山羊地方性鼻内肿瘤病毒ENTV/CH/GT/2015株全基因组cDNA文库的构建及生物信息学分析

Complete genomic cloning and bioinformatics analysis of goat enzootic nasal tumor virus ENTV/CH/GT/2015

为获得山羊地方性鼻内肿瘤病毒(ENTV-2)古田株(暂命名为:ENTV/CH/GT/2015株)全基因组序列。通过PCR方法从山羊鼻内肿瘤组织中分段扩增获得ENTV-2全基因组7个片段,目的片段经胶回收后分别连接至pMD19-T载体上,并转化至基因工程菌DH5a,提取阳性质粒进行测序,利用生物学软件对获得的序列进行拼接分析。获得ENTV/CH/GT/2015株全基因组序列长度为7506 bp,包含相互重叠的gag-pro-pol-env编码区及5′端非编码区,并上传至GenBank上获得登录号为MK210250.1;与国外NC004994.2株和AY197548.2株核苷酸同源性均为87.5%,与国内ENTV-2CHN8株核苷酸同源性为97.7%。氨基酸序列分析结果显示,gag蛋白在124~126 aa插入GVE 3个氨基酸和229~232 aa插入APPP 4个氨基酸,env蛋白在590~593 aa处存在AESL 4个氨基酸的缺失;gag和env蛋白抗原表位分析结果显示,尽管存在氨基酸的突变和缺失,但抗原表位并没有明显差异。糖基化位点预测结果显示:gag、pro、pol和env蛋白分别含有1,1,4,6个糖基化位点,均可能不包含信号肽。遗传进化树分析结果推测,该毒株是国内ENTV-2CHN2株和ENTV-2CHN10株变异毒株。本研究获得ENTV/CH/GT/2015株全基因序列并明确其生物学特性,不仅丰富了ENTV-2全基因序列库,还为今后研究ENTV-2快速检测方法及致病机理提供了研究素材。

In order to obtain the complete genome of enzootic nasal tumor virus of goats(ENTV-2)Gutian strain(tentatively named ENTV/CH/GT/2015 strain).In this study,7 pairs of primers were designed based on the whole genome sequence of 16 strains of ENTV-2 included in GenBank,and 7 fragments of ENTV-2 whole genome were obtained by segmenting and amplifying the tumor tissue in goats nose by PCR method.After gel recycling,the target fragments were connected to pMD19-T vector and transformed into E.coli DH5 a.The extracted positive plasmids were sent to sequencing.DNAStar software was used to splice the obtained sequences.In this study,a genome sequence of ENTV/CH/GT/2015 strain with a length of 7506 bp was obtained,including overlapping gag-pro-pol-env coding region and 5’-end non-coding region,and was uploaded to GenBank to obtain the login number Mk210250.1.It has 87.5%nucleotide similarity with NC004994.2 and AY197548.2,and 97.7%with domestic ENTV-2 CHN8 strain.Amino acid sequence analysis showed that gag protein was inserted into 3 amino acids of GVE at 124-126 aa and 4 amino acids of APPP at 299-232 aa,while env protein has 4 amino acids of AESL deletion at 590-593 aa.The result of antigenic index analysis of gag and env protein showed that there were no significant difference in antigen epitoes despite the mutation and deletion of amino acids.The predicted result of glycosylation sites showed that gag,pro,pol and env proteins contained 1,1,4 and 6 glycosylation sites respectively,and all of them might not contain signal peptide.Genetic evolutionary tree analysis indicated that the strain was the mutant strain of domestic ENTV-2 CHN2 strain and ENTV-2 CHN10 strain.In this study,the whole gene sequences of ENTV/CH/GT/2015 was obtained and its bioinformatics were clarified,which not only enrich the whole ENTV-2 gene sequence library,but also provide research materials for the future research on the rapid detection method and pathogenic mechanism of ENTV-2.