牦牛源BVDV非结构蛋白NS5A的原核表达及抗原表位分析

doi:10.16431/j.cnki.1671-7236.2022.08.034

摘要/Abstract

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1 488 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—358、365—373和414—422位氨基酸处。【结论】成功表达并鉴定了牦牛源BVDV的非结构蛋白NS5A,系统发育进化树表明BVDV GSTZ株基因型属于BVDV-1型,NS5A蛋白具有良好的抗原性,为深入解析BVDV非结构蛋白NS5A的自身结构功能、免疫学特性以及进一步研究非结构蛋白对病毒复制的影响提供参考。

关键词: 牛病毒性腹泻病毒; 非结构蛋白; NS5A; 原核表达

Abstract: 【Objective】 The NS5A gene of Bovine viral diarrhea virus (BVDV) was expressed in vitro by prokaryotic expression system to obtain the non-structural protein NS5A.The nucleotide and amino acid sequences were analyzed to analyze the function of BVDV non-structural protein NS5A.【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-28a(+) vector,and the recombinant prokaryotic expression vector pET28a-NS5A 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 NS5A protein were predicted by DNAStar software,and the B-cell antigen epitope of NS5A protein was predicted combined with the prediction of secondary structure.【Result】 The target gene fragment of NS5A amplified by PCR was 1 488 bp,which was consistent with the expectation.The results of double enzyme digestion and sequencing showed that the recombinant plasmid pET28a-NS5A 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 NS5A belonged to BVDV-1 in genetic evolutionary characteristics. The hydrophilicity of NS5A 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, 348-378, 389-447, 455-463, 469-495. The surface plasticity were mainly located at amino acids 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. And there were many flexible regions, mainly located at amino acids 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 and 472-483. The B-cell antigen epitopes of NS5A protein were mainly located at amino acids 15-18,76-81,154-158,169-178,218-228,297-309,348-358,365-373 and 414-422.【Conclusion】 The non-structural protein NS5A of BVDV from yak was successfully expressed and identified.The phylogenetic tree showed that the BVDV GSTZ isolate was classified into BVDV-1.NS5A protein had good antigenicity.This study provided a basic plateform which provided reference for function and immunological characteristics of BVDV non-structural protein NS5A,and further study on the effect of NS5A protein on viral replication and relationship between biological function and protein structure.

Key words: Bovine viral diarrhea virus; non-structural protein; NS5A; prokaryotic expression

中图分类号:

Q939.94

王慧慧, 冯茜莉, 蒲飞洋, 李易聪, 汪梦竹, 周小凯, 赵泽阳, 马忠仁, 李倬, 马晓霞. 牦牛源BVDV非结构蛋白NS5A的原核表达及抗原表位分析[J]. 中国畜牧兽医, 2022, 49(8): 3180-3189.

WANG Huihui, FENG Xili, PU Feiyang, LI Yicong, WANG Mengzhu, ZHOU Xiaokai, ZHAO Zeyang, MA Zhongren, LI Zhuo, MA Xiaoxia. Prokaryotic Expression and Antigen Epitope for Non-structural Protein NS5A of BVDV Isolated from Yak[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3180-3189.

参考文献

[1] 高亚桃,李妍,刘立元,等.1株牛病毒性腹泻病毒河北株的分离鉴定与遗传演化分析[J].畜牧与兽医,2022,54(1):100-105. GAO Y T,LI Y,LIU L Y,et al.Isolation,identification and phylogenetic analysis of a strain of Bovine viral diarrhea virus isolated in Hebei province[J].Animal Husbandry&Veterinary Medicine,2022,54(1):100-105.(in Chinese)
[2] 杨德新,王新庄,闫磊,等.牛病毒性腹泻/粘膜病研究进展[J].中国牛业科学,2021,47(4):36-39. YANG D X,WANG X Z,YAN L,et al.Research progress of bovine viral diarrhea/mucosal disease[J].China Cattle Science,2021,47(4):36-39.(in Chinese)
[3] 韦雪华,张向东,谢之景,等.双重RT-qPCR鉴别CSFV和BVDV方法的建立及初步应用[J].中国兽医杂志,2018,54(5):28-31. WEI X H,ZHANG X D,XIE Z J,et al.Establishment and preliminary application of dual fluorescence quantitative RT-PCR in identifying CSFV and BVDV methods[J].Chinese Journal of Veterinary Medicine,2018,54(5):28-31.(in Chinese)
[4] 徐磊,曾亮明,张体银,等.福建省猪源BVDV流行病学检测分析及其对CSFV抗体的影响[J].中国兽医学报,2021,41(6):1031-1037. XU L,ZENG L M,ZHANG T Y,et al.Epidemiological detection and analysis of Bovine viral diarrhea virus (BVDV) from swine and its effect on CSFV antibody in Fujian province[J].Chinese Journal of Veterinary Science,2021,41(6):1031-1037.(in Chinese)
[5] CHANG L,QI Y,LIU D,et al.Molecular detection and genotyping of Bovine viral diarrhea virus in Western China[J].BMC Veterinary Research,2021,17(1):66.
[6] 温树波,宋扬,蒙小刚,等.一株进口胎牛血清源BVDV毒株的分离与鉴定[J].中国病原生物学杂志,2021,16(7):743-746. WEN S B,SONG Y,MENG X G,et al.Isolation and identification of an BVDV strain from imported fetal bovine serum[J].Journal of Pathogen Biology,2021,16(7):743-746.(in Chinese)
[7] 才冬杰.牛病毒性腹泻病毒基因组分析及LDLR敲除对其侵染牛肾细胞的影响[D].北京:中国农业大学,2018. CAI D J.Genomic analysis of Bovine viral diarrhea virus and effects of LDLR knockout on its infection of MDBK cells[D].Beijing:China Agricultural University,2018.(in Chinese)
[8] YESILBAG K,ALPAY G,BECHER P,et al.Variability and global distribution of subgenotypes of Bovine viral diarrhea virus[J].Viruses,2017,9(6):128.
[9] TIAN B,CAI D J,LI W Q,et al.Identification and genotyping of a new subtype of Bovine viral diarrhea virus 1 isolated from cattle with diarrhea[J].Archives of Virology,2021,166(4):1259-1262.
[10] 周子恒,张哲,肖盛中,等.牛病毒性腹泻病毒NS3蛋白原核表达纯化及多克隆抗体的制备[J].动物医学进展,2020,41(10):21-25. ZHOU Z H,ZHANG Z,XIAO S Z,et al.Prokaryotic expression and purification of Bovine viral diarrhea virus NS3 protein and preparation of polyclonal antibody[J].Progress in Veterinary Medicine,2020,41(10):21-25.(in Chinese)
[11] 邵红,张林雁,夏立勇,等.奶牛黏膜病的BVDV HJ-1株全基因组序列分析[J].黑龙江八一农垦大学学报,2021,33(4):21-28. SHAO H,ZHANG L Y,XIA L Y,et al.Complete genome sequence analysis of BVDV HJ-1 strains from mucosal disease in dairy coes[J].Journal of Heilongjiang Bayi Agricultural University,2021,33(4):21-28.(in Chinese)
[12] 韩慧,郑源强,石艳春.牛病毒性腹泻病毒研究进展[J].畜禽业,2019,30(7):6-7. HAN H,ZHENG Y Q,SHI Y C.Research progress on Bovine viral diarrhea virus[J].Livestock and Poultry Industry,2019,30(7):6-7.(in Chinese)
[13] SHENG C,KOU S M,JIANG Q Y,et al.Characterization of the C-terminal sequence of NS5A necessary for the assembly and production of Classical swine fever virus infectious particles[J].Research in Veterinary Science,2014,97(2):449-454.
[14] ISKEN O,LANGERWISCH U,SCHONHERR R,et al.Functional characterization of Bovine viral diarrhea virus nonstructural protein 5A by reverse genetic analysis and live cell imaging[J].Journal of Virology,2014,88(1):82-98.
[15] JI C Y,ZHANG Y,SUN R N,et al.Isolation and identification of type F Bovine enterovirus from clinical cattle with diarrhoea[J].Viruses,2021,13(11):2217.
[16] 李晓燕.BVDV-1 H2017分离株全基因组序列测定及遗传进化分析[D].呼和浩特:内蒙古农业大学,2019. LI X Y.Sequencing and genetic evolution analysis of BVDV-1 H2017 strain[D].Hohhot:Inner Mongolia Agricultural University,2019.(in Chinese)
[17] GIANGASPERO M,STEINBACH F,STRONG R,et al.Characterization of internal ribosome entry sites according to secondary structure analysis to Classify border disease virus strains[J].Journal of Virological Methods,2019,113704.
[18] 郭富城,金丽,苏强,等.PEDV N蛋白的原核表达纯化及B细胞抗原表位预测分析[J].浙江农业学报,2020,32(5):762-769. GUO F C,JIN L,SU Q,et al.Prokaaryotic expression and purification of PEDV N protein and analysis of B cell antigen epitope[J]. Acta Agriculturae Zhejiangensis,2020,32(5):762-769.(in Chinese)
[19] 尹纪营,刁乃超,田甜,等.牛病毒性腹泻病毒蛋白与宿主蛋白相互作用的研究进展[J].中国预防兽医学报,2021,43(11):1228-1233. YIN J Y,DIAO N C,TIAN T,et al.Research progress of the interaction between Bovine viral diarrhea virus proteins and host proteins[J].Chinese Journal of Preventive Veterinary Medicine,2021,43(11):1228-1233.(in Chinese)
[20] TU H,GAO L,SHI S T,et al.Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein[J]. Virology,1999,263(1):30-41.
[21] ISKEN O,LANGERWISCH U,SCHONHERR R,et al.Functional characterization of Bovine viral diarrhea virus nonstructural protein 5A by reverse genetic analysis and live cell imaging[J].Journal of Virology,2013,88(1):82-98.
[22] RAJPUT M K S,ABDELSALAM K,DARWEESH M F,et al.Both cytopathic and non-cytopathic Bovine viral diarrhea virus (BVDV) induced autophagy at a similar rate[J].Veterinary Immunology and Immunopathology,2017,193-194:1-9.
[23] CASTRO E F,CASAL J J,DE MARCO M J E,et al.Identification of potent Bovine viral diarrhea virus inhibitors by a structure-based virtual screening approach identification of potent Bovine viral diarrhea virus inhibitors by a structure-based virtual screening approach[J].Bioorganic&Medicinal Chemistry Letters,2018,29(2):262-266.
[24] FU Y,DOMINISSINI D,RECHAVI G,et al.Gene expression regulation mediated through reversible m⁶A RNA methylation[J].Nature Reviews Genetics,2014,15(5):293-306.
[25] 黄雯静.m⁶A甲基化修饰调控牛病毒性腹泻病毒NS5A的表达[D].大庆:黑龙江八一农垦大学,2021. HUANH W J.m⁶A-methyladenosine modification rugulatesin NS5A expression of Bovine viral diarrhea virus[D].Daqing:Heilongjiang Bayi Agricultural University,2021.(in Chinese)