非洲猪瘟病毒结构蛋白CD2v重组核酸疫苗的制备与免疫原性评估  

Preparation and immunogenicity evaluation of recombinant vaccine against structural CD2v protein of African swine fever virus

作　　者：张霁辉 徐娅玲 牛熙 黄世会 冉雪琴 王嘉福 ZHANG Ji-hui;XU Ya-ling;NIU Xi;HUANG Shi-hui;RAN Xue-qin;WANG Jia-fu(College of Life Sciences/Institute of Agro-Bioengineering,Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region(Ministry of Education),Guizhou University,Guiyang 550025,China;College of Animal Science,Key Laboratory o of Animal Genetics,Breeding and Reproduction in Plateau Mountainous Region(Ministry of Education),Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州大学生命科学学院/农业生物工程研究院山地植物资源保护与种质创新教育部重点实验室,贵州贵阳550025 [2]贵州大学动物科学学院/高原山地动物遗传育种与繁殖教育部重点实验室,贵州贵阳550025

出　　处：《中国兽医科学》2022年第10期1215-1222,共8页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31960641);贵州省科技创新人才团队项目(黔科合平台人才[2019]5615);贵州省科技支撑计划项目(黔科合支撑[2017]2585,黔科合支撑[2017]2587)

摘　　要：构建针对非洲猪瘟病毒(ASFV)CD2v蛋白的重组核酸疫苗并评价其免疫原性。通过PCR扩增目的基因CD2v、LTB、CD2v+LTB,分别连接至pc DNA3.1(-)载体,获得重组表达质粒,经双酶切和测序验证正确后,提取无内毒素质粒转染293T细胞,通过Western-blot验证重组蛋白的表达,并用其注射小鼠进行免疫,对免疫效力进行评价。Western-blot结果表明,构建的重组表达质粒可以在体外表达出目的蛋白。注射小鼠后,外周血CD4^(+)T淋巴细胞的比例升高;ELISA检测发现,与pc DNA3.1(-)和PBS组相比,pc DNA3.1-CD2v+LTB组的INF-γ、IL-2、IL-4水平均极显著升高(P<0.01)。pc DNA3.1-CD2v和pc DNA3.1-CD2v+LTB组相比,pc DNA3.1-CD2v+LTB的INF-γ、IL-2水平差异显著(P<0.05),而IL-4含量有差异但不显著,无统计学意义(P>0.05)。注射pc DNA3.1-CD2v质粒的小鼠血清效价约为1∶12800,注射pc DNA3.1-CD2v+LTB质粒的小鼠血清效价约为1∶25600。上述结果表明,本试验构建的重组核酸疫苗pc DNA3.1-CD2v、pc DNA3.1-CD2v+LTB、pc DNA3.1-LTB及抗原CD2v能够显著刺激小鼠的体液免疫和细胞免疫,LTB能够显著提高核酸疫苗的免疫应答。The recombinant nucleic acid vaccine against the CD2v protein of the African swine fever virus was constructed,and its immunogenicity was evaluated.The DNA of CD2v was synthesized according to the sequences of ASFV/pig/China/CAS19-01/2019.And then,the CD2v,LTB and CD2v+LTB were amplified by PCR method,and connected to the pc DNA3.1(-)vector respectively to construct recombinant expression plasmids.The endotoxin-free plasmids were transfected into 293T cells after double-enzyme digestion and sequencing verification.and the expression of the recombinant protein was verified with ern blot.Right after,the mice were injected with a vaccine to elicit immunity and evaluate the immune efficacy.The results showed that the recombinant plasmids could express the target protein in vitro by Western-blot detection.The CD4^(+)T lymphocytes in peripheral blood were increased after vaccination of mice.The results of ELISA indicated that the levels of INF-γ,IL-2 and IL-4 in pc DNA3.1-CD2v+LTB group were increased significantly(P<0.01).The levels of INF-γand IL-2 in pc DNA3.1-CD2v+LTB were significantly different from those in pc DNA3.1-CD2v group(P<0.05),while there was no significant difference in the ranks of IL-4(P>0.05).The serum titers of mice injected with pc DNA3.1-CD2v plasmid and pc DNA3.1-CD2v+LTB plasmid were about 1∶12800 and 1∶25600 respectively.In conclusion,the recombinant nucleic acid vaccines and the expressed protein of CD2v could sign ifi cantly stimulate the humoral and cellular immunity of mice,and LTB could dramatically improve the immune response of the nucleic acid vaccines.

关 键 词：非洲猪瘟病毒 CD2v蛋白 大肠杆菌不耐热肠毒素B亚基 核酸疫苗 

分 类 号：S859.797[农业科学—临床兽医学]