摘要
为构建刚地弓形虫微线体蛋白3(MIC3)原核表达体系,获得高纯度MIC3蛋白用于制备单克隆抗体,评价MIC3蛋白在临床诊断方面的应用价值,根据刚地弓形虫MIC3基因编码的已知序列设计引物,应用PCR技术从刚地弓形虫NT株的基因组DNA中扩增出去除信号肽的MIC3基因,将该基因克隆入pET-32a(+)表达载体,构建pET-32a(+)表达载体,并转化入大肠杆菌(Escherichia.coli)Trans 5α感受态细胞。扩增的MIC3基因与GenBank中相应基因序列(AF509564.1)的同源性达99.9%。将阳性重组表达质粒转化入E.coli BL21-codon plus,经IPTG20℃低温诱导表达,SDS-PAGE分析结果显示,MIC3融合蛋白分子量约为56 000。Western-blotting分析鉴定结果表明,MIC3融合蛋白可被猪抗弓形虫免疫血清所识别。获得的MIC3融合蛋白具有一定的反应原性,为下一步利用重组蛋白建立弓形虫的诊断方法和研制弓形虫的亚单位疫苗奠定了基础。
This study was aimed to construct a prokaryotic expression system for MIC3 gene of Toxoplasma gondii in order to produce a greater-yield and highly-purified recombinant MIC3 protein,therefore evaluate its potential for diagnosis of Toxoplasma gondii infection.According to the sequence of Toxoplasma gondii MIC3 reported in GenBank,primers were designed and synthesized,which were used to amplify partial fragments of MIC3 gene from the genomic DNA of T.gondii NT strain.The PCR product was then inserted into pET-32a（＋） expression vector and sequenced.The recombinant plasmid pET-32a（＋）-MIC3 was transformed into Escherichia coli BL21 competent cells and induced with IPTG to express in high level at low temperature to reduce formation of inclusion body.The recombinant MIC3（rTgMIC3） was analyzed by SDS-PAGE and Western blotting with anti-sera from pig infected with the T.gondii NT strain.The sequence of the amplified MIC3 was identical to the MIC3 sequence in GenBank（Accession no.AF509564.1）.The measured molecular mass of rTgMIC3 agreed with the theoretical molecular mass and the rTgMIC3 could be recognized by sera from pig infected with the T.gondii NT strain.Results suggest that rTgMIC3 is potential for developing a serodiagnostic test for T.gondii infection.
出处
《江苏农业学报》
CSCD
北大核心
2011年第6期1295-1299,共5页
Jiangsu Journal of Agricultural Sciences
基金
江苏省科技支撑计划(社会发展)项目(BE2010758)
