小一致,并成功连接到克隆载体上,测序结果显示HN基因序列与GenBank上已发表的序列完全一致。其编码蛋白由571个氨基酸组成,分子式为C2766H4316N752O852S24,分子质量单位为62.5ku,等电点理论值为6.24。HN蛋白为跨膜蛋白,富含无规则卷曲(Cc)和α-螺旋(Hh),其主要定位在线粒体和胞质中。该蛋白共有12个亲水性较高区域(参数得分≥1.9),17个表面可及性较高区域(参数得分≥1.9),9个柔韧性较高区域(参数得分≥2.0),36个翻译后修饰位点,17个潜在的B细胞表位,12个CTL表位和辅助性T细胞的联合表位。结论成功克隆了HN基因并分析了其编码蛋白的主要特性,为后期HN蛋白的分子克隆及其抗肿瘤特性研究奠定了基础。Objectives To clone the hemagglutinin-neuraminidase（HN）gene of the HBNU/LSRC/F3 strain of the Newcastle disease virus and to analyze its main characteristics using bioinformatics software. Methods The total RNA of NDV was extracted.Primers were designed in accordance with the open reading frame of the HN gene,and the gene was amplified using reverse transcription PCR（RT-PCR）.The gene was ligated to a cloning vector.The major characteristics and T/B cell epitopes of the HN protein were analyzed and predicted using Web-based programs for bioinformatic analysis such as ProtParam,protscale,TMpred,COILS,MotifScan,and SWISS-MODEL provided by an expert protein analysis system（ExPASy）,CDD from the NCBI,SMART,SignalP 4.0,Bepipred 1.0,NetCTL,BcePred,ABCpred,SOPMA,and SYFPEITHI along with bioinformatic software such as PSORT II Prediction and rasmol. Results The HN gene was amplified with RT-PCR,and the product was consistent with the expected size.The gene was ligated to a cloning vector.Sequencing indicated that the sequence of the HN gene was exactly the same as that published in GenBank.The protein consisted of 571 amino acids,its molecular formula was C2766H4316N752O852S24,it had a molecular mass of 62.5ku,and its theoretical isoelectric point was 6.24.The HN protein is a transmembrane protein replete with random coils（Cc）and alpha helices（Hh）.The protein is mainly located in mitochondria and the cytoplasm.The protein has 12 highly hydrophilic regions（corresponding score≥1.9）,17 highly surface-accessible regions（corresponding score≥1.9）,9highly flexible regions（corresponding score≥2.0）,36post-translational modification sites,7potential B-cell epitopes,and 12 CTL and helper T-cell epitopes. Conclusion The HN gene was cloned and the main characteristics of the protein it encodes were analyzed.This work has laid the foundation for later molecular cloning of the HN protein and study of its anti-tumor properties.

关 键 词：HN蛋白 生物信息学 主要特性 抗肿瘤 

分 类 号：R383.33[医药卫生—医学寄生虫学]