Study On Effect Of Bacillus Thuringiensis Insecticidal Crystal Proteins On Ascaris Suum And Immune Protection With Recombinant Ascaris Suum As37 Protein

Ascaris roundworm are gastrointestial nematodes that widely distributed in both humans and animals wordwide. One of roundworms, Ascaris suum, was originally identified as a ubiquitous, pathogenic parasite of swine. It is a severe threat to the growing pigs which can cause diarrhea, anaemia even death. Despite the existence of efficient drugs such as Mebendazole, Ivermectin and Albendazole, but these drugs cause side effect, drug-resistence and drug-remaining. It is necessary to find a new drug to treating Ascaridasis. Bacillus thuringiensis (Bt) Insesticidal Crystal Proteins (ICPs) are highly toxic to a wide variety of animal and plant parasites such as Trichostrongylus colubriformis, Haemonchus contortus and Schistosoma japonicum. It is a new field treating A. suum with ICPs toxin, in this research, author studied on the effect of Bt ICPs toxin to A. suum. Furthermore, the immune on Ascaris suum were making progress these years. Recently, some Escherichia coli-expressed recombinant antigens from A. suum were reported to be suitable vaccine candidates for the control of Ascaris infection. Here we attempted to clone rAs37 gene by reverse transcription-polymerase chain reaction (RT-PCR) using primers based on the published cDNA sequence of As37 gene. The Escherichia coli-expressed recombinant protein showed highly immunoreactive by Western-blot analysis and can be considered as a potential vaccine candidate. The results of research are summarized as following: 1. Screening of Bt which are high toxic to A.suum larvaeA.suum L3 (third stage larvae) and L4 (fourth stage larvae), obtained from the livers and lungs of mice which were challenged with 2,500 infective eggs of A.suum, were cultivated at 37℃ with medium-RPIM1640 in 5% CO2 incubator. Different quantities of Bt YBT-1517, YBT-1518, YBT-1521,YBT-1532,YBT-1537, R1, R2 ICPs toxins were added in every hole of cell plate respectively. Seven kinds of ICPs were effective to L3 in vitro. The LD50 values of these toxins to L3 at 24h and 48h were about 0.875mg/mL and 0.434mg/mL;0.934 mg/mL and 0.452 mg/mL; 1.375 mg/mL and 0.843mg/mL; 1.023 mg/mL and 0.623 mg/mL;1.112 mg/mL and 0.674 mg/mL ;0.951mg/mL and 0.458 mg/mL;0.958 mg/mL and 0.460 mg/mL respectively. YBT-1517 ICPs was the most effective toxin as well as YBT-1518. The mortality of A.suum increased with the consistency of crystal protein increasing and the time prolonging. LD50 values decreased with treating time prolonging.Four strains such as Bt YBT-1517, YBT-1518, R1, R2 ICPs were effective to A.suum L4.The LD50 values of these toxins at 24h and 48h were 0.935mg/mL and 0.452mg/mL;0.875mg/mL and 0.415mg/mL; 0.904mg/mL and 0.453mg/mL;0.886...