非洲猪瘟病毒CP312R基因编码蛋白的原核表达及多克隆抗体的制备与应用
Prokaryotic expression of protein encoded by CP312R gene of African swine fever virus & application and preparation of its polyclonal antibody
刘佳晨 1郭子强 1陈金霞 1曹云雷 1童武 2乔思娜 1刘长龙 2赵冉 3郑海红 1童光志 2李丽薇 2高飞2
作者信息
- 1. 中国农业科学院上海兽医研究所,上海 200241
- 2. 中国农业科学院上海兽医研究所,上海 200241;扬州大学 江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州 225009
- 3. 厦门市动物疫病预防控制中心,福建厦门 361009
- 折叠
摘要
为制备ASFV CP312R 基因编码蛋白的多克隆抗体,利用PCR方法从非洲猪瘟病毒(African swine fever vi-rus,ASFV)的灭活样品中扩增出924 bp的CP312R基因全长序列,利用同源重组法构建出原核表达质粒pCold-Ⅰ-ASFV-CP312R,将其转化至原核表达感受态细胞BL21中,经1 mmol/L的IPTG低温条件下诱导表达CP312R编码蛋白并经过镍柱亲和层析纯化后,利用SDS-PAGE对重组蛋白进行表达鉴定和反应原性分析,表达蛋白的相对分子质量约为34 kDa,通过 Western blot分析,该蛋白能被ASFV抗体特异性识别.将所得的蛋白经处理纯化后免疫小鼠3次,得到含多克隆抗体的血清.利用该抗体检测实验室构建并拯救的表达ASFV CP312R基因编码蛋白的重组猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV),结果显示该多抗具有良好的反应原性和特异性,并且证明了 ASFV CP312R基因编码蛋白可在PRRSV活载体中稳定表达.
Abstract
To prepare polyclonal antibody against protein encoded by African swine fever virus(AS-FV)CP312R,the 924 bp full-length sequence of CP312R gene was amplified from inactivated samples of ASFV by PCR,and the prokaryotic expression plasmid pCold-Ⅰ-ASFV-CP312R was constructed by homologous recombination and was transformed into competent cell BL21.The ex-pression of pCP312R protein was induced by 1 mmol/L IPTG at low temperature for 16 h,and then the recombinant protein was identified and analyzed by SDS-PAGE.The molecular weight of the expressed protein was about 34 kDa,which was specifically recognized by ASFV antibody by Western blot analysis.The obtained protein was purified by nickel column affinity chromatogra-phy,and the mice were immunized for 3 times to obtain sera containing polyclonal antibody.Then,the antibody was used to detect the recombinant porcine reproductive and respiratory syndrome vi-rus(PRRSV)expressing the protein encoded by ASFV CP312R gene which was constructed and rescued in our lab.The experimental results showed that the polyclonal antibody had good reactivi-ty and specificity,and it was also proved that ASFV pCP312R could be stably expressed in PRRSV live vector.
关键词
非洲猪瘟病毒/CP312R基因/原核表达/多克隆抗体Key words
African swine fever virus/CP312R gene/prokaryotic expression/polyclonal antibody引用本文复制引用
基金项目
国家重点研发计划(2021YFD1801401)
国家自然科学基金ASFV专项(31941017)
中央级公益性科研院所基本科研业务费专项(Y2020YJ15)
上海市自然科学基金资助项目(21ZR1476900)
上海市自然科学基金资助项目(21ZR1477200)
出版年
2024
NETL
NSTL
万方数据