Construction, identification and sequence analysis of eukaryotic expression reco mbinant plasmid contai-ning dense granules protein 8 gene from Toxoplasma gondii in BALB/c mice / 中华传染病杂志

ABSTRACT

Objective To clone the coding dense granules protein 8(GRA8) from Toxoplasma gondii RH isolate for the potential use in the development of DNA vaccination. Methods Amplifing gene fragment coding GRA8 from the genomic DNA of Toxoplasma gondii RH isolate by means of ploymerase chain reaction (PCR), the gene is inserted into cloning vector pUC-19 digesting with restrictive enzymes and linking react ions. The positive colon is screed on LB plates containing ampicilline and IPTG, Xgal identified by blue-white and restrictive enzyme digestion. The inserted GR A8 gene was recombined with pcDNA3.1(+) eukaryotic expression vector by digestion with restrictive enzyme and linking reactions. The positive coloe is screene d o n LB plates containing ampicillin and identified by restrictive enzymes and link ing reactions. Results The GRA8 gene with about 804 base is specifically amplified from genomic DNA of Toxoplasma GRA8/RH and pcDNA3.1(+)-GRA8 recombinant is successfull y constructed. The sequencing results showed that GRA8 gene of isolate RH and R H from genebank shares quite high homology. Conclusion The gene encoding GRA8 is amplified from genomic DNA of Toxoplasma gene isolate GRA8/RH and pcDNA3.1 (+)-GRA8 recombinant is successfully constructed.