Expressing F. Hepatica Protective Antigenic Gene In BCG

Fascioliasis is a serious parasitic disease over the world. Fasciola hepatica are a widely spread parasite in many kinds of economic herbivores such as sheep, yak, cattle, horse, deer, camel,... etc., and hinder the development of the productive force of livestock husbandry. Moreover , the economic losses caused by Fascioliasis can be very huge. So, it is very important to develop an effective vaccine against Fascioliasis. Expressing outside morbid antigenic gene in Bacille Calmette-Guerin (BCG) of Mycobacterium and developing BCG live vaccine or multivalent vaccine has become one of the most important things in new-style vaccine research all over the world. This thesis describes the constructing of a recombinant plasmid including F. hepatica primary protective antigenic gene FH3 and its expressing in BCG.The protective antigenic gene FH3 of F. hepatica was cut out from recombinant plasmid pUC18/FH3 using EcoR\ , sequenced and it is 0.89kb. Then using T4DNA ligase, the FH3 fragment was inserted in the E. coli-Mycobacteria shuttle expression plasmid vector pMV261 which was digested by EcoR\ too in correct reading frame to construct a recombinant shuttle plasmid including the protective antigenic gene of F. hepatica named pMV261-FH3. Using electroporation, the recombinant plasmidpMV261~FH3 was introduced into BCG and the recombinant BCG was resistive to kanamycin. The most fitted electroporation parameters were: 6.25kv/cm , 25 u F , 1000 Q . A number of factors that affected transformation efficiency were investigated, such as glycine concentration, age of BCG, and so on. By dot blotting it was confirmed that the pMV261-FH3 had been inserted in recombinant BCG chromosome . Under heat induced (45℃) , the expression recombinant protein was analyzed by SDS-PAGE and its relative molecular mass is 22kD. Our results may lay a foundation for constructing recombinant BCG vaccine against F. hepatica.