苏云金杆菌晶体蛋白对猪蛔虫的作用及猪蛔虫重组As37蛋白的免疫保护研究
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摘要
猪蛔虫病是仔猪常见的多发性寄生虫病，可导致仔猪的腹泻、消瘦、贫血，严重的导致仔猪死亡，严重危害养猪业。目前猪蛔虫病的防治以咪唑类药物驱虫为主，这些药物在治疗猪蛔虫的同时对动物机体均有不同程度的副作用，且不同程度的残留，危害人类健康、污染环境，所以需要寻找防治猪蛔虫病的一种新药。自1972年发现苏云金芽胞杆菌(Bacillus thuringiensis，Bt)对植物寄生线虫具有杀虫活性以来，随后发现Bt伴胞晶体毒素对蛇形毛圆线虫、捻转血矛线虫、日本血吸虫等动物寄生虫的毒性。本研究旨在寻找高毒力的Bt菌株对猪蛔虫进行有效治疗。另外，关于猪蛔虫的免疫研究近年来取得了一定的进展，国外先后报道了猪蛔虫重组抗原的免疫活性；国内在猪蛔虫的免疫上还是空白，尚未见到商用猪蛔虫疫苗。为了更好地防治猪蛔虫，本研究克隆了猪蛔虫rAs37基因，构建原核表达载体进行高效表达，并对表达蛋白的免疫活性进行了分析，为开发猪蛔虫亚单位疫苗打下基础。主要研究工作和结果如下：
1．Bt伴胞晶体毒素对体外培养的猪蛔虫幼虫的毒力
将感染性猪蛔虫卵对小白鼠进行人工感染，获得三期、四期蛔虫幼虫，经体外培养，用Bt YBT-1517，YBT-1518，YBT-1521，YBT-1532，YBT-1537，R_1，R2伴胞晶体毒素分别作用于幼虫，结果发现七种毒素均对猪蛔虫三期幼虫有毒杀作用。随着作用时间的延长，毒素浓度的增加，虫体运动缓慢，死亡率增加，其在24h、48h时的半数致死剂量分别为0.875mg／mL，0.434mg／mL；0.934mg／mL，0.452mg／mL；1.375mg／mL，0.843mg／mL；1.023mg／mL，0.623mg／mL；1.112mg／mL，0.674mg／mL；0.951mg／mL，0.458mg／mL；0.958mg／mL，0.460mg／mL。YBT-1517对猪蛔虫三期幼虫杀虫效果最好，YBT-1518与其毒力相当，而YBT-1521的半数致死量较其他菌株大。毒素含量越高，幼虫死亡率越高，毒素作用时间的延长，其半数致死剂量降低。
YBT-1517，YBT-1518，R_1，R2四种菌株的晶体蛋白对四期幼虫有毒杀作用。其在24h、48h时的半数致死剂量分别为0.935mg／mL，0.452mg／mL；0.875mg／mL，0.415mg／mL；0.904mg／mL，0.453mg／mL；0.886mg／mL，0.448mg／mL。YBT-1518、YBT-1517、R_1、R_2四种晶体蛋白毒力相当，其中YBT-1518晶体毒素对猪蛔虫四期幼虫的杀虫效果略高于其它三种毒素。
2．Bt伴胞晶体毒素对小鼠体内猪蛔虫幼虫不同作用方式的研究及其免疫定位
感染猪蛔虫的小鼠经静脉注射、肌肉注射、灌胃、腹腔注射YBT-1517，YBT-1518晶体毒素，结果发现毒素均对小鼠体内蛔虫幼虫有显著驱杀效果，静脉注射减虫率最高，效果极显著，高于其它三种方式，而肌肉注射则次之，灌胃与腹腔注射无差异。YBT-1517减虫率分别为72.7％、19.1％、14.6％、14.36％；YBT-1518减虫率为70.87％、32.83％、19.10％、20.51％。YBT-1517与YBT-1518晶体蛋白毒力相当。
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 larvae
A.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
1．Bt伴胞晶体毒素对体外培养的猪蛔虫幼虫的毒力
将感染性猪蛔虫卵对小白鼠进行人工感染，获得三期、四期蛔虫幼虫，经体外培养，用Bt YBT-1517，YBT-1518，YBT-1521，YBT-1532，YBT-1537，R_1，R2伴胞晶体毒素分别作用于幼虫，结果发现七种毒素均对猪蛔虫三期幼虫有毒杀作用。随着作用时间的延长，毒素浓度的增加，虫体运动缓慢，死亡率增加，其在24h、48h时的半数致死剂量分别为0.875mg／mL，0.434mg／mL；0.934mg／mL，0.452mg／mL；1.375mg／mL，0.843mg／mL；1.023mg／mL，0.623mg／mL；1.112mg／mL，0.674mg／mL；0.951mg／mL，0.458mg／mL；0.958mg／mL，0.460mg／mL。YBT-1517对猪蛔虫三期幼虫杀虫效果最好，YBT-1518与其毒力相当，而YBT-1521的半数致死量较其他菌株大。毒素含量越高，幼虫死亡率越高，毒素作用时间的延长，其半数致死剂量降低。
YBT-1517，YBT-1518，R_1，R2四种菌株的晶体蛋白对四期幼虫有毒杀作用。其在24h、48h时的半数致死剂量分别为0.935mg／mL，0.452mg／mL；0.875mg／mL，0.415mg／mL；0.904mg／mL，0.453mg／mL；0.886mg／mL，0.448mg／mL。YBT-1518、YBT-1517、R_1、R_2四种晶体蛋白毒力相当，其中YBT-1518晶体毒素对猪蛔虫四期幼虫的杀虫效果略高于其它三种毒素。
2．Bt伴胞晶体毒素对小鼠体内猪蛔虫幼虫不同作用方式的研究及其免疫定位
感染猪蛔虫的小鼠经静脉注射、肌肉注射、灌胃、腹腔注射YBT-1517，YBT-1518晶体毒素，结果发现毒素均对小鼠体内蛔虫幼虫有显著驱杀效果，静脉注射减虫率最高，效果极显著，高于其它三种方式，而肌肉注射则次之，灌胃与腹腔注射无差异。YBT-1517减虫率分别为72.7％、19.1％、14.6％、14.36％；YBT-1518减虫率为70.87％、32.83％、19.10％、20.51％。YBT-1517与YBT-1518晶体蛋白毒力相当。
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 larvae
A.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
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