摘要
为了研制廉价高效的猪圆环病毒2型(PCV2)亚单位疫苗,根据大肠杆菌密码子的偏好性合成PCV2b(GenBank:AY969004.1)cap基因全序列,利用PCR技术,分别将PCV2的2个B细胞表位(226LKDPPLNP233和195HVGLGTAF202)以单独和串联的方式连接到Cap的C端,成功构建至pE-SUMO表达载体,将3个重组载体转化表达菌株BL21(DE3)后诱导表达,经SDS-PAGE分析和Western Blotting鉴定,3种重组蛋白均以可溶形式表达且表达正确。对3种重组蛋白分别进行镍柱亲和层析纯化,再用SUMO蛋白酶除去融合标签,获得无标签的Cap-e1、Cap-e2和Cap-e12蛋白,并分别与相应的弗氏佐剂等体积乳化,将20只6周龄大小的雌性Balb/c小鼠随机分成5组,分别免疫3种重组蛋白,同时设置疫苗组和PBS组作为对照组,并进行免疫评价,比较C末端连接的不同B细胞表位对Cap免疫原性的影响。抗体检测结果表明,除第2周外,各试验组的抗体水平均显著高于PBS组,且连接2个表位的Cap-e12蛋白产生的抗体水平高于只连接1个表位的蛋白(Cap-e1、Cap-e2),而在第6周时,Cap-e12蛋白抗体水平与疫苗组无显著差异。综上Cap的免疫原性与连接表位数量有关,连接2个表位后能显著提高Cap的免疫原性。
To explore inexpensive and effective porcine circovirus type 2(PCV2)vaccine,the optimized cap gene of PCV2b(GenBank:AY969004.1)was synthesized according to codon usage of E.coli.Two B cell epitopes of PCV2(226 LKDPPLNP 233和195 HVGLGTAF 202)were individually and/or tandemly conjugated at the C-terminal of Cap by PCR technology,then the final segments were cloned into pE-SUMO vector,3 recombinant vectors were induced after transformed into expression strain BL21(DE3),identification by SDS-PAGE and Western Blotting,all three recombinant proteins are expressed in soluble form and correctly.The three recombinant proteins were purified by nickel column affinity chromatography,and then SUMO protease was used to remove the fusion tags to obtain the unlabeled Cap-e1,Cap-e2 and Cap-e12 proteins,which were respectively emulsified with equal volume corresponding Freund's adjuvant,twenty 6-week-old female Balb/c mice were randomly divided into 5 groups,immunized with 3 kinds of recombinant proteins,the vaccine group and the PBS group were set as the control group.An immunological evaluation was performed to compare the impact of different B-cell epitopes linked to the C-terminal on the immunogenicity of Cap,the results showed that,except for the second week,the antibody level of each test group was significantly higher than PBS group,and the antibody level produced by the Cap-e12 protein conjugated with 2 epitopes was higher than that of the protein linked only 1 epitope(Cap-e1,Cap-e2),in the sixth week,the antibody level of Cap-e12 protein had no significant difference with the vaccine group.In conclusion,the immunogenicity of Cap is related to the number of linked epitopes,the conjugation of two epitopes can significantly improve the immunogenicity of Cap.
作者
刘晴坤
冯华
任春晓
魏蔷
刘运超
张改平
LIU Qingkun;FENG Hua;REN Chunxiao;WEI Qiang;LIU Yunchao;ZHANG Gaiping(College of Life Sciences,Henan Agricultural University,Zhengzhou 450002,China;Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;School of Life Sciences,Zhengzhou University,Zhengzhou 450001,China)
出处
《华北农学报》
CSCD
北大核心
2020年第3期233-238,共6页
Acta Agriculturae Boreali-Sinica
基金
河南省重点研发与推广专项(182102110087)。
