Construction Of Yeast Two-hybrid CDNA Library Of Toxoplasma Gondii And Screen Of AMA1C-terminal Interacting Proteins

Toxoplasma gondii is a pathogen of Toxoplasmosis and belong to the Apicomplexaprotozoa. The growth and replication of this pathogen needs to invade host cells. At the sametime, it also evaded the host immune response. The host cell invasion and appreciation is veryimportant for life cycle and pathogenicity of T.gondii. Recently, the proliferation of T.gondii canonly be effective blocked after treatment with an anticoccidial drug. But it will also promote thedevelopment of the parasite, and therefore does not prevent chronic infection. In order todevelop treatment drug we urgent need insight into molecular mechanisms of parasitereplication and development.The complete encoding gene of AMA1c was amplified from a cDNA by PCR and it wascloned into prokaryotic expression vector pColdIII and vector pGBKT7of eukaryoticexpression system respectively. The results of enzyme digestion analysis and sequencingshowed that the recombinant plasmid pColdIII-AMA1c and pGBKT7-AMA1c were constructedsuccessfully. The pColdIII-AMA1c was transformed into E. coli DH5a competent cells, thenpurification was exerted on GST Resin Column. It was demonstrated by SDS-PAGE that asoluble fusion protein of33ku was expressed. Mices were immunized with the protein toprepare its antiserum. Western blot showed that the serum retained good specificity. IFA wasconducted by using sera prepared in mice agaist purified protein and T.gondii. The results showAMA1c positioned at the top of T.gondii.To screen the interaction factor for C-terminal fragment of AMA1(AMA1c) in T.gondii,Yeast two-hybrid cDNA library of T.gondii was construced by Clontech’s Matchmaker GoldYeast Two-Hybrid System kit in this study. The titer of the amplified library was4.26×107cfu/ml. The bait vector (pGBKT7-AMA1c) was transformed into the yeast strain Y2H byPEG/LiAc method and its self-activation was tested by the phenotype assay, as well as to detectthe bait protein expression by Western blot. The constructed library was screened then asdescribed in the manufacturer’s instruction. The results showed that the bait protein can besuccessfully expressed in yeast cells, pGBKT7-AMA1c had no toxicity to the yeast cells andcould not induce self-activation in yeast two-hybrid system. Moreover, a total of13AMA1cinteracting proteins were screened from the library by this system with AMA1c as bait. Thisstudy laid a solid foundation for the further research on the function of AMA1c.