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

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; 谢跃

doi:10.16829/j.slxb.150643

兽类学报 >

2022 , Vol. 42 >Issue 3: 304 - 311

DOI: https://doi.org/10.16829/j.slxb.150643

研究论文

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

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 ,
谢跃

展开

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

黄文俊(1986-),男,本科,兽医师,主要从事大熊猫疫病防控研究.

收稿日期: 2021-09-13

修回日期: 2022-02-23

网络出版日期: 2022-06-02

基金资助

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)

收起

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

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

Expand

1. Veterinary Hospital, Chengdu Research Base of Giant Panda Breeding, Chengdu 610081, China;
2. College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, China

Received date: 2021-09-13

Revised date: 2022-02-23

Online published: 2022-06-02

Fold

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，Bt YBT-1518 Cry5B由1 246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Bt PS86Q3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ED₅₀值为14.5 μg/mL，第7天的ED₅₀值为0.16 μg/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。

关键词： 大熊猫; 苏力菌; 西氏贝蛔虫; Cry5B; 杀虫活性

本文引用格式

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 , 谢跃 . 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[J]. 兽类学报, 2022 , 42(3) : 304 -311 . DOI: 10.16829/j.slxb.150643

Abstract

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 Bt YBT-1518 Cry5B protein consisted of 1 246 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 ED₅₀ of 14. 5 μg/mL on day 3 and 0. 16 μg/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.

Key words： Giant panda; Bacillus thuringiensis; Baylisascaris schroederi; Cry5B; Antinematode activity

参考文献

Charuchaibovorn S, Sanprasert V, Sutthanont N, Hu Y, Abraham A, Os troff G R, Aroian R V, Jaleta T, Lok J B, Nuchprayoon S. 2019.
Bacillus thuringiensis Cry5B is active against Strongyloides ster coralis in vitro. The American Journal of Tropical Medicine and Hygiene, 101 (5):1177-1182.
Dementiev A, Board J, Sitaram A, Hey T, Kelker M S, McCaskill D, Wang N X, Hung S C, Chan M K, Lee M M, Hughes J, Wegener A, Aroian R V, Narva K E, Berry C. 2016. The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins. BMC Biology, 14:71.
Durmaz E, Yan H, Aroian R V, Klaenhammer T R. 2015. Intracellular and extracellular expression of Bacillus thuringiensis crystal pro tein Cry5B in Lactococcus lactis for use as an anthelminthic. Ap plied & Environmental Microbiology, 82 (4):1286-1294.
Frieal C H, Lochnit G, Zahringer U, Bahr U, Geyer R. 2003. Struc tural elucidation of zwitterionic carbohydrates derived from glyco sphingolipids of the porcine parasitic nematode Ascaris suum.The Biochemical Journal, 369:89-102.
Griffitts J S, Haslam S M, Yang T, Garczynski S F, Mulloy B, Morris H, Cremer P S, Dell A, Adang M J, Aroian R V. 2005. Glycolip ids as receptors for Bacillus thuringiensis crystal toxin. Science, 307:922-925.
Hu Y, Aroian R V. 2012. Bacterial pore-forming proteins as anthel mintics. Invertebrate Neuroscience, 12:37-41.
Hu Y, Miller M M, Derman A I, Ellis B L, Monnerat R G, Pogliano J, Aroian R V. 2013. Bacillus subtilis strain engineered for treat ment of soil-transmitted helminth diseases. Applied & Environ mental Microbiology, 79 (18):5527-5532.
Hu Y, Miller M, Zhang B, Nguyen T T, Nielsen M K, Aroian R V. 2018a. In vivo and in vitro studies of Cry5B and nicotinic acetyl choline receptor agonist anthelmintics reveal a powerful and unique combination therapy against intestinal nematode parasites.PLoS Neglected Tropical Diseases, 12 (5):e0006506.
Hu Y, Nguyen T T, Lee A C, Urban Jr J F, Miller M M, Zhan B, Koch D J, Noon J B, Abraham A, Fujiwara R T, Bowman D D, Ostroff G R, Aroian R V. 2018b. Bacillus thuringiensis Cry5B protein as a new pan-hookworm cure. International Journal for Parasitol ogy:Drugs and Drug Resistance, 8 (2):287-294.
Hu Y, Zhan B, Keegan B, Yiu Y Y, Miller M M, Jones K, Aroian R V. 2012. Mechanistic and single-dose in vivo therapeutic studies of Cry5B anthelmintic action against hookworms. PLoS Neglected Tropical Diseases, 6 (11):e1900.
Hui F, Scheib U, Hu Y, Sommer R J, Aroian R V, Ghosh P. 2012.
Structure and glycolipid binding properties of the nematicidal pro tein Cry5B. Biochemistry, 51 (49):9911-9921.
Li D S, He Y, Deng L H, Chen Z Q, Cheng Y X, Xie Y, Han H Y, Yang G Y, Wang C D. 2015. Effect of ivermectin and thiazil dihy droxynaphthoate on Baylisascaris schroederi from captive giant pandas. Animal Husbandry & Veterinary Medicine, 47 (6):87 - 89. (in Chinese)
Li J Q, Karim M R, Li J, Zhang L P, Zhang L X. 2020. Review on parasites of wild and captive giant pandas (Ailuropoda melano leuca):diversity, disease and conservation impact. International Journal for Parasitology:Parasites and Wildlife, 13:38-45.
Qin Z Y, Liu S R, Bai M H, Geng Y, Su X Y. 2021. First report of fa tal baylisascariasis-induced acute pancreatitis in a giant panda.Parasitology International, 84:102380.
Riet E V, Wuhrer M, Wahyuni S, Rrtra K, Deelder A M, Tielens A G M, Kleij D, Yazdanbakhsh M. 2006. Antibody responses to Ascaris-derived proteins and glycolipids:the role of phosphoryl choline. Parasite Immunology, 28:363-371.
Sanders J, Xie Y, Gazzola D, Li H C, Abraham A, Flanagan K, Rus F, Miller M, Hu Y, Guynn S, Draper A, VAkalapudi S, Petersson K H, Zarlenga D, Li R W, Urban Jr J F, Ostroff G R, Zajac A, Aroian R V. 2020. A new paraprobiotic-based treatment for control of Haemonchus contortus in sheep. International Journal for Para sitology:Drugs and Drug Resistance, 14:230-236.
Urban J F, Hu Y, Miller M M, Scheib U, Yiu Y Y, Aroian R V. 2013.Bacillus thuringiensis-derived Cry5B has potent anthelmintic ac tivity against Ascaris suum. PLoS Neglected Tropical Diseases, 7(6):e2263.
Urban J F, Nielsen M K, Gazzola D, Xie Y, Beshah E, Hu Y, Li H C, Rus F, Flanagan K, Draper A, Vakalapudi S, Li R W, Ostroff G R, Aroian R V. 2021. An inactivated bacterium (paraprobiotic) ex pressing Bacillus thuringiensis Cry5B as a therapeutic for Ascaris and Parascaris spp. infections in large animals. One Health, 12:100241.
Vercruysse J, Claerebout E. 2001. Treatment vs non-treatment of hel minth infections in cattle:defining the threshold. Veterinary Para sitology, 98 (1-3):195-214.
Wang L, Li X F, Xu B R, Wu Q J, Zhang Y J. 2006. Advances in re search on molecular structure and function of Bt (Bacillus thuringiensis) receptors in insect. Acta Entomological Sinica, 49(6):1009-1016. (in Chinese)
Wang T, Xie Y, Zheng Y L, Wang C D, Li D S, Koehler A V, Gasser R B. 2018. Parasites of the giant panda:a risk factor in the conser vation of a species. Advances in Parasitology, 99:1-33.
Xie Y, Chen S J, Yan Y B, Zhang Z H, Li D S, Hua Y, Wang C D, Nong X, Zhou X, Gu X B, Wang S X, Peng X R, Yang G Y. 2013. Po tential of recombinant inorganic pyrophosphatase antigen as a new vaccine candidate against Baylisascaris schroederi in mice.
Veterinary Research, 44:1-6.
Xiong L, Zhang H T, Wang C D, Li C W, Lan J C, Luo L, Liu L, Yang G Y. 2019. Killing effect of the disinfectant Neopredisan 135-1
on Baylisascaris schroederi eggs of giant panda in vitro. Progress in Veterinary Medicine, 40 (1):125-130. (in Chinese)
Yang G, Zhang Z. 2013. Parasitic Diseases of Wildlife. Beijing:Sci ence Press. (in Chinese)
Zhang J S, Daszak P, Huang H L, Yang G Y, Kilpatrick A M, Zhang S. 2008. Parasite threat to panda conservation. Ecohealth, 5:6-9.
Zhu Y, Grueber C, Li Y D, He M, Hu L, He K, Liu H, Zhang H M, Wu H L. 2020. MHC-associated Baylisascaris schroederi load in forms the giant panda reintroduction program. International Jour nal for Parasitology:Parasites and Wildlife, 12:113-120.
王莉, 李学锋, 徐宝仁, 吴青君, 张友军. 2006. Bt杀虫晶体蛋白受体分子的结构与功能. 昆虫学报, 49 (6):1009-1016.
李德生, 何燕, 邓林华, 陈祖琴, 成彦曦, 谢跃, 韩洪应, 杨光友, 王承东. 2015. 伊维菌素和双羟萘酸噻嘧啶对圈养大熊猫西氏贝蛔虫驱除试验. 畜牧与兽医, 47 (6):87-89.
杨光友, 张志和. 2013. 野生动物寄生虫病学. 北京:科学出版社.
熊浪, 张浩杰, 王承东, 李才武, 兰景超, 罗娌, 刘礼, 杨光友. 2019.
消毒剂 Neopredisan135-1 对大熊猫西氏贝蛔虫卵的体外杀灭效果观察. 动物医学进展, 40 (1):125-130.

Options

文章导航

模态框（Modal）标题

摘要

本文引用格式

Abstract

参考文献