Construction Of CDNA Library Of Orientobilharzia Turkestanicum And Cloning And Expression Of Tropomyosin Gene

Orientobilharziasis, a kind of schistosomiasis, is caused by the trematodes of genus Orientobilharzia parasitizing in the portal veins and mesentery veins of many animals including cattle and sheep. In addition, Orientobilharzia cercariae may also make human suffer from cercarial dermatitis. Hence Orientobilharziasis is an important zoonose parasitic disease, Orientobilharzia turkestanicum is a main species in China. Orientobilharziasis is widely distributed in China and prevailed in some regions and seasons. Not only does it seriously endanger people's health, but brings animal husbandry a great economic loss. It is because of the special development process of Orientobilharzia after infecting human that people pay a widely attention to Orientobilharzia. At present, study on it falls behind other schistosome species. So it is very important for us to deeply research Orientobilharzia.In this thesis, cDNA library for mature worm of Orientobilharzia turkestanicum was successfully constructed using the SMART cDNA library construction kit. Besides, the full-length cDNA of tropomyosin (TM) of Orientobilharzia turkestanicum was cloned using RT-PCR, RACE and cDNA library. The cDNA sequence has been submitted to GeneBank and its accession number is AY560898. In addition, The partial coding sequence of TM gene was subcloned into expression vector pET28a ( + ) , the recombinant plasmid pET28-TM was transformed into BL21 and expressed the dissoluble fusion proteins induced by IPTG. The molecular weight of the fusion protein is around 37.5KD. The fusion proteins were purified using Ni-NTA chromagraphy column. Western-blotting results showed that the purified proteins can react with serum of goat infected naturally by Orientobilharzia and stimulate the rabbit to produce the relevant antibody recognized specifically by the antiserum. The purified proteins were mixed with Montanide ISA 206 adjuvant (the ratio of the antigen to oil adjuvant =1:1) . The prepared mixture immunized the rabbit and the serum of the rabbit was collected from first week to 10th week. ELISA results show that the level of TM antibody reached the highest titer on the 5th week, and kept a higher liter from 7th week to 1 Oth week. Better immunogenicity of TM suggests that it has a good prospect in developing vaccine and diagnostic reagent. Our results lay a foundation to research the immunity of the Orientbilharziasis further.