摘要
目的筛选肝片吸虫cDNA表达文库,获得肝片吸虫感染候选检测抗原基因,经对筛选结果分析后选取假定蛋白D915_000128基因进行表达和反应原性鉴定。方法利用绵羊肝片吸虫感染阳性血清对肝片吸虫cDNA表达文库进行筛选,以阳性噬菌斑为模板进行PCR扩增,对PCR产物测序并进行生物信息学分析和比较。通过PCR技术将选取的候选检测抗原基因片段进行扩增,并与原核表达载体pET-32a连接,构建重组表达质粒,经酶切鉴定正确后转化BL21感受态细胞,以异丙基-β-D硫代半乳糖苷(IPTG)诱导表达,应用SDS-PAGE和Western blot对重组蛋白的大小及其反应原性进行分析和鉴定。结果通过对肝片吸虫cDNA表达文库的筛选,成功获得了肝片吸虫候选检测抗原基因12个,其中2个为已报道的检测抗原基因(GenBank:PIS79729.1、ABC66278.1),10个为新发现的可用于肝片吸虫感染检测的候选抗原基因(GenBank:AYA57790.1、THD19001.1、TPP64335.1、PIS88126.1、AAA80273.1、THD26809.1、THD19926.1、TPP60711.1、THD26810.1、THD29021.1)。成功构建假定蛋白D915_000128基因原核表达载体,其诱导表达产物经SDS-PAGE分析为85×10^(3)的重组融合蛋白,Western blot分析该重组蛋白可被绵羊肝片吸虫自然感染阳性血清识别,即具有反应原性。结论筛选获得12个肝片吸虫候选检测抗原基因,分析比较选取了肝片吸虫假定蛋白D915_000128基因,构建的原核表达载体表达的重组蛋白具有良好的反应原性。
Objective The cDNA expression library of Fasciola hepatica was screened to obtain candidate antigen genes for detection of F.hepatica infection.After analysis of screening results,the hypotheticalprotein D915_000128 gene was selected for expression and reactogenicity identification.Methods A sheep F.he patica infection positive serum was used to screen the F.hepatica cDNA expression library.Positive phage spots were used as templates for PCR amplification,and the PCR products were sequenced and subjected to bioinformatic analysis and comparison.The selected candidate detection antigen gene fragment was amplified by PCR and ligated with the prokaryotic expression vector pET-32a to construct a recom binant expression plasmid,which was transformed into BL21 competent cells after correct enzymatic identification and expression was induced with isopropyl-3-D thiogalactopyranoside(IPTG).The molecular weight and reactogenicity of the recombinant protein were analyzed and characterized by applying SDS-PAGE and Western blot.Results Through screening of the F.hepatica cDNA expression library,12 candidate antigen genes for detection of F.hepatica were successfully obtained,of which 2 were reported detection antigen genes(GenBank:PIS79729.1,ABC66278.1)and 10 were newly discovered candidate antigen genes that could be used for detection of F.he patica infection(GenBank:AYA57790.1,THD19001.1,TPP64335.1,PIS88126.1,AAA80273.1,THD26809.1,THD19926.1,TPP60711.1,THD26810.1,THD29021.1).We successfully constructed a prokaryotic expression vector for the hypothetical protein D915_000128 gene.The recombinant protein was analyzed by SDS-PAGE as 85×10^(3),and the Western blot showed that the recombinant protein could be recognized by sheep F.he patica positive sera.Conclusion Twelve candidate antigen genes of F.he patica were selected,and the hypothetical protein D915_000128 gene of F.hepatica was selected.The recombinant protein expressed by the constructed prokaryotic expression vector has good reactivity.
作者
刘少雄
张楠
王晓岑
李新
李建华
宫鹏涛
张西臣
LIU Shao-xiong;ZHANG Nan;WANG Xiao-cen;LI Xin;LI Jian-hua;GONG Peng-tao;ZHANG Xichen(Key Laboratory of Zoonosis Research,Ministry of Education,College of Veterinary Medicine,Jilin University,changchun130062,China)
出处
《中国病原生物学杂志》
CSCD
北大核心
2023年第3期293-296,302,共5页
Journal of Pathogen Biology
基金
吉林省科技发展计划项目(No.20200402044NC,20190103075JH)
关键词
肝片吸虫
免疫学筛选
候选检测抗原基因
原核表达
Fasciola hepatica
immunological screening
candidate detection antigen gene
prokaryotic expression
