Cloning Of Toxoplasma Gondii Calpain-related CDNA And Recombinant Protein Expression

Objective: To clone and identify a cDNA fragment of Toxoplasma gondii calpain-related gene, and to construct the recombinant expression vector pET32a-calpain-related and express the recombinant protein for the preparation of a vaccine against T. gondii infection.Methods: Toxoplasma gondii tachyzoites were isolated from BALB ?c mice infected by successive inoculation with the RH strain of the protozoon. Total RNA was prepared from the tachyzoites. RT-PCR was performed to clone a fragment of calpain-related cDNA. The cDNA fragment was ligated into pGEM-T vector which was then comfirmed by Sal Iand Bam HI digestion and DNA sequencing, and then subcloned into the expression vector pET32a. The enzymatic digestion and sequencing analysis were performed to verify the construction. The cells bacterial host E. coli BL 21 were transformed by the recombinant plasmid pET32a-calpain-related. The fusion protein was expressed in the host bacteria by the induction or IPTG and purified through a Ni-NTA sepharose column. RNA Dot-blot and Northern blotting were performed to further verify the expression and mRNA sequence of the gene in the protozoon.Results: Gel analysis showed that the total RNA isolated from T. gondii tachyzoites had a proper quality for performing a RT-PCR. A cDNA fragment of about 500 bp was obtained by the technique of RT-PCR from the total RNA with a pair of specific primers for T. gondii calpain-related mRNA. The cDNA fragment was inserted into the vector pGEM-T which was then verified by Sal I and Bam HI digestion and DNA sequencing. The result of DNA sequencing demonstrated that the cDNA fragment of T. gondii calpain-related contained 500 bp of nucleotides which is identical to the predicted mRNA sequence previously reported in the internet site (http://www.toxodb.org). The recombinant expression vector pET32a-calpain-related was constructed and confirmed by enzyme digestin and DNA sequencing. The recombinant protein of calpain-related was expressed in E. coli BL21 cells. SDS-PAGE analysis showed that the recomninant protein had a molecular weight of 26kD which was identical to the expected. The result of RNA Dot-blot indicated that the calpain-related gene was expressed in this protozoon. The result of Northern blot suggested that the mRNA of T. gondii calpain-related had a length of about 6800 bp.Conclusion: A cDNA fragment of T. gondii calpain-related gene was successfully cloned and subloned into the expression vector pET32a. The recombinant protein of calpain-related with a molecular weight of 26 kD was expressed in E. coli BL21 cells and will be used as immunogen in the study for a preparation of a vaccine against T. gondii infection.