摘要
为了解新疆北疆地区牛轮状病毒VP7基因的结构与功能,参照GenBank中所收录的牛轮状病毒DQ株序列设计了一对特异性引物,利用生物信息学技术分析VP7蛋白基因的同源性、密码子偏爱性、信号肽、跨膜区、糖基化位点、磷酸化位点及二级结构等,利用RT-PCR扩增出1080bp的VP7全长基因,测序结果与参考的核苷酸同源性达到90%左右,克隆到pMD19-T载体中,获得pMD19-T-VP7阳性质粒。以阳性质粒为模板进行PCR鉴定,定向克隆到表达载体28a上,经酶切、PCR鉴定阳性质粒转化到大肠杆菌BL21(DE3)中,经IPTG(终浓度为1.5mmol/mL)37℃诱导4—5h。结果表明,SDS-PAGE电泳显示表达了大小约40KD的带His标签的融合蛋白,经WesternBlot分析表达的VP7蛋白可与BRV阳性血清反应。VP7基因氨基酸密码子偏爱以G、C结尾的密码子;VP7蛋白可能存在4个糖基化位点,无信号肽,2个跨膜区,VP7蛋白二级结构预测,a-螺旋区域85个氨基酸占26.15%;β折叠区域91个氨基酸占28.00%;无规则卷区域149个氨基酸占45.85%。本研究结果对牛轮状病毒VP7基因相关信息进一步了解,为VP7蛋白作为诊断用抗原做了铺垫,对于诊断试剂以及疫苗的相关开发具有参考意义。
In order to understand the structure and function of gene VP7 of bovine rotavirus in northern Xinjiang,a pair of specific primers were designed according to the bovine rotavirus DQ strain sequence included in GenBank,using the bioinformatics analysis of VP7 protein gene homology,codon preference,signal peptide,transmembrane region,sugar glycosylation sites,phosphorylation sites and two level structure of science and technology,the usage of RT-PCR amplified VP7 1080bp gene,sequencing results and reference nucleotide homology reached 90%,by cloning into pMD19-T vector,PMD19-T-VP7 positive plasmid was acquired.The positive plasmid was identified by PCR template,cloned into expression vector 28a,after enzyme digestion and PCR identification of positive plasmids were transformed into Escherichia coli BL21 (DE3),IPTG (final concentration 1.5mmol/mL) induced by 37 DEG C for 4-5 hours.The results showed that SDS-PAGE electrophoresis showed a fusion protein with a His tag of about 40KD in size.The VP7 protein expressed by Western Blot analysis could react with BRV positive sera.VP7 gene amino acid codon preference to G and C codons; VP7 protein may exist 4 glycosylation sites,no signal peptide,2 transmembrane regions,two predicted the secondary structure of VP7 protein,a-helix region:85 amino acids accounted for 26.15%,beta sheet region of 91 amino acids accounted for 28%,no rules volume area:149 amino acids accounted for 45.85% The results of this study can further understand the related information of bovine rotavirus VP7 gene,and pave the way for VP7 protein as a diagnostic antigen,which has reference significance to the development of diagnostic reagents and vaccines.
作者
陆亚冬
LU Ya-dong(College of animal science and technology,Shihezi University,Xinjiang,Shihezi 832003)
出处
《绿洲农业科学与工程》
2018年第2期18-25,共8页
Oasis Agriculture Science and Engineering
基金
兵团重大科技项目“肉牛高效养殖关键技术集成与示范”(2014AA001-3)。
