Construction And Identification Of Recombinant Mycobacterium Smegmatis Expressing ROP18 Of Toxoplasm Gondii

Objective:(1)To amplify of the gene of rhoptry protein 18(ROP18)from toxoplasma gondii.(2)To construct the recombinant shuttle-plasmid pMV261-ROP18.(3)To construct and identify the recombinant Mycobacterium smegmatis expressing ROP18 of toxoplasm gondii.Method:(1)Toxoplasm gondii RH strain was injected into abdominal cavity of mice by intraperitoneal injection method.After three generation of subculture,the tachyzoite of toxoplasma gondii RH strain were gained from the abdominal cavity of mice.(2)The tachyzoites of toxoplasma gondii resuspended by physiological saline were repeated freezing and thawing three times and Ultrasonic broken,then the protein of Toxoplasma gondii tachyzoites was extracted.The mice were immuned by the Toxoplasma gondii tachyzoite protein respectively mixed with freund’s complete adjuvant and incomplete freund’s adjuvant.Then the mice serum inclouding the antibody to the Toxoplasma gondii tachyzoite protein was collected.(3)According to the full gene sequence of toxoplasma gondii rhoptry protein ROP18 in NCBI gene database,the primers used to amplify ROP18 gene were designed and synthesised.The HindⅢ and BamH I restriction enzyme site were designed in the front of the primers respectively.The genomic DNA of the tachyzoite Toxoplasm gondii RH strain was extracted from the tachyzoite of Toxoplasm gondii RH strain collected.The full gene sequence of toxoplasma gondii rhoptry protein ROP18 was amplified from the genomic DNA of the tachyzoite Toxoplasm gondii RH strain by PCR.Then the ROP18 gene which was amplified were inserted into shuttle-plasmid pMV261 to construct recombinant recombinant shuttle-plasmid pMV261-ROP18.The recombinant recombinant shuttle-plasmid pMV261-ROP18 constructed were confirmed by sequencing after identification using PCR and restriction enzyme digestion.(4)The recombinant recombinant shuttle-plasmid pMV261-ROP18 was transformed into Mycobacterium smegmatis by electroporation to recombinant Mycobacterium smegmatis.The ROP18 protein expression of the recombinant Mycobacterium smegmatis after heat induced was identified with SDS-PAGE and Western blotting.Result:A large amount of tachyzoites of toxoplasma gondii RH strain were gained from the abdominal cavity of mice.And the serum inclouding the antibody to the Toxoplasma gondii tachyzoite protein were obtained from the mice immuned by the Toxoplasma gondii tachyzoite protein tracted from the tachyzoites of Toxoplasma gondii.A DNA sequence with an open reading frame of 1665bp was successfully amplified from the genome DNA of Toxoplasma gondii by PCR.The recombinant plasmids of the pMV261-ROP18 were constructed successfully identified by restrictionenzyme digestion showing DNA bands at about 4500bp and 1665bp.And the result of sequencing showed that the inserted gene was consistent with the ROP18 gene sequence of toxoplasma gondii RH strain in NCBI gene database,and the homologies was 100%.A 66KD exogenous protein expressed from recombinant Mycobacterium smegmatis transformed with recombinant pMV261-ROP18 was displayed with SDS-PAGE.And the 66KD exogenous protein could be recognised by the serum inclouding the antibody to the Toxoplasma gondii tachyzoite protein.Conclusion:(1)The gene of rhoptry protein 18(ROP18)was successfully amplified from toxoplasma gondii.(2)The recombinant shuttle-plasmid pMV261-ROP18 was constructed successfully.(3)The recombinant Mycobacterium smegmatis expressing ROP18 of toxoplasm gondii was constructed successfully.