弓形虫GRA7基因的克隆表达及免疫分析

o construct prokaryotic expression plasmid pET28a-GRA7 and pET32a-GRA7 containing the dense granulesantigen 7(GRA7) gene of T.gondii RH strain, and analyze the recombinant protein. The gene fragment GRA7 was amplified by PCR from genomic DNA of T.gondii RH strain. The PCR product was analyzed by sequencing. The GRA7 gene was digested by the restriction endonuclease EcoRⅠ and BamHⅠ, and was further subcloned into plasmid pET28a(+) and pET32a(+). The plasmid pET28a-GRA7 and pET32a-GRA7 were then transformed into BL21(DE3) to express the recombinant protein with IPTG induction. The expression product was identified by SDS-PAGE and Western blotting. Specific fragments of the GRA7 gene about 710 bp were obtained by PCR. The plasmid pET28a-GRA7 and pET32a-GRA7 were confirmed with Enzyme digestion and PCR analysis. With IPTG induction, the recombinant protein GRA7 was expressed in a fusional form in pET32a-GRA7/ BL21(DE3), while pET28a-GRA7/ BL21(DE3) was fail to express the recombinant protein. Western blotting showed that the recombinant protein could be specifically recognized by sera from mice chronically infected by T. gondii. A prokaryotic expression vector for GRA7 has been successfully constructed and the expressed recombinant GRA7 shows a specific immunoreactivity, those built a foundation for the further studies on T. gondii diagnosis and vaccine.