猪细小病毒AV30 VP2基因在昆虫细胞中表达及其免疫原性鉴定  

Expression and identification of porcine parvovirus AV30 VP2 gene in insect cells

作　　者：李茜 代军飞 丁耀忠[1] ASHENAFI Kiros Wubshe 李国秀 马炳[1] 张杰[1] LI Qian;DAI Jun-fei;DING Yao-zhong;ASHENAFI Kiros Wubshe;LI Guo-xiu;MA Bing;ZHANG Jie(State Key Labotorary of Veterinary Etiological Biology/Lanzhou Veterinary Reasearch Insttitute,Chinese Acmiemy of Argricultural Science,Lanzhou 730046,China)

机构地区：[1]中国农业科学院,兰州兽医研究所,家畜疫病病原生物学国家重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2020年第7期852-860,共9页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2017YFD0501800;2016YFD0501500);甘肃省国际科技特派员项目(17JR7WA030)。

摘　　要：本研究选用Bac-to-Bac昆虫杆状病毒表达系统构建含有猪细小病毒AV30未优化、优化后衣壳蛋白VP2基因的重组杆状病毒,感染sf9、HF细胞表达VP2蛋白。首先,扩增PPV AV30得到VP2基因,测序后由公司根据昆虫细胞嗜性进行基因优化。同时构建并收获未优化、优化重组杆状病毒r BacVP2。其感染HF细胞后,进行Western-blot及IFA实验分析表明,未优化、优化VP2蛋白在昆虫细胞中正确表达,分子质量约为64 ku,并具有良好的反应原性。电镜观察,未优化、优化VP2蛋白均自我组装成VLP,与PPV AV30病毒粒子大小形状相似。最后,用VP2-VLP免疫BALB/c小鼠,能刺激小鼠产生抗PPV的特异性抗体。免疫后的小鼠脾脏淋巴细胞在PPV AV30病毒的刺激后,出现显著的增殖情况,同时产生了Th1和Th2型相关细胞因子。本研究成功构建了VP2-VLP,并在小鼠实验中显示了良好的免疫效果,为以VP2为表位载体的嵌合型病毒样颗粒疫苗进一步研制奠定了基础。Recombinant baculovirus containing unoptimizing、optimizing VP2 gene of PPV AV30 was constructed using Bac-to-Bac baculovirus expression system,and recombinant VP2 protein was expressed by infecting sf9,HF cells with recombinant baculovirus.Firstly,the coding sequences of PPV AV30 VP2 gene was analyzed using bioinformatics software.After sequencing,the company conducted genetic optimization according to the insect cytotropism.Finally,obtain the production of recombinant viral r Bac VP2.After the HF cells were infected,the expressed unoptimizing,optimizing VP2 protein was analyzed by Western blot and indirect immunofluorescence assay(IFA).The results showed that the recombinant VP2 protein with a 64 k Da molecular weight was expressed successfully,TEM observation showed that unoptimizing、optimizing VP2 protein all self-assembled into VLP.Then VP2-VLP was used to immunize BALB/c mice.After immunizing mice,the protein VP2-VLP can stimulate the production of specific antibodies against PPV AV30 in mice.The spleen lymphocytes of immunized mice can proliferate significantly after stimulation of the PPV AV30 and efficiently produce Th1 or Th2 type cytokines.In this study,VP2-VLP was successfully constructed and showed good immune effect in mouse experiments,laying a foundation for further development of chimeric virus-like particle vaccine with VP2 as epitope carrier.

关 键 词：猪细小病毒 VP2基因 昆虫杆状系统 VLP 免疫原性 

分 类 号：S852.65[农业科学—基础兽医学]