鴨源沙門氏菌與鴨源大腸桿菌融合菌株的構(gòu)建及鑒定分析
發(fā)布時間：2018-07-31 12:17
【摘要】：鴨沙門氏菌病(又名鴨副傷寒)和鴨大腸桿菌病是鴨場普遍存在的兩種細(xì)菌病,給養(yǎng)鴨業(yè)造成了極大的經(jīng)濟(jì)損失。生產(chǎn)中往往通過分別注射兩種細(xì)菌疫苗進(jìn)行免疫預(yù)防,費時,成本也高,因而構(gòu)建鴨源沙門氏菌與鴨源大腸桿菌融合菌株可為預(yù)防鴨沙門氏菌病、鴨大腸桿菌病提供制備二聯(lián)疫苗所需的優(yōu)良菌株,對預(yù)防這兩種細(xì)菌病具有重要意義。本實驗首先對實驗室保存的、分離自四川、重慶地區(qū)不同鴨場的15株鴨源沙門氏菌和10株鴨源大腸桿菌進(jìn)行血清型鑒定及17種抗生素藥敏試驗；篩選出一株血清型為6,7：1,v：e,n,x,并對慶大霉素(Gentamicin, Gen)耐藥、四環(huán)素(Tetracycline,Tcy)敏感的鴨源沙門氏菌S7 (6,7:l,v:e,n,x, GenR, Tcys),以及一株血清型為078,并對慶大霉素敏感、四環(huán)素耐藥的鴨源大腸桿菌D2 (O78, GenS, TcyR)作為親本菌株；通過繪制S7、D2兩親本菌株生長曲線,確定S7、D2原生質(zhì)體制備的最佳時間分別為14～16 h、10～12 h;同時通過對S7、D2兩親本菌株的最低抑菌濃度(MIC)測定,確定高滲再生選擇性培養(yǎng)基中應(yīng)加入的慶大霉素濃度為16 ug/mL,四環(huán)素濃度為32 ug/mL。抗生素濃度應(yīng)用試驗結(jié)果表明S7在含16 ug/mL慶大霉素的普通瓊脂培養(yǎng)基上生長良好,而在含32 ug/mL四環(huán)素的普通瓊脂培養(yǎng)基上完全不生長；D2在含32 ug/mL四環(huán)素的普通瓊脂培養(yǎng)基上生長良好,而在含16 ug/mL慶大霉素的普通瓊脂培養(yǎng)基上完全不生長；并且S7、D2在同時含16 ug/mL慶大霉素和32 ug/mL四環(huán)素的普通瓊脂培養(yǎng)基上均完全不生長。采用溶菌酶(Lysozyme,又稱胞壁質(zhì)酶)法制備S7、D2原生質(zhì)體,在原生質(zhì)體制備過程中添加EDTA至終濃度為0.01 mol/L。通過溶菌酶濃度試驗,最終選擇3.0 ug/mL的溶菌酶濃度作為制備S7原生質(zhì)體的最佳溶菌酶濃度,在此濃度下S7能夠得到62.88%的原生質(zhì)體制備率及33.55%的原生質(zhì)體再生率；而制備D2原生質(zhì)體的最佳溶菌酶濃度為2.5 ug/mL,能夠得到51.69%的原生質(zhì)體制備率及27.79%的原生質(zhì)體再生率。進(jìn)行原生質(zhì)體融合并根據(jù)條件篩選后最終得到7株能夠穩(wěn)定遺傳的融合菌株。融合菌株呈革蘭氏陰性桿菌,均能凝集鴨源沙門氏菌和鴨源大腸桿菌陽性血清；硫化氫生化試驗結(jié)果為陽性,其余生化試驗項目結(jié)果為陰性。根據(jù)外膜蛋白基因保守區(qū)域分別設(shè)計一對引物,通過雙重PCR檢測融合菌株部分基因片段,其中3株能夠同時檢測到鴨源沙門氏菌的目的條帶,3株僅能夠檢測到某一個親本菌株目的條帶,還有一株則檢測不到任何的目的條帶,測序結(jié)果同源性為100%。本實驗還提取了融合菌株及兩親本菌株外膜蛋白,通過SDS-PAGE電泳進(jìn)行外膜蛋白成分變化分析,有3株融合菌株能夠同時表達(dá)鴨源沙門氏菌和鴨源大腸桿菌雙親本菌株外膜蛋白。
[Abstract]:Duck salmonellosis (also known as duck paratyphoid fever) and duck Escherichia coli (E. coli) are two common bacterial diseases in duck farms. In production, two kinds of bacterial vaccines are injected separately to prevent the disease, which is time-consuming and costly. Therefore, the construction of the fusion strain of salmonella and Escherichia coli from duck can be used to prevent duck salmonella. Duck colibacillosis provides excellent strains for the preparation of dual vaccine, which is of great significance for the prevention of these two bacterial diseases. In this experiment, 15 strains of duck salmonella and 10 strains of Escherichia coli isolated from different duck farms in Sichuan and Chongqing were tested for serotype identification and antibiotic susceptibility test. A strain of salmonella S7 (6: 7: v: eNX), a strain resistant to gentamicin (Gentamicin, Gen), a strain of salmonella (S7) sensitive to tetracycline (Tcy), and a strain of serotype (GenR, Tcys),) of 078 were screened out, and the strain was resistant to gentamicin, and was sensitive to gentamicin. Tetracycline resistant Duck Escherichia coli D2 (O78, GenS, TcyR) as parent strain). The optimum preparation time for protoplast of S7 / D 2 was 14 ~ 16 h ~ 10 ~ 12 h, respectively, and the minimum inhibitory concentration (MIC) of S7 / D _ 2 strain was determined by (MIC). The concentration of gentamicin and tetracycline were determined to be 16ugmL and 32ugmL respectively. The results of antibiotic concentration test showed that S7 grew well on the ordinary Agar medium containing 16 ug/mL gentamicin, but not on the normal Agar medium containing 32 ug/mL tetracycline. D2 grew well on the ordinary Agar medium containing 32 ug/mL tetracycline, but not on the normal Agar medium containing 16 ug/mL gentamicin. Moreover, S7 D 2 did not grow on Agar medium containing both 16 ug/mL gentamicin and 32 ug/mL tetracycline. The protoplasts of S _ 7N _ 2 were prepared by lysozyme (lysozyme) method. The final concentration of EDTA was 0.01mol / L when EDTA was added to the protoplasts. Through the lysozyme concentration test, the lysozyme concentration of 3.0 ug/mL was chosen as the best concentration of lysozyme to prepare S7 protoplast. Under this concentration, 62.88% protoplast preparation rate and 33.55% protoplast regeneration rate were obtained. The optimal concentration of lysozyme for the preparation of D2 protoplasts was 2.5 ugmL, and 51.69% protoplast preparation rate and 27.79% protoplast regeneration rate were obtained. After protoplast fusion and screening according to the conditions, 7 fusion strains with stable inheritance were obtained. The fusion strains were Gram-negative bacilli, which could agglutinate the positive sera of Salmonella duck-origin and Escherichia coli from ducks, and the results of hydrogen sulfide biochemistry test were positive, and the results of other biochemical tests were negative. According to the conserved region of the outer membrane protein gene, a pair of primers were designed to detect the partial gene fragments of the fusion strain by double PCR. Three of them could simultaneously detect the target band of salmonella from duck. 3 strains could detect only one parent strain, and one strain could not detect any target band. The homology of the sequencing results was 100%. The outer membrane proteins of the fusion strain and two parent strains were extracted and analyzed by SDS-PAGE electrophoresis. Three fusion strains were able to express the outer membrane proteins of Salmonella duck-origin and Escherichia coli parents at the same time.
【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61
本文編號：2155560
[Abstract]:Duck salmonellosis (also known as duck paratyphoid fever) and duck Escherichia coli (E. coli) are two common bacterial diseases in duck farms. In production, two kinds of bacterial vaccines are injected separately to prevent the disease, which is time-consuming and costly. Therefore, the construction of the fusion strain of salmonella and Escherichia coli from duck can be used to prevent duck salmonella. Duck colibacillosis provides excellent strains for the preparation of dual vaccine, which is of great significance for the prevention of these two bacterial diseases. In this experiment, 15 strains of duck salmonella and 10 strains of Escherichia coli isolated from different duck farms in Sichuan and Chongqing were tested for serotype identification and antibiotic susceptibility test. A strain of salmonella S7 (6: 7: v: eNX), a strain resistant to gentamicin (Gentamicin, Gen), a strain of salmonella (S7) sensitive to tetracycline (Tcy), and a strain of serotype (GenR, Tcys),) of 078 were screened out, and the strain was resistant to gentamicin, and was sensitive to gentamicin. Tetracycline resistant Duck Escherichia coli D2 (O78, GenS, TcyR) as parent strain). The optimum preparation time for protoplast of S7 / D 2 was 14 ~ 16 h ~ 10 ~ 12 h, respectively, and the minimum inhibitory concentration (MIC) of S7 / D _ 2 strain was determined by (MIC). The concentration of gentamicin and tetracycline were determined to be 16ugmL and 32ugmL respectively. The results of antibiotic concentration test showed that S7 grew well on the ordinary Agar medium containing 16 ug/mL gentamicin, but not on the normal Agar medium containing 32 ug/mL tetracycline. D2 grew well on the ordinary Agar medium containing 32 ug/mL tetracycline, but not on the normal Agar medium containing 16 ug/mL gentamicin. Moreover, S7 D 2 did not grow on Agar medium containing both 16 ug/mL gentamicin and 32 ug/mL tetracycline. The protoplasts of S _ 7N _ 2 were prepared by lysozyme (lysozyme) method. The final concentration of EDTA was 0.01mol / L when EDTA was added to the protoplasts. Through the lysozyme concentration test, the lysozyme concentration of 3.0 ug/mL was chosen as the best concentration of lysozyme to prepare S7 protoplast. Under this concentration, 62.88% protoplast preparation rate and 33.55% protoplast regeneration rate were obtained. The optimal concentration of lysozyme for the preparation of D2 protoplasts was 2.5 ugmL, and 51.69% protoplast preparation rate and 27.79% protoplast regeneration rate were obtained. After protoplast fusion and screening according to the conditions, 7 fusion strains with stable inheritance were obtained. The fusion strains were Gram-negative bacilli, which could agglutinate the positive sera of Salmonella duck-origin and Escherichia coli from ducks, and the results of hydrogen sulfide biochemistry test were positive, and the results of other biochemical tests were negative. According to the conserved region of the outer membrane protein gene, a pair of primers were designed to detect the partial gene fragments of the fusion strain by double PCR. Three of them could simultaneously detect the target band of salmonella from duck. 3 strains could detect only one parent strain, and one strain could not detect any target band. The homology of the sequencing results was 100%. The outer membrane proteins of the fusion strain and two parent strains were extracted and analyzed by SDS-PAGE electrophoresis. Three fusion strains were able to express the outer membrane proteins of Salmonella duck-origin and Escherichia coli parents at the same time.
【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61
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