摘要
为比较非洲猪瘟病毒(African swine fever virus,ASFV)p30蛋白氨基酸序列中不同片段的反应原性,探究p30蛋白氨基酸序列中可能的抗原优势片段。本研究通过PCR、克隆技术将p30蛋白氨基酸序列中不同片段的编码基因分别插入表达质粒pET32a,经IPTG诱导、Ni柱纯化、透析后,获得p30蛋白氨基酸序列中8~101 aa片段(P-1#)、58~101 aa片段(P-2#)、101~158 aa片段(P-3#)、8~194 aa片段(P-4#),用酶联免疫吸附试验(ELISA)分析p30蛋白氨基酸序列中不同片段与p30蛋白免疫兔血清和ASFV阳性猪血清的反应原性。结果显示,上述片段均获得诱导表达及纯化,表达形式有可溶性表达(P-1#、P-3#)和包涵体表达(P-2#、P-4#)。各片段以1.0 mg/L包被时,p30免疫兔血清与p30蛋白氨基酸序列中4种片段均能发生免疫反应,ASFV阳性猪血清与P-4#、P-1#反应较佳,与P-3#反应中等,与P-2#反应最弱。综上,p30蛋白氨基酸序列中不同片段反应原性的差异为认识p30抗原优势区域提供了数据,这将有助于非洲猪瘟血清学诊断抗原的科学筛选。
The purpose of this study was to compare the reactogenicity of different fragments of the p30 protein amino acid sequence of African swine fever virus(ASFV),and to explore the possible antigen-dominant fragments in the p30 protein amino acid sequence.In this study,the coding genes of different fragments of p30 amino acid sequence were inserted into the expression plasmid pET32a by PCR and cloning techniques.After IPTG induction,Ni column purification and dialysis,8-101 aa fragment(P-1#),58-101 aa fragment(P-2#),101-158 aa fragment(P-3#)and 8-194 aa fragment(P-4#)in the amino acid sequence of p30 protein were obtained.The reactogenicity of different fragments of p30 protein amino acid sequence with p30 protein immunized rabbit serum and ASFV positive pig serum was analyzed by enzyme linked immunosorbent assay(ELISA).The results showed that the above fragments were induced and purified.The expression forms were soluble expression(P-1#,P-3#)and inclusion body expression(P-2#,P-4#).When each fragment was coated at 1.0 mg/L,the p30 immunized rabbit serum and the four fragments in the amino acid sequence of p30 protein could react with each other.The reaction of ASFV-positive pig serum with P-4#and P-1#was better,with P-3#was moderate,and with P-2#was the weakest.In summary,the differences in the reactogenicity of different fragments in the amino acid sequence of p30 protein provide data for understanding the dominant fragments of p30 antigen,which will help the scientific screening of serological diagnostic antigens of African swine fever.
作者
周俊明
倪艳秀
范宝超
祝昊丹
朱雪蛟
王丹丹
胡屹屹
李彬
ZHOU Junming;NI Yanxiu;FAN Baochao;ZHU Haodan;ZHU Xuejiao;WANG Dandan;HU Yiyi;LI Bin(Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture and Rural Affairs,Nanjing 210014,China)
出处
《江苏农业学报》
CSCD
北大核心
2024年第10期1875-1881,共7页
Jiangsu Journal of Agricultural Sciences
基金
国家重点研发计划项目(2022YFD1800601)
国家自然科学基金项目(31941013)
江苏省农业科技自主创新基金项目[CX(21)3137]
河北省省级科技计划资助项目(21322401D)
苏北科技专项(SZ-LYG202109)。
关键词
非洲猪瘟病毒
p30蛋白
氨基酸序列片段
反应原性
African swine fever virus
p30 protein
amino acid sequence fragment
reactogenicity
