弓形虫GRA1基因的克隆与原核表达

目的:克隆并构建PET-28a-GRA1重组表达载体,转化人大肠杆菌E.coli BL21,诱导表达并鉴定,为进一步研究GRA1的生物学特性和免疫保护作用奠定实验基础.方法:根据GenBank中编码GRA1的已知基因序列设计并合成一对引物,应用PCR技术扩增GRA1基因,插入原核表达载体PET-28a中,构建重组表达质...

Cloning and prokaryotic expression of GRA1 gene of Toxoplasma gondii

Objective To clone the GRA1 gene and construct the prokaryotic expression vector PET-28a-GRA1,and to transform the expression vector into E.coli BL21,then induce and identiy,and to lay a foundation for the study on the biological characteristics and immune protection of GRA1.Methods A pair of primers were designed according to the sequence of GRA1 from GenBank.The GRA1 gene was amplified by PCR.The amplified GRA1 gene was inserted into prokaryotic expression vector PET-28a,and the constructed recombinant plasmid PET-28a-GRA1 was transformed to E.coli BL21 for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blotting.Results The sequencing result showed the homology of 100% of the cloned GRA1 gene to that reported in GenBank.The positive recombinant plasmids PET-28a-GRA1 were identified by restriction endonuclease digestion and PCR,the objective gene fragment with the size of 573 bp was acquired,in accordance with the expected results,and the recombinant plasmid PET-28a-GRA1 was constructed successfully.The results of SDS-PAGE revealed that the molecular weight of recombinant protein PET-28a-GRA1 was 24 000 and Western blotting proved that the recombinant protein was recognized by murine antisera against Toxoplasma gondii.Conclusion The GRA1 gene has been successfully cloned,the recombinant plasmid PET-28a-GRA1 is generated and expressed highly in prokaryote.