兔出血症病毒衣壳蛋白VP60抗原区的原核表达及其免疫保护效果  

Prokaryotic expression and immune protection effect of antigenic region of rabbit hemorrhagic disease virus capsid protein VP60

作　　者：杨柳[1,2] 林永润 牟豪 许国洋 余远迪 沈克飞 付利芝[1,2] YANG Liu;LIN Yong-run;MU Hao;XU Guo-yang;YU Yuan-di;SHEN Ke-fei;FU Li-zhi(Chongqing Academy of Animal Sciences,Chongqing 402460,China)

机构地区：[1]重庆市畜牧科学院,重庆402460 [2]重庆市兽用生物制品工程技术研究中心,重庆402460 [3]西南大学荣昌校区,重庆402460

出　　处：《中国生物制品学杂志》2021年第6期671-677,共7页Chinese Journal of Biologicals

基　　金：重庆市农业发展基金项目(20309);重庆荣昌农牧高新技术产业研发专项(19256);新兽药研发创新团队(18201)。

摘　　要：目的原核表达兔出血症病毒(rabbit hemorrhagic disease virus,RHDV)衣壳蛋白VP60抗原区,并检测其免疫保护效果。方法采用RT-PCR法从感染RHDV死兔肝脏中扩增RHDV VP60基因,克隆基因并进行测序和生物信息学分析。用柔性连接肽(Gly4Ser)3串联VP60蛋白两抗原区域,根据E.coli密码子偏好性优化合成编码串联蛋白的DNA序列,插入至原核表达载体pET30a(+),构建重组表达质粒,转化感受态E.coli BL21(DE3)。挑选阳性克隆菌培养,用终浓度1 mmol/L的IPTG诱导表达,产物经Ni^(2+)-NTA金属螯合蛋白层析纯化后进行SDS-PAGE和Western blot检测。将纯化蛋白经背部皮下免疫家兔,1.0 mg/只,共免疫3次,每次间隔2周。分别于免疫前、首次免疫后14、28、42 d经耳静脉采血,分离血清,ELISA法检测RHDV抗体水平;末次免疫2周,经家兔腿部肌肉注射RHDV SZ/2013强毒株病毒液,2 mL/只,每天观察家兔症状,攻毒后10 d剖解观察脏器病理变化。结果扩增的VP60基因序列与RHDV山东分离株VP60基因的核苷酸相似性最高,达95.86%,将该毒株命名为RHDV Chongqing/2016,明确了两抗原区段为VP60氨基酸序列的N-末端L区和C-末端H区。重组表达质粒经菌落PCR及测序鉴定,证明构建正确。纯化蛋白相对分子质量约50000,与兔抗RHDV多克隆抗体可发生特异性结合。家兔免疫14 d即产生了抗RHDV特异性抗体,随时间延长呈逐渐升高趋势,且较稳定;攻毒后10 d内家兔无死亡,采食正常,无病症出现,保护率达100%;剖检可见家兔肝、肺和肾等脏器均正常,未见出血症状。结论原核表达了RHDV衣壳蛋白VP60抗原区,且具有良好的免疫保护作用,本实验为相应疫苗的研发提供了实验依据。Objective To express the antigenic region of capsid VP60 of rabbit hemorrhagic disease virus(RHDV)in prokaryotic cells and determine its immune protection effect.Methods VP60 gene was amplified from the liver of rabbits which died from RHDV infection by RT-PCR,cloned and subjected to sequencing and bioinformatics analysis.Two antigenic regions were connected via a flexible linker(Gly4Ser)3.The DNA sequence encoding tandem protein was optimized and synthesized according to the coding preference of E.coli and inserted into prokaryotic expression vector p ET30 a(+).The constructed recombinant plasmid pET30 a(+)-LH was transformed to competent E.coli BL21(DE3).Positive clones were screened,cultured and induced with 1.0 mmol/L IPTG.The expressed product was purified by Ni^(2+)-NTA metal chelating chromatography,and identified by SDS-PAGE and Western blot,with which rabbits were immunized s.c.at a dosage of 1.0 mg for 3 times each at an interval of 2 weeks.Serum samples were collected before and 14,28 and 42 d after the first dose and determined for specific RHDV antibody level by ELISA.Two weeks after the last dose,the rabbits were challenged i.m.with virulent RHDV SZ/2013 strain,2 mL for each,observed for clinical symptoms every day,and subjected to autopsy 10 d later.Results The homology of nucleotide sequence of amplified VP60 gene sequence from RHDV strain from the liver of rabbits,named as RHDV Chongqing/2016 strain,was 95.86%to that of RHDV strains isolated in Shandong Province,China.The two antigenic regions,L region at N-terminus and H region at C-terminus of VP60 amino acid of RHDV Chongqing/2016 strain,were identified.Colony PCR and sequencing proved that recombinant plasmid pET30 a(+)-LH was constructed correctly.The purified protein,with a relative molecular mass of about 50000,showed specific binding to rabbit polyclonal antibody against RHDV.Specific RHDV antibody was induced in rabbits 14 d after immunization,of which the level showed an increasing tendency as time went on was stable.No rabbits died wit

关 键 词：兔出血症病毒 VP60蛋白 基因重组 原核表达 免疫保护 

分 类 号：S855.3[农业科学—临床兽医学]