Cloning and expression of recombinant Fasciola antigen¿

Fasciolosis is a liver disease caused by Fasciola hepatica or F. gigantica that causes significant economic loss in cattles, other animal species and man. The appearance of Fasciola hepatica populations that are resistant to common flukicidal drugs make a need for the development of anti-liver fluke vaccines. Though we selected a target Fasciola gene that expresses (rFhp) protein, in which its native form shows significant induction to lymphoproliferative response of Peripheral blood mononuclear cells. Then we amplified the gene through Polymerase chain reaction process using ¿gt11 specific primers. This gene was cloned through TOPO (TA) cloning vector in XL10-Gold competent cells, then positive colonies that contain rTOPO were used for the excision of the gene to be expressed in expression vectors. ECORI digestions of the purified plasmids were done to detect the existence of the target insert. The results showed the excision of Fh¿400 from recombinant PQE32 vector and its size determined at 500bp. Expression and screening of small cultures was done and a 6xHis-tagged protein was purified & stained on SDS-PAGE that appeared at about 14.5 kDa.