CDV强毒BJ16B2株H基因在昆虫杆状病毒中的表达与鉴定

doi:10.16431/j.cnki.1671-7236.2018.08.001

摘要/Abstract

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pFastBac^TMHTA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pFastBac^TMHTA-H转化大肠杆菌DH10Bac^TM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pFastBac^TMHTA-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

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 pFastBac^TMHTA 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 DH10Bac^TM 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.

Key words: canine distemper virus (CDV); H gene; insect baculovirus expression system

中图分类号:

S855.3

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.

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]. , 2018, 45(8): 2041-2048.

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