Cloning Of Actin Gene And Cloning And Expression Of Triosephosphate Isomerasegene Gene From Orientobilhazia Turkestanicum

Orientobilharziasis, a kind of schistosomiasis, is caused by the trematodes of genus Orientobilharzia parasitizing in portal veins and mesentery veins of mammals such as catties and sheep. It is widely distributed in China and brings animal husbandry a great economic loss. In addition, Orientobilharzia cercariae may make human suffer from cercarial dermatitis and seriously endanger people'health, so Orientobilharziasis is an important zoonoses parasitic disease.At present, it is difficult in prevention and treatment of Orientobilharziasis. Firstly, the preventive effect is not remarkable by killing snails, the middle host of schistosomes. Secondly, although Praziquantel is very effective for cure of Orientobilharziasis, there are some weakness such as high cure cost, resistence and repeated infection. It is widely believed that development of preventive vaccine against schistosomiasis is very necessary.In this research, the full-length cDNA of Actin and Triosephosphate Isomerase gene(TPI) of Orientobilharzia turkestanium were cloned, and the TPI was highly expressed in Picha Pastoris GS115 cells. At first, the partial coding region of Actin and TPI were cloned using RT-PCR. The 3'end fragment and 5'end fragment of Actin and TPI were obtained by 3RACE and 5RACE. The full-length cDNA sequence was gotten by splicing sequences of the three fragments. The cDNA sequences have been submitted to GeneBank and accession number of ACTIN is DQ017265 and accession number of TPI is DQ092331. Then, the full-length TPI was cloned by RT-PCR and it was cloned into pMD18-T vector, and the recombinant plasmid pMD18-T/TPI was digested with Not I and EcoK I and the digested full-length TPI was subcloned into espression vector pPIC9k.After electroporation into the GS115 cells, the recombinant plasmids lined by Sal I were screened out by His~- medium and G418. The protein was induced in the medium contains methanol. The supernatant was collected on the 3rd day, SDS-PAGE indicated that the target protein is around 43KD. Western-blot shows it can react with serum of goat naturally infected by orientobilharzia. The optimization experiments indicated that highest yield could be gotten by 0.5% methanol, and on the 72th hour. The TPI protein was purified by Superdex 75 column and protein concentration is around 1200mg/L.At last, the rabbits were vaccined by the purified protein mixed with Montanide ISA 206 adjuvant(the ratio of the antigen to adjuvant is 1:1). ELISA result showed that the level of TPI antibody reached the highest titer on the 4th week, and the vaccined effect of enhanced vaccined group is better than one time vaccined group.The study above facilitates that TPI is immunogenic and can become one of candidate genes for prevention of Orientobilharziasis.