摘要
根据编码MIC3的已知基因序列设计并合成一对引物,应用PCR技术从弓形虫RH株的基因组DNA中扩增编码MIC3的全长基因,克隆入pGEX-KG表达载体,转化入E.coliDH5α感受态细胞,经含氨苄青霉素琼脂平板筛选,小量抽提质粒进行酶切、PCR及DNA测序鉴定。然后阳性重组质粒转化入E.coliBL21-CodonPlus,IPTG诱导,表达产物经SDS-PAGE和Western-blot分析鉴定。结果显示,扩增的MIC3基因与GenBank中相应基因序列(AJ132530)的同源性达99.6%,表达的MIC3融合蛋白表观分子量约为66 ku,且可被兔抗弓形虫免疫血清识别。说明所获得的表达蛋白质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。
A pair of primers was designed according to the sequence of MIC3 from GenBank. The entire ORF encoding MIC3 sequence was obtained by amplification from genomic DNA of Toxoplasma gondii RH strain and cloned into the plasmid pGEX-KG. The constructed recombinant plasmid was transferred into E. coli DH5α susceptible cell and identified by enzyme digesting, PCR and sequencing. Then the target recombinant plasmid was transferred into E. coli BL21- Codonplus susceptible cell, the expression of the recombinant pGEX-KG-MIC3 was induced by IPTG. The expressed products were analysed by SDS-PAGE and Western-blot. The results showed that the sequence identities were 99.6% between amplified MIC3 gene and that from GenBank（AJ132530）. The size of recombinant MIC3 protein was about 66 ku. It could be specifically recognized by immune serum against Toxoplasma gondii from rabbit.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2006年第6期587-591,共5页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
教育部高等学校博士点科研基金项目(20040504016)
教育部重点科研项目(105120)
