牦牛源BVDV非结构蛋白NS5A的原核表达及抗原表位分析  

Prokaryotic Expression and Antigen Epitope for Non-structural Protein NS5A of BVDV Isolated from Yak

作　　者：王慧慧 冯茜莉 蒲飞洋 李易聪 汪梦竹 周小凯[1,2] 赵泽阳 马忠仁[1] 李倬[1] 马晓霞[1] WANG Huihui;FENG Xili;PU Feiyang;LI Yicong;WANG Mengzhu;ZHOU Xiaokai;ZHAO Zeyang;MA Zhongren;LI Zhuo;MA Xiaoxia(Biomedical Research Center,Northwest Minzu University,Lanzhou 730030,China;College of Life Science and Engineering,Northwest Minzu University,Lanzhou 730010,China)

机构地区：[1]西北民族大学生物医学研究中心,兰州730030 [2]西北民族大学生命科学与工程学院,兰州730010

出　　处：《中国畜牧兽医》2022年第8期3180-3189,共10页China Animal Husbandry & Veterinary Medicine

基　　金：甘肃省自然科学基金(20JR5RA505);校企横向科研项目(2021西民合(科研)第175号)。

摘　　要：【目的】利用原核表达系统体外表达牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)NS5A基因,获得非结构蛋白NS5A,对其进行核苷酸、氨基酸序列分析,以解析BVDV非结构蛋白NS5A的功能。【方法】参考BVDV-1型毒株V006的NS5A基因序列(GenBank登录号:KX170647)设计并合成1对特异性引物,以分离到的牦牛BVDV GSTZ毒株cDNA为模板,PCR扩增NS5A基因片段,并克隆至表达载体pET-28a(+)中,构建重组原核表达载体pET28a-NS5A。经酶切初步鉴定及测序鉴定正确后,转化大肠杆菌BL21(DE3)感受态细胞,然后利用IPTG诱导表达。经10%SDS-PAGE电泳及Western blotting分析鉴定重组蛋白的表达,并根据NS5A基因的序列构建遗传发育进化树,利用DNAStar软件预测NS5A蛋白的亲水性、表面可塑性和抗原性等特性,并结合二级结构的预测对NS5A蛋白的B细胞抗原表位进行预测。【结果】PCR扩增NS5A目的基因片段为1488 bp,双酶切和测序鉴定结果证明,重组质粒pET28a-NS5A构建成功。经10%SDS-PAGE电泳及Western blotting鉴定重组蛋白,表达出了大小为55 ku的目的蛋白,大小与预期结果相符。通过对不同BVDV毒株NS5A基因序列构建遗传发育进化树,显示GSTZ毒株NS5A在遗传进化特征上属于BVDV-1型。NS5A蛋白的亲水性主要位于12—21、32—69、75—113、120—135、143—147、152—163、165—180、215—230、265—274、296—340、348—378、389—447、455—463、469—495位氨基酸处,表面可塑性主要位于14—18、37—42、76—81、86—109、154—160、169—178、218—228、297—309、348—358、365—373、414—442、430—437、454—460位氨基酸处,柔性区域较多,主要位于14—21、37—43、67—82、86—93、97—110、152—158、169—179、218—231、240—255、296—310、313—328、344—359、364—373、413—422和472—483位氨基酸处。NS5A蛋白的B细胞抗原表位主要位于15—18、76—81、154—158、169—178、218—228、297—309、348�【Objective】The NS5A gene of Bovine viral diarrhea virus(BVDV)was expressed in vitro by prokaryotic expression system to obtain the non-structural protein NS5 A.The nucleotide and amino acid sequences were analyzed to analyze the function of BVDV non-structural protein NS5 A.【Method】A pair of primers were designed and synthesized according to the NS5A gene sequence of BVDV-1 V006 strain(GenBank accession No.:KX170647)deposited in GenBank.The NS5A gene was amplified by PCR using the cDNA of BVDV GSTZ strain from yak as a template and was inserted into the pET-28 a(+)vector,and the recombinant prokaryotic expression vector pET28 a-NS5 A was constructed.After preliminary identification by enzyme digestion and sequencing,E.coli BL21(DE3)competent cells were transformed,and then induced by IPTG.The expression of the recombinant protein was identified by 10%SDS-PAGE electrophoresis and Western blotting analysis,and the genetic development and evolution tree was constructed according to the sequence of NS5A gene.The hydrophilicity,surface plasticity and antigenicity of NS5 A protein were predicted by DNAStar software,and the B-cell antigen epitope of NS5 A protein was predicted combined with the prediction of secondary structure.【Result】The target gene fragment of NS5A amplified by PCR was 1488 bp,which was consistent with the expectation.The results of double enzyme digestion and sequencing showed that the recombinant plasmid pET28 a-NS5 A was successfully constructed.The recombinant protein was identified by 10%SDS-PAGE electrophoresis and Western blotting,and the target protein with the size of 55 ku was expressed.The size was consistent with the expected results.Through the construction of genetic development evolutionary tree for different BVDV strain NS5A gene sequences,it showed that GSTZ strain NS5 A belonged to BVDV-1 in genetic evolutionary characteristics.The hydrophilicity of NS5 A protein were mainly located at amino acids 12-21,32-69,75-113,120-135,143-147,152-163,165-180,215-230,265-274,296-340

关 键 词：牛病毒性腹泻病毒 非结构蛋白 NS5A 原核表达 

分 类 号：Q939.94[生物学—微生物学]