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| CDV强毒BJ16B2株H基因在昆虫杆状病毒中的表达与鉴定 | |
| 引用本文： | 陈美荣,林伟东,宋天琪,鑫婷,郭晓宇,黄克和,贾红. CDV强毒BJ16B2株H基因在昆虫杆状病毒中的表达与鉴定[J]. 中国畜牧兽医, 2018, 45(8): 2041-2048. DOI: 10.16431/j.cnki.1671-7236.2018.08.001 |
| 作者姓名： | 陈美荣 林伟东 宋天琪 鑫婷 郭晓宇 黄克和 贾红 |
| 作者单位： | 1. 中国农业科学院北京畜牧兽医研究所, 北京 100193; 2. 南京农业大学动物医学院, 南京 210095 |
| 基金项目： | 国家重点研发计划（2016YFD0501003）；农业科技创新计划（ASTIP-IAS-11） |
| 摘 要： | 试验旨在获得具有天然构象且抗原性良好的犬瘟热病毒（canine distemper virus，CDV）强毒株的H蛋白。以分离的CDV强毒株BJ16B2为模板，RT-PCR特异扩增H基因，克隆至表达载体pFastBacTM HTA上，酶切及测序鉴定正确后比对分析H基因同源性，并将重组质粒pFastBacTMHTA-H转化大肠杆菌DH10BacTM感受态细胞，同源重组构建穿梭质粒rBacmid-H，将重组穿梭质粒转染Sf9昆虫细胞，拯救重组杆状病毒。利用IFA和Western blotting对获得的重组蛋白进行鉴定及抗原性分析。结果显示，重组质粒pFastBacTMHTA-H酶切测序鉴定正确，该H基因属于Asia-1强毒株，与参考毒株的核苷酸和氨基酸同源性分别为89.8%~99.3%和89.6%~99.5%。在杆状病毒中所表达的H蛋白能与CDV阳性血清及His-tag抗体发生特异性反应，IFA鉴定能够产生特异性绿色荧光，且在68 ku左右出现蛋白条带，表明H蛋白成功表达且具有良好的反应原性。本研究获得了具有天然构象及良好抗原性的CDV重组H蛋白，为后续蛋白结构和抗原特性差异分析、单抗制备、CDV亚单位疫苗的开发奠定了基础。 |
| 关 键 词： | 犬瘟热病毒(CDV) H基因 昆虫杆状病毒表达系统 |
Expression and Identification of H Gene of CDV BJ16B2 Strain in Insect Baculovirus Expression System |
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| CHEN Meirong,LIN Weidong,SONG Tianqi,XIN Ting,GUO Xiaoyu,HUANG Kehe,JIA Hong. Expression and Identification of H Gene of CDV BJ16B2 Strain in Insect Baculovirus Expression System[J]. China Animal Husbandry & Veterinary Medicine, 2018, 45(8): 2041-2048. DOI: 10.16431/j.cnki.1671-7236.2018.08.001 | |
| Authors: | CHEN Meirong LIN Weidong SONG Tianqi XIN Ting GUO Xiaoyu HUANG Kehe JIA Hong |
| Affiliation: | 1. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China |
| Abstract: | To obtain the native conformation and antigenicity of hemagglutinin (H) protein of canine distemper virus (CDV) virulent strain,the H gene which referred to BJ16B2 strain was accurately amplified by RT-PCR and cloned into pFastBacTMHTA vector.The recombinant plasmid was confirmed by restriction enzyme digestion and DNA sequencing,H gene was analyzed and the recombinant plasmid was transformed into E.coli DH10BacTM competent cell,and constructed shuttle vector,which was transfected into Sf9 cell to save baculovirus expressing H protein.To determine the antigenicity of recombinant protein,Western blotting and IFA analysis were used to detect the interesting protein.The results demonstrated that H gene belongs to Asia-1 virulent strain,and the homology of nucleotide and amino acid among the reference strains was 89.8% to 99.3% and 89.6%to 99.5%,respectively.The fusion protein generated by recombinant baculovirus was reacted with both His-tag monoclonal antibodies and CDV positive serum by Western blotting test,and presented specific green fluorescence in IFA test (68 ku),which showed that this protein had sufficient antigenicity.The resluts served as a strong foundation for the the subsequent analysis of protein structure and antigenicity differences,the preparation of monoclonal antibodies and the development of CDV subunit vaccine. |
| Keywords: | canine distemper virus (CDV) H gene insect baculovirus expression system |
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