非洲猪瘟病毒E199L-CP204L-E183L串联序列在杆状病毒表达系统中的表达及免疫效果评价  

Expression of African swine fever virus E199L-CP204L-E183L in baculovirus expression system and assessment of its immunogenicity

作　　者：张寅寅 李智晟 王晨晓 倪洁 刘静宜 陈鸿军[2] 孙耀贵[1] ZHANG Yinyin;LI Zhisheng;WANG Chenxiao;NI Jie;LIU Jingyi;CHEN Hongjun;SUN Yaogui(College of Veterinary Medicine,Shanxi Agricultural University,Jinzhong 030801,China;Shanghai Veterinary Research Institute,Chinese Academy of Agriculture Sciences,Shanghai 200241,China)

机构地区：[1]山西农业大学动物医学学院,山西晋中030801 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国兽医科学》2024年第11期1458-1464,共7页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2021YFD1801401)。

摘　　要：本研究将非洲猪瘟病毒E199L-CP204L-E183L串联序列,克隆到pFastBac1中,获得重组质粒pFastBac-CMV-EGFP-P2A-E199L-CP204L-E183L-HA,将其转化至DH10Bac感受态细胞中,经抗性、蓝白斑筛选及PCR鉴定,获得重组杆粒。将重组杆粒转染sf9细胞,细胞出现明显病变后进行传代,获得重组杆状病毒rBac-E199L-CP204L-E183L。利用蛋白质免疫印迹(Western-blot)及间接免疫荧光法(IFA)对重组杆状病毒的蛋白表达情况进行检测。结果显示,IFA检测到明显荧光,Western-blot检测到大小符合目的蛋白的条带,表明重组杆状病毒表达成功。将构建的重组杆状病毒免疫BALB/c小鼠,通过检测血清特异性抗体和T淋巴细胞增殖水平验证重组杆状病毒的免疫效果。结果表明,二免后第14天,在免疫小鼠中检测到抗pE199L、p30、p54蛋白的特异性抗体,免疫组小鼠的淋巴细胞增殖水平均显著高于对照组小鼠。以上结果表明,本研究成功构建了能够表达串联抗原的重组杆状病毒,免疫小鼠后能够诱导小鼠产生体液免疫反应及刺激淋巴细胞增殖。In this study,African swine fever virus tandem sequence of E199L,CP204L and E183L were cloned into p Fast Bac1 to obtain recombinant plasmids p Fast Bac-CMV-EGFP-P2A-E199L-CP204L-E183L-HA.The recombinant plasmids were transformed into DH10Bac competent cells.Following antibiotic resistance and blue-white screening,and PCR verification,recombinant bacmids were obtained.Recombinant baculoviruses r Bac-E199L-CP204L-E183L were obtained by transfecting sf9 cells with recombinant bacmids.Western-blot and indirect immunofluorescence assay(IFA)were used to detect protein expression of recombinant baculoviruses in sf9 cells.The results showed that IFA detected obvious fluorescence and Western-blot detected bands matching the size of the target proteins,indicating that the constructed recombinant baculoviruses were successfully expressed.Subsequently,the immunogenicity of r Bac-E199L-CP204L-E183L was investigated by detecting the serum specific antibodies and T lymphocyte proliferation.The results showed that antibodies against p E199L,p30,and p54 were detected on 14days post second immunization,respectively.The proliferation of T lymphocyte of the immunized mice is significantly higher than that of control mice.Overall,three tandem proteins were successfully expressed in baculovirus expression system and immunization of the recombinant baculovirus induced humoral immune responses,and stimulated lymphocyte proliferation.

关 键 词：非洲猪瘟病毒 E199L CP204L E183L 杆状病毒 免疫评价 

分 类 号：S852.659.1[农业科学—基础兽医学]