Cloning And Expression Of Hypoxanthine-xanthine-guanine Phosphoribosyltransferase Gene And Production Of Multiclonal Antibodies Against HXGPRT And AK Of Toxoplasma Gondii

The Department of Parasitology and Institute of Zoonoses,Anhui Medical University; The Key Laboratory of Gene Resource Utilization for Severe Diseases(Anhui Medical University) Ministry of Education,Hefei 230032,PR ChinaObjective:To clone and express hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase(AK)gene from Toxoplasma gondii RH strain,and to produce multiclonal antibodies against HXGPRT and AK of Toxoplasma gondii.Methods:RH strain tachyzoites,by laboratory mouse passage, were harvested from ascites of mice and genomic RNA was prepared.Fragments of HXGPRT and AK encoding genes were generated by RT-PCR amplification from genomic RNA of T.gondii.The PCR products were ligated to vectors of pMD18-T and pGEM-T respectively.The recombinants were confirmed by BamHI/XhoI digestion, PCR,and DNA sequencing and were cloned into expression vector pET28a.The recombinants were transformed into E.coli BL21(DE3).Fusion expressions were induced by isopro-pyl-beta-D-thiogactoside(IPTG),purified through the collumn of affinity chromatography with Ni2+(nickel sulfate,NiSO4).identified through SDS-PAGE and Western blotting.The concentration of recombinant proteins were tested by improved Bradford method.New Zealand white rabbits were immuned with recombinant protein of T.gondii HXGPRT and AK and sera were collected ten days after the second enhanced immunization.Multiclonal antibodies were verified with antigen of recombinant HXGPRT and recombinant AK respecativly by Western blotting and the tites were test by ELISA.Results T.gondii HXGPRT encoding genes with a molecular size of 693 bp,T.gondii AK encoding gene with a molecular size of 1092bp which are highly homologous to the sequence previously reported were obtained.The expressions were confirmed by SDS-PAGE and Western blotting. Multiclonal antibodies against T.gondii HXGPRT and T.gondii AK which tites are1: 10~7 andl:10~6 were produced and comfirmed by Western blotting.Conclusion The recombinant constructions of T.gondii HXGPRT and T.gondii AK were generated and expressions were induced.Multiclonal antibodies were gained.