【摘要】：雞大腸桿菌病(Chicken Colibacillosis)是由禽致病性大腸桿菌(avian pathogenic Escherichia coli, APEC)引起的雞體局部或全身性感染的傳染病,是造成養(yǎng)禽業(yè)嚴重經(jīng)濟損失的主要傳染病之一。目前,抗菌藥物仍是防治雞大腸桿菌病的主要措施,由此帶來的生物安全問題受到廣泛關(guān)注。針對雞大腸桿菌病的防控,采取疫苗預(yù)防與合理用藥相結(jié)合的綜合措施是最為行之有效的方法。因此,開發(fā)強免疫效力且具有廣譜保護性的新型APEC基因工程疫苗是解決該現(xiàn)狀的一種有效途徑。本研究選擇減毒雞傷寒沙門氏菌SG9R株為宿主菌,以asd基因為營養(yǎng)缺陷標志構(gòu)建減毒雞傷寒沙門氏菌染色體-質(zhì)粒平衡致死系統(tǒng)表達禽源大腸桿菌Ⅰ型菌毛抗原,以探討該重組口服疫苗對預(yù)防雞大腸桿菌病的保護效果。1.禽源大腸桿菌Ⅰ型菌毛操縱子fim基因的克隆、表達及生物活性以禽源大腸桿菌分離株CE2基因組DNA為模板,采用長PCR技術(shù)擴增出編碼Ⅰ型菌毛操縱子fim基因,克隆至表達質(zhì)粒載體pBR322,構(gòu)建和篩選出含fim完整基因并正確插入的pBR322-fim重組質(zhì)粒,將該重組質(zhì)粒轉(zhuǎn)化至無菌毛的宿主大腸桿菌SE5000。該表達重組菌能分別與雞紅細胞、酵母細胞發(fā)生明顯的凝集反應(yīng)且均能被D-甘露糖所抑制；重組菌與鼠抗Ⅰ型菌毛亞單位蛋白FimA高免血清產(chǎn)生明顯的凝集反應(yīng)。電鏡觀察到重組菌表面長滿菌毛,利用該重組菌免疫小鼠制備抗血清經(jīng)交叉吸附純化后獲得Ⅰ型菌毛單因子血清,可有效用于禽源大腸桿菌Ⅰ型菌毛表達的檢測。用重組菌pfim進行人肺腺上皮細胞A549體外粘附試驗和粘附抑制試驗,結(jié)果表明：重組菌pfim和野生株CE2均具有粘附肺腺上皮細胞的能力,且重組菌的粘附特性明顯強于野生菌株,而D-甘露糖能有效地抑制上述重組菌或野生菌株對人肺腺上皮細胞的粘附結(jié)合。這為開發(fā)以Ⅰ型菌毛作為免疫原的基因工程疫苗的研制奠定了基礎(chǔ)。2.禽傷寒沙門氏菌SG9R株asd缺失株平衡致死系統(tǒng)平臺的構(gòu)建禽傷寒沙門氏菌SG9R弱毒株在預(yù)防禽傷寒中發(fā)揮著較好作用,為將SG9R弱毒株開發(fā)為能攜帶外源基因的口服活疫苗載體且保持其原有免疫原性,利用Red同源重組系統(tǒng)對SG9R株基因組中編碼天冬氨酸β-半醛脫氫酶的asd基因進行敲除,獲得SG9RΔasd缺失突變株。該突變株在缺乏外源性DAP培養(yǎng)條件下發(fā)生溶菌死亡,而在添加DAP的培養(yǎng)基或?qū)霐y帶asd基因的互補質(zhì)粒后才能恢復(fù)生長,以此為基礎(chǔ)建立以asd基因為營養(yǎng)缺陷標志的禽傷寒沙門氏菌染色體-質(zhì)粒平衡致死系統(tǒng)。選擇表達綠色熒光蛋白(GFP)基因作為報告基因,以攜帶鏈球菌asd基因的pYA3342質(zhì)粒為表達載體,構(gòu)建了表達綠色熒光蛋白的重組菌SG9R (pYA3342-GFP)。該重組菌在體外傳至40代后,經(jīng)熒光顯微鏡觀察和流式細胞儀分析表明仍可穩(wěn)定高效表達綠色熒光蛋白；同時,將該重組菌口服接種20日齡仔雞,發(fā)現(xiàn)其廣泛分布于雞的脾臟、肝臟及腸道組織等,體內(nèi)穩(wěn)定性試驗表明重組菌可在雞體內(nèi)停留2-3周。以上結(jié)果表明該缺失株可以用來作為宿主載體平衡致死系統(tǒng)來高效穩(wěn)定表達外源基因,為弱毒禽傷寒沙門氏菌作為載體的基因工程活疫苗研制提供了優(yōu)良的操作平臺。3.禽源大腸桿菌Ⅰ型菌毛抗原在雞傷寒沙門氏菌SG9RΔasd缺失株中的表達及免疫保護效果研究利用PCR技術(shù)擴增出禽源大腸桿菌表達Ⅰ型菌毛的fim操縱子基因并將其克隆至攜帶asd基因的組成型表達載體pYA3342,構(gòu)建重組表達質(zhì)粒pYA3342-fim,重組質(zhì)粒經(jīng)電轉(zhuǎn)化至減毒雞傷寒沙門氏菌SG9RΔasd缺失株,普通LB平板篩選陽性克隆,命名為SG9R (pYA3342-fim)。經(jīng)甘露糖敏感性血凝試驗與抗甘露糖血凝試驗、酵母細胞凝集試驗表明重組菌在普通培養(yǎng)條件下(37℃C,振蕩培養(yǎng))能夠很好的表達Ⅰ型菌毛。同時,對重組疫苗的生長特性、遺傳穩(wěn)定性及口服安全性等生物學(xué)特性進行了評定。通過對3周齡非免疫雞口服接種重組菌苗SG9R (pYA3342-fim)作為免疫組,SG9R (pYA3342)為空載體對照組,PBS為空白組,分別對各組試驗雞于不同時間段采集血清利用間接ELISA法測定抗Ⅰ型菌毛特異性IgG抗體動態(tài)水平,一次免疫14d后,免疫組血清抗體水平顯著高于空載體對照組和空白組(P0.05),而空載體對照組與空白組之間無顯著性差異(P0.05)；在整個免疫周期內(nèi)(4周),免疫組雞血清中特異性抗體水平均呈上升趨勢。用禽致病性大腸桿菌強毒株QD2對各試驗組雞群經(jīng)后胸氣囊攻毒,實驗觀察10d內(nèi),免疫組、空載體對照組及空白對照組的死亡率分別為40.0%、73.3%、80.0%。試驗結(jié)果表明,本實驗構(gòu)建重組活菌苗SG9R (pYA3342-fim)對禽大腸桿菌病的預(yù)防可以起到一定的保護效果。
[Abstract]:Chicken colibacis is an infectious disease caused by avian pathogenic Escherichia coli (APEC) or systemic infection, which is one of the major infectious diseases causing serious economic loss in poultry industry. At present, the anti-bacteria medicine is still the main measure to control the chicken colibacteremia, and the biological safety problem brought by the antibacterial medicine is widely concerned. According to the prevention and control of chicken colibacs, comprehensive measures combining vaccine prevention and rational drug administration are the most effective methods. Therefore, it is an effective way to develop a new APEC genetic engineering vaccine with strong immunopotency and broad-spectrum protection. In this study, the S. typhimurium SG9R strain was selected as host strain, and the asd gene was used as a nutrition defect marker to construct the Salmonella typhimurium chromosome-plasmid balanced lethal system to express avian source E. coli type I fimbriae antigen. To investigate the protective effect of the recombinant oral vaccine on the prevention of E. coli in chickens. The cloning, expression and biological activity of the Escherichia coli type I fimbrium gene of avian source are the template of CE2 genomic DNA of the avian source E. coli isolate, and the encoding type I fimbrium gene is amplified by using the long PCR technology, and cloned to the expression plasmid vector pBR322. A recombinant plasmid pBR322-fim containing fim complete gene was constructed and screened, and the recombinant plasmid was transformed into sterile wool host E. coli SE5000. The recombinant bacteria can react with chicken red blood cells and yeast cells respectively, and can be inhibited by D-mannose. Electron microscopy showed that the surface of recombinant bacteria was full of fimbriae, and the serum of type 鈪，

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