摘要
为获得免疫原性好的Ⅰ型猫疱疹病毒(FHV-1)gB截短蛋白和制备特异性的多克隆抗体,以用于检测gB蛋白的表达水平变化及FHV-1的血清学诊断技术研究,本试验截取gB蛋白的优势抗原区并插入pET-30a载体,构建原核表达质粒pET-30a-gB,测序正确后转入BL21(DE3)感受态细胞进行诱导表达。通过亲和层析纯化蛋白,将获得的重组蛋白免疫6周龄雌性新西兰大白兔制备多克隆抗体。采用固定病毒-稀释血清法测定多抗中和效价,使用FHV-1感染后的细胞样本进行间接免疫荧光和West-blotting检测其反应性和特异性。结果显示,成功构建了表达FHV-1截短gB蛋白的重组质粒pET-30a-gB,在37℃诱导8 h后主要以包涵体形式表达。中和试验结果显示,制备FHV-1 gB截短蛋白多克隆抗体中和效价为1∶48。间接免疫荧光和West-blotting试验结果表明,所制备多克隆抗体具有良好的反应性和特异性,可特异性检测出FHV-1感染细胞中的gB蛋白。结果表明,原核表达的gB截短重组蛋白具有良好的免疫原性,制备的多克隆抗体具有较高的中和效价以及较好的反应性和特异性,为进一步解析gB蛋白的生物学功能和建立FHV-1血清学诊断方法提供了生物材料保障。
In order to obtain good immunogenicity truncated gB protein of type Ⅰ feline herpesvirus(FHV-1) and prepare specific polyclonal antibodies for detection of the gB expression level and establish FHV-1 serological diagnosis methods.The dominant antigenic region of gB protein was intercepted and inserted into the p ET-30a vector to construct the recombination prokaryotic expression plasmid p ET-30a-gB.After sequencing verification,it was transferred into BL21(DE3) for induced expression.The protein was purified by affinity chromatography,and the obtained recombinant protein was immunized with 6-week-old female New Zealand white rabbits to prepare polyclonal antibodies.The neutralizing titer of polyclonal antibody was determined by fixed virus-dilution serum method,and the reactivity and specificity was detected by indirect immunofluorescence and West-blotting test using FHV-1 infected samples.In result,the recombinant plasmid p ET-30a-gB expressing FHV-1 truncated gB protein was successfully constructed,and it was mainly expressed in the form of inclusion bodies after induction at 37 ℃ for 8 h.The neutralization test results showed that the neutralization titer of the prepared polyclonal antibody was 1 ∶ 48.The indirect immunofluorescence and West-blotting test results showed that the polyclonal antibody had good reactivity and specificity,and could specifically detect the gB protein after FHV-1 infection.In conclusion,the gB truncated recombinant protein obtained by prokaryotic expression had good immunogenicity,and the prepared polyclonal antibody had high neutralization titer,fair reactivity and specificity.This study provided guarantee for further analysis of the biological function of gB protein and establish FHV-1 serological diagnosis methods.
作者
李娜
王真真
杜汉宇
贾楠楠
宋若楠
朱杰
李传峰
程松
梁留存
刘光清
孟春春
LI Na;WANG Zheng-zheng;DU Han-yu;JIA Nan-nan;SONG Ruo-nan;ZHU Jie;LI Chuan-feng;CHENG Song;LIANG Liu-cun;LIU Guang-qing;MENG Chun-chun(Shanghai Veterinary Research Institute,Chinese Academy of A gricultural Sciences,Shanghai 200241,China;Xinjiang Agricultural University,Urumqi 830052,China;Laboklin Laboratory for Clinic Diagnostic,Shanghai 200241,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2022年第12期1531-1537,共7页
Chinese Veterinary Science
基金
上海市市级科技重大专项(ZD2021CY001)
中央级公益性行业科院所基本科研业务费(2021JB08、2022JB01)。
