牛传染性鼻气管炎病毒多表位嵌合蛋白的表达及免疫原性分析  

Expression and immunogenicity of recombinant multiple-epitope chimeric protein of infectious bovine rhinotracheitis virus

作　　者：仝晓丹 王俊 冉旭华[1,2] 倪宏波 闻晓波[1,2] TONG Xiao-dan;WANG Jun;RAN Xu-hua;NI Hong-bo;WEN Xiao-bo(College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319,Heilongjiang Province,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319 [2]海南大学动物科技学院海南省热带动物繁育与疫病研究重点实验室,海南海口570228

出　　处：《中国生物制品学杂志》2021年第2期141-145,151,共6页Chinese Journal of Biologicals

基　　金：黑龙江省农垦总局攻关课题(HNK125B-11-08A,HNK125B-11-10A)。

摘　　要：目的表达牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)多表位嵌合蛋白,并分析其免疫原性。方法将已鉴定出的IBRV的gB、gC和gD抗原表位及破伤风毒素通用T细胞表位P2分别以GGGGS及AAYAAY为linker进行串联连接,用DNASTAR Protean软件分析其抗原性,选择最佳连接组合化学合成嵌合蛋白基因序列。将合成的目的基因克隆至载体pET-28a(+),构建重组质粒pET-28a-P2-gB/gC/gD(GGGGS)和pET-28a-P2-gB/gC/gD(AAYAAY),转化至感受态E.coli BL21(DE3),表达重组蛋白,采用Ni-NTA Purification System纯化。将纯化蛋白与ISA206佐剂乳化,经后腿肌肉注射免疫中国白兔,100μg/(mL·只),共免疫3次,每次间隔3周。初次免疫后21、42和63 d采血,分离血清,间接ELISA法检测抗体水平。结果两种重组质粒经双酶切及测序鉴定,构建正确。重组蛋白P2-gB/gC/gD(GGGGS)和P2-gB/gC/gD(AAYAAY)在E.coli中均以包涵体形式表达,纯化后产量分别可达12.4和15.3 mg/L。重组蛋白P2-gB/gC/gD(AAYAAY)诱导白兔的血清抗体水平显著高于重组蛋白P2-gB/gC/gD(GGGGS),且差异有统计学意义(P <0.05)。结论成功表达了IBRV多表位嵌合蛋白,且具有良好的免疫原性。Objective To express a multiple-epitope chimeric protein of infectious bovine rhinotracheitis virus(IBRV)and analyze its immunogenicity. Methods The identified IBRV gB,gC,gD epitopes and universal tetanus toxin T cell epitope P2 were linked using GGGGS or AAYAAY as a linker,of which the antigenicity was analyzed by DNASTAR Protean software. The optimal combination of gene fragments was synthesized and cloned into expression vector pET-28a(+). The constructed recombinant plasmid pET-28a-P2-gB/gC/gD(GGGGS)or pET-28a-P2-gB/gC/gD(AAYAAY)was transformed to competent E. coli BL21(DE3). The expressed recombinant proteins were purified by using Ni-NTA Purification System and emulsified with ISA206 adjuvant,with which Chinese white rabbits were immunized for 3 times each at an interval of 3 weeks,100 μg/mL for each. Serum samples were collected 21,42 and 63 d after the first immunization and determined for antibody level by indirect ELISA. Results Colony PCR and sequencing proved that recombinant plasmids pET-28a-P2-gB/gC/gD(GGGGS) and pET-28a-P2-gB/gC/gD(AAYAAY) were constructed correctly. Both the recombinant proteins P2-gB/gC/gD(GGGGS)and P2-gB/gC/gD(AAYAAY)were expressed in a form of inclusion body in E. coli,of which the yields reached 12. 4 and 15. 3 mg/L respectively after purification. The serum antibody level induced by recombinant protein P2-gB/gC/gD(AAYAAY)was higher than that by P2-gB/gC/gD(GGGGS)(P < 0. 05). Conclusion Recombinant multiple-epitope chimeric protein of IBRV was successfully expressed,which showed good immunogenicity.

关 键 词：牛传染性鼻气管炎病毒 抗原表位 嵌合蛋白 免疫原性 

分 类 号：S852.4+3[农业科学—基础兽医学] R392-33[农业科学—兽医学]