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

Abstract

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.

CLC Number:

S852.616.3

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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The Study on Cytotoxicity of Recombinant NetB Toxin of Clostridium perfringens

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