【摘要】：金黃色葡萄球菌腸毒素B(SEB)是由金黃色葡萄球菌分泌產(chǎn)生的一種外毒素,也是一種超抗原。SEB能夠非特異性地激活免疫細(xì)胞,尤其是T細(xì)胞。產(chǎn)生眾多細(xì)胞因子,能夠提高特異性抗原的免疫原性。因而SEB具備成為免疫佐劑的潛力。但由于SEB對(duì)人和哺乳動(dòng)物具有較大的毒副作用,因此其佐劑活性在畜牧獸醫(yī)領(lǐng)域的應(yīng)用受到限制。為了減輕或消除SEB的毒性作用,我們通過PCR定點(diǎn)突變技術(shù),構(gòu)建SEB突變體(N23S,H32N和K172E),篩選出超抗原活性較好的突變體。同時(shí)研究SEB及其突變體在肉雞體內(nèi)的安全性及其佐劑活性,為促進(jìn)SEB作為免疫佐劑在獸醫(yī)領(lǐng)域的應(yīng)用奠定基礎(chǔ)。本試驗(yàn)主要包括三個(gè)部分工作:1.獲得SEB突變重組蛋白利用PCR方法對(duì)SEB的3個(gè)關(guān)鍵抗原位點(diǎn)進(jìn)行突變,包括N23S,H32N和K172E,并構(gòu)建了原核表達(dá)質(zhì)粒p ET-28a-SEB,p ET-28a-SEBM23(N23S),p ET-28a-SEBM32(H32N),p ET-28a-SEBM172(K172E),p ET-28a-SEBM23/172(N23S+K172E),p ET-28a-SEBM32/172(H32N+K172E),p ET-28a-SEBM23/32/172(N23S+H32N+K172E)。在大腸桿菌中誘導(dǎo)表達(dá)并純化獲得7種重組蛋白。2.SEB及其突變體SEBM32/172對(duì)AIV-H9/NDV二聯(lián)滅活苗的佐劑作用分別用重組蛋白SEB和SEBM32/172配合H9亞型禽流感、新城疫二聯(lián)滅活苗免疫肉雞,通過HI實(shí)驗(yàn)和MTT實(shí)驗(yàn)評(píng)價(jià)SEB及其突變體SEBM32/172對(duì)滅活疫苗的佐劑作用。結(jié)果顯示在免疫后第14-28天5μg SEBM32/172能不同程度的提高滅活疫苗中AIV-H9的免疫效果,對(duì)NDV的作用不明顯;1μg或10μg SEBM32/172和SEB的對(duì)滅活疫苗的佐劑效應(yīng)不穩(wěn)定。淋巴細(xì)胞活性檢測(cè)結(jié)果顯示1μg SEB和5μg SEBM32/172在免疫后第7天顯著高于疫苗對(duì)照組,其他時(shí)間段存在一定差異。3.SEB及其突變體SEBM32/172在肉雞體內(nèi)的安全性評(píng)價(jià)為了研究突變SEBM32/172后能否降低SEB對(duì)機(jī)體毒性作用,我們用10倍佐劑劑量(50μg/只)的重組蛋白SEB和SEBM32/172分別肌肉注射肉雞,分別在接種后第7、14、21天進(jìn)行剖檢取樣制備組織病理切片,進(jìn)行病理學(xué)觀察。在試驗(yàn)過程中肉雞表現(xiàn)正常,臟器沒有明顯的肉眼可見的組織病變,但在顯微鏡下,實(shí)驗(yàn)組肉雞的腎臟存在不同程度的病理?yè)p傷,如腎小管變性壞死、腎小球腫大、腎小球腎炎等,病理?yè)p傷程度隨時(shí)間的增加而加重,而SEBM32/172組的病理?yè)p傷比SEB組的病理?yè)p傷程度低,表明雙突變SEBH32NK172E能降低SEB對(duì)肉雞的毒性作用。
[Abstract]:Staphylococcus aureus enterotoxin B (SEB) is an exotoxin secreted by Staphylococcus aureus. It is also a superantigen. SEB can activate immune cells, especially T cells. The production of many cytokines can improve the immunogenicity of specific antigens. Therefore, SEB has the potential to become an immune adjuvant. However, the application of SEB adjuvant in animal husbandry and veterinary field is limited because of its toxicity and side effects to human and mammal. In order to reduce or eliminate the toxicity of SEB, we constructed SEB mutants (N23SnH32N and K172E) by PCR site-directed mutagenesis, and screened out the mutants with better superantigen activity. At the same time, the safety and adjuvant activity of SEB and its mutants in broilers were studied, which laid a foundation for the application of SEB as immune adjuvant in veterinary field. The experiment consists of three parts: 1. 鑾峰緱SEB紿佸彉閲嶇粍铔嬬櫧鍒╃敤PCR鏂規(guī)硶瀵筍EB鐨，

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