产气荚膜梭菌重组NetB毒素细胞毒性作用研究

doi:10.11843/j.issn.0366-6964.2016.05.020

畜牧兽医学报 ›› 2016, Vol. 47 ›› Issue (5): 1018-1025.doi: 10.11843/j.issn.0366-6964.2016.05.020

产气荚膜梭菌重组NetB毒素细胞毒性作用研究

王莹莹¹，乔艺然¹，赵蕾¹，刘鹏¹，任玉东²，李广兴^1*，黄小丹¹，张瑞莉¹，杨贵君¹

（1.东北农业大学动物医学学院，哈尔滨 150030；2.东北农业大学电气与信息学院，哈尔滨 150030）

收稿日期:2015-10-27 出版日期:2016-05-23 发布日期:2016-05-23
通讯作者: 李广兴， Tel：0451-55190723，E-mail：ligx@neau.edu.cn
作者简介:王莹莹(1989-)，女，黑龙江绥化人，硕士生，主要从事动物病理学研究， E-mail：286249234@qq.com
基金资助:
鸡传染性支气管炎病毒S蛋白介导的细胞侵染机制研究（31272569）；部分革兰氏阴性细菌耐药普遍规律的数字化研究（31370140）

The Study on Cytotoxicity of Recombinant NetB Toxin of Clostridium perfringens

WANG Ying-ying¹，QIAO Yi-ran¹，ZHAO Lei¹，LIU Peng¹，REN Yu-dong²，LI Guang-xing^1* ，HUANG Xiao-dan¹，ZHANG Rui-li¹，YANG Gui-jun¹

(1.College of Veterinary Medicine，Northeast Agricultural University，Harbin 150030，China；2.College of Engineering，Northeast Agricultural University，Harbin 150030，China)

Received:2015-10-27 Online:2016-05-23 Published:2016-05-23

摘要/Abstract

摘要：

坏死性肠炎B样毒素（NetB）是新近发现的由产气荚膜梭菌产生与鸡坏死性肠炎发生密切相关的成孔毒素。本研究通过观察NetB毒素对HeLa细胞以及小鼠肠道的毒性损伤作用，进一步阐明该毒素在鸡坏死性肠炎发病过程中的作用。采用PCR方法扩增NetB毒素基因片段，构建重组表达质粒pGEX-NetB并转化E.coli BL21（DE3），经IPTG诱导表达获得重组NetB蛋白，纯化鉴定后用复性的蛋白质进行HeLa细胞毒性试验。结果如下：光镜观察可见，NetB蛋白作用2 h后，部分细胞出现肿胀变圆，少数细胞破裂，细胞质外溢；12 h后，细胞已无正常形态，细胞圆缩，细胞因坏死脱落溶解空隙增大，偶见病变细胞聚集成团；48 h后，细胞坏死明显，只残存细胞溶解后的碎片。HE染色后观察可见病变细胞染色不均，细胞膜碎裂而不完整，细胞质外溢。电镜观察可见，病变细胞缺少完整的细胞膜结构，细胞质外溢，细胞核形状不规则，核膜膨出，核仁边集，亚细胞器也出现不同程度的病变，其中线粒体病变最为明显。通过对小鼠病理模型观察可见，接种NetB毒素小鼠发病迅速，腹部膨大，肠道臌气明显；组织学观察表明接种小鼠内脏器官出现不同程度的病理变化，以小肠最为明显，表现为肠黏膜损伤，绒毛断裂、脱落等。NetB毒素可引起HeLa细胞细胞膜损伤而呈现细胞毒性；小鼠体内试验NetB毒素可复制出NE典型病变，表明NetB毒素与NE的发病密切相关。

Abstract:

Necrotic enteritis B-like (NetB) toxin is a new found pore forming toxin produced by Clostridium perfringens，and is closely related to the occurrence of necrotic enteritis in chickens.In order to study the role of NetB toxins in the pathogenesis of necrotic enteritis of chickens.We studied the cytotoxicity of the toxin on HeLa cells，and the intestine of mice by inoculating with recombinant NetB toxin.Polymerase chain reaction (PCR) was utilized to amplify NetB toxin gene，recombinant expression plasmid pGEX-NetB was constructed and transformed into E.coli BL21 (DE3) to obtain recombinant protein NetB by IPTG induction.After purification and identification，the refolded protein was used in cell toxicity test.Two hours post inoculation (PI) of NetB protein，some HeLa cells became swollen and round，a few cells ruptured and cytoplasm spill.HeLa cells at 12 h PI shrank and lost normal form，necrotized，shed，dissolved，and the gap between cells increased，residual cells occasionally gathered into groups.HeLa cells at 48 h PI nectrotized and disappeared significantly，only cell fragments left.After HE staining，HeLa cells were found stained uniformly，cell membrane ruptured and incompleted，cytoplasm spillover.Under electron microscopy，the diseased cells lost the intact structure of cytomembrane，and cytoplasm spill，nucleus shape became irregular for nuclear membrane bulging，nucleolus rim set.Other organelles appeared varying degrees of damage，however，mitochondrial abnormality was most obvious.Observation of pathological model of mouse established in this study showed that the symptoms developed quickly，abdominal swelling for intestinal pep.Histological observations indicated that internal organs of inoculated mice presented different degrees of pathological changes，and small intestine had remarkable manifestations of intestinal mucosal damage with villus breakage and shedding.In conclusions，NetB toxin caused the membrane damage of HeLa cells and exerted its cytotoxic effect；NetB toxin-inoculated mice presented the typical NE pathological lesions and demonstrated close relationship with NE pathogenesis.

中图分类号:

S852.616.3

王莹莹，乔艺然，赵蕾，刘鹏，任玉东，李广兴，黄小丹，张瑞莉，杨贵君. 产气荚膜梭菌重组NetB毒素细胞毒性作用研究[J]. 畜牧兽医学报, 2016, 47(5): 1018-1025.

WANG Ying-ying，QIAO Yi-ran，ZHAO Lei，LIU Peng，REN Yu-dong，LI Guang-xing，HUANG Xiao-dan，ZHANG Rui-li，YANG Gui-jun. The Study on Cytotoxicity of Recombinant NetB Toxin of Clostridium perfringens[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(5): 1018-1025.

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参考文献

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产气荚膜梭菌重组NetB毒素细胞毒性作用研究

The Study on Cytotoxicity of Recombinant NetB Toxin of Clostridium perfringens

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