重组Bt伴胞晶体蛋白Cry5B对大熊猫西氏贝蛔虫的离体杀灭活性观察  

Observation on antinematode activity of the recombinant Bacillus thuringiensis-derived Cry5B against the giant panda Baylisascaris schroederi in vitro

作　　者：黄文俊 罗娌 陈欣 刘礼 廖礼慧 王小兰 李碧 李明喜 陈敏 易得娇 李翰 张皓 卓贵富 刘云健 李莹馨 陈奕君 周璇[2] 谢跃[2] HUANG Wenjun;LUO Li;CHEN Xin;LIU Li;LIAO Lihui;WANG Xiaolan;LI Bi;LI Mingxi;CHEN Min;YI Dejiao;LI Han;ZHANG Hao;ZHUO Guifu;LIU Yunjian;LI Yingxin;CHEN Yijun;ZHOU Xuan;XIE Yue(Veterinary Hospital,Chengdu Research Base of Giant Panda Breeding,Chengdu 610081,China;College of Veterinary Medicine,Sichuan Agricultural University,Wenjiang 611130,China)

机构地区：[1]成都大熊猫繁育研究基地兽医院,成都610081 [2]四川农业大学动物医学院,温江611130

出　　处：《兽类学报》2022年第3期304-311,共8页Acta Theriologica Sinica

基　　金：成都大熊猫繁育研究基地自立课题(2020CPB-B20);四川省国际科技创新合作/港澳台科技创新合作项目(2019YFH0065)。

摘　　要：大熊猫是中国特有的珍稀物种,西氏贝蛔虫是危害大熊猫最为严重的一种肠道寄生性线虫。针对目前长期药物驱虫导致的大熊猫西氏贝蛔虫耐药及生态污染问题,苏力菌(Bacillus thuringiensis,Bt)晶体蛋白Cry5B因其特异的线虫杀灭活性,是一种理想的新型抗蛔虫病药物候选。本研究拟通过原核表达方式产生Bt YBT-1518重组Cry5B蛋白,并评价其对大熊猫西氏贝蛔虫的离体杀灭活性。结果表明,BtYBT-1518 Cry5B由1246个氨基酸组成,分子量(MW)为139.889 kDa,包含Endotoxin_N、δ-Endotoxin_C、Endotoxin_C、Endotoxin_C2、Cry1Ac_D5结构域,与BtPS86Q3晶体蛋白关系最近。重组Cry5B蛋白在IPTG浓度为1.4 mmol/L时表达量最大,表达于菌体上清液。离体杀灭活性实验表明,重组Cry5B蛋白的大熊猫西氏贝蛔虫抑杀效果具有显著的剂量依赖性,对肠道L4期幼虫第3天的ED_(50)值为14.5μg/mL,第7天的ED_(50)值为0.16μg/mL;但对成虫更敏感,仅作用2 d便可显著下降虫体活性,直至死亡,证实重组Cry5B蛋白对大熊猫蛔虫具有较强的毒杀作用。这些结果为后续论证Bt YBT-1518重组Cry5B蛋白可以作为一种新型、环保的抗大熊猫西氏贝蛔虫病药物提供了数据参考。The roundworm Baylisascaris schroederi is one of the most serious intestinal parasitic nematodes found in the giant panda,a rare species endemic to China.Given the current situation of the long-term anthelmintic treatment-causing resistance and ecological pollution,Bacillus thuringiensis(Bt)-producing crystal protein Cry5B is an ideal and novel antiascariasis drug candidate due to its specific antinematode activity.This study was designed to prokaryotically produce the recombinant protein Cry5B from the Bt YBT-1518 and to evaluate its antinematode activity on the intestinal fourthstage larvae(L4s)and adults of B.schroederi.The results showed that the recombinant BtYBT-1518 Cry5B protein consisted of 1246 amino acids with a molecular weight(MW)of 139.889 kDa,contained Endotoxin_N,δ-Endotoxin_C,Endotoxin_C,Endotoxin_C2,and Cry1Ac_D5 domains,and shared the closest relationship with that of Bt PS86Q3.After optimization,the recombinant Cry5B protein was expressed in the supernatant with the maximum yield when IPTG was up to 1.4 mmol/L.Further analysis of antinematode activity indicated a significant dose-dependent response to the recombinant Cry5B protein with ED_(50)of 14.5μg/mL on day 3 and 0.16μg/mL on day 7 for L4s.It appeared that adults were more sensitive to this protein than L4s as all worms became immotile on day 2 and even dead on day 7.Combined,these findings suggested that the recombinant Bt YBT-1518 Cry5B protein is able to completely intoxicate B.schroederi.These results provided insights into the development and clinical application of Bt YBT-1518 recombinant Cry5B protein as a new drug against the giant panda baylisascariasis.

关 键 词：大熊猫 苏力菌 西氏贝蛔虫 Cry5B 杀虫活性 

分 类 号：S864.7[农业科学—野生动物驯养]