豬大腸桿菌重組腸毒素蛋白之免疫效力評估

Evaluation the efficacy immunity of swine enterotoxin Escherichia coli recombinant protein

王笠荏(Wang Li-Ren)

指導教授 ： 朱純燕

國立屏東科技大學/獸醫學院/動物疫苗科技研究所/碩士(2019年)

https://doi.org/10.6346/NPUST201900301

摘要

新生仔豬及哺乳豬常因感染腸毒素型大腸桿菌 (Enterotoxigenic Escherichia coli, ETEC) 及產志賀毒素的大腸桿菌(Shiga toxin-producing Escherichia coli, STEC) 導致下痢、衰弱甚至死亡，造成豬場嚴重的經濟損失。ETEC與STEC皆為革蘭氏陰性菌、具菌毛黏附因子，可促進細菌黏附小腸黏膜上皮細胞，如EHEC之F18黏附因子，當菌體黏附於小腸上皮細胞後產生腸毒素導致宿主下痢及水腫甚至於死亡等。忌熱型腸毒素 (Heat-labile enterotoxin, LT) 與志賀毒素 (verotoxins, VT) 皆為AB毒素 (AB toxin)， A次單位結構為毒力因子，B次單位結構主要與小腸上皮細胞受體結合。忌熱型腸毒素會經由活化腺苷酸環化酶 (adenylate cyclase) 造成細胞毒性，並提高cAMP濃度，導致豬隻產生水樣腹瀉 (rice water diarrhea) 與脫水之病徵。本研究已成功轉殖ETEC之LT-B基因及STEC之VT-B基因並結合F18黏附因子之FedF基因，於原核表現系統pET32a上生產蛋白，並以SDS-PAGE和Western blot確認產物大小和抗原性。我們將重組蛋白免疫於小鼠，以酵素免疫分析法 (Enzyme-linked immunosorbent assay, ELISA) 證實重組蛋白pET32a / VT-FedF-LT可引起抗體反應且與對照組具有顯著之差異(p < 0.05) ; 細胞激素方面可以引起IFN-γ、IL-2、IL-4、IL-6及IL-12的表現，證明細胞性免疫反應的表現較為平衡。綜合上述結果，pET32a / VT-FedF-LT具有發展為豬大腸桿菌次單位疫苗之潛力。

關鍵字

腸毒素型大腸桿菌 ； 出血性大腸桿菌 ； 忌熱型腸毒素 ； 志賀毒素 ； 次單位疫苗

並列摘要

Newborn and post-weaning piglets are often infected with Enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing Escherichia coli (STEC), causing squatting, debilitation and even death, lead to a serious economic losses in farms. ETEC and STEC are Gram-negative bacteria, with fimbriae adhesion factor, which can increase the adhesion to small intestinal mucosal epithelial cells. Such as F18 adhesion factor of EHEC. As long as the bacteria adhere to the small intestinal epithelial cells, it will release enterotoxin which gives rise to diarrhea and edema even death or other symptoms. Heat-labile enterotoxin (LT) and verotoxins (VT) are AB toxin. The AB toxin was completed with a subunit A and five subunits B. A subunit is the major virulence factor. The AB toxin will bind to the receptor on small intestinal epithelial cells. LT can cause the cytotoxicity through the activation of adenylate cyclase and increased cAMP concentration. With the increase of cAMP will lead to rice water diarrhea and dehydration of infected hosts. In this study we have successfully clone LT-B gene of ETEC, VT-B gene of STEC and FedF gene of F18 construct into pET32a to develop subunit vaccine. The product size and antigenicity was verified by SDS-PAGE and Western blot respectively. We used recombinant protein immunize in mice. Evaluate the efficacy of immunity with enzyme-linked immunosorbent assay (ELISA) prove pET32a / VT-FedF-LT protein can enhance antibody titer was significantly higher than the control group (p < 0.05). The pET32a / VT-FedF-LT also can induce IFN-γ、IL-2、IL-4、IL-6 and IL-12 showed that Th1 and Th2 cellular immune response is balanced. Regarding to the results, our approach may be feasible for developing an effective subunit vaccine against Enterotoxigenic Escherichia coli.

並列關鍵字

Enterotoxigenic Escherichia coli ； Enterohemorrhagic Escherichia coli ； Heat-labile enterotoxin (LT) ； verotoxin (VT) ； subunit vaccine

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