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

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2h后,部分细胞出现肿胀变圆,少数细胞破裂,细胞质外溢;12h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;48h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。

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 BL 21 （DE3） to obtain recombinant protein NetB by IPTG induction. After purification and identi- fication,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 ceils occasionally gathered into groups. HeLa cells at 48 h PI nec- trotized and disappeared significantly,only cell fragments left. After HE staining, HeLa cells werefound stained uniformly, cell membrane ruptured and incompleted, cytoplasm spillover. Under e- lectron 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 or- ganelles appeared varying degrees of damage,however,mitochondrial abnormality was most obvi- ous. Observation of pathological model of mouse established in this study showed that the symp- toms 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 eytotoxic effect NetB toxin-inoculated mice presented the typical NE pathological lesions and demonstrated close relationship with NE pathogenesis.