鸭源沙门氏菌与鸭源大肠杆菌融合菌株的构建及鉴定分析
发布时间：2018-07-31 12:17
【摘要】：鸭沙门氏菌病(又名鸭副伤寒)和鸭大肠杆菌病是鸭场普遍存在的两种细菌病,给养鸭业造成了极大的经济损失。生产中往往通过分别注射两种细菌疫苗进行免疫预防,费时,成本也高,因而构建鸭源沙门氏菌与鸭源大肠杆菌融合菌株可为预防鸭沙门氏菌病、鸭大肠杆菌病提供制备二联疫苗所需的优良菌株,对预防这两种细菌病具有重要意义。本实验首先对实验室保存的、分离自四川、重庆地区不同鸭场的15株鸭源沙门氏菌和10株鸭源大肠杆菌进行血清型鉴定及17种抗生素药敏试验；筛选出一株血清型为6,7：1,v：e,n,x,并对庆大霉素(Gentamicin, Gen)耐药、四环素(Tetracycline,Tcy)敏感的鸭源沙门氏菌S7 (6,7:l,v:e,n,x, GenR, Tcys),以及一株血清型为078,并对庆大霉素敏感、四环素耐药的鸭源大肠杆菌D2 (O78, GenS, TcyR)作为亲本菌株；通过绘制S7、D2两亲本菌株生长曲线,确定S7、D2原生质体制备的最佳时间分别为14～16 h、10～12 h;同时通过对S7、D2两亲本菌株的最低抑菌浓度(MIC)测定,确定高渗再生选择性培养基中应加入的庆大霉素浓度为16 ug/mL,四环素浓度为32 ug/mL。抗生素浓度应用试验结果表明S7在含16 ug/mL庆大霉素的普通琼脂培养基上生长良好,而在含32 ug/mL四环素的普通琼脂培养基上完全不生长；D2在含32 ug/mL四环素的普通琼脂培养基上生长良好,而在含16 ug/mL庆大霉素的普通琼脂培养基上完全不生长；并且S7、D2在同时含16 ug/mL庆大霉素和32 ug/mL四环素的普通琼脂培养基上均完全不生长。采用溶菌酶(Lysozyme,又称胞壁质酶)法制备S7、D2原生质体,在原生质体制备过程中添加EDTA至终浓度为0.01 mol/L。通过溶菌酶浓度试验,最终选择3.0 ug/mL的溶菌酶浓度作为制备S7原生质体的最佳溶菌酶浓度,在此浓度下S7能够得到62.88%的原生质体制备率及33.55%的原生质体再生率；而制备D2原生质体的最佳溶菌酶浓度为2.5 ug/mL,能够得到51.69%的原生质体制备率及27.79%的原生质体再生率。进行原生质体融合并根据条件筛选后最终得到7株能够稳定遗传的融合菌株。融合菌株呈革兰氏阴性杆菌,均能凝集鸭源沙门氏菌和鸭源大肠杆菌阳性血清；硫化氢生化试验结果为阳性,其余生化试验项目结果为阴性。根据外膜蛋白基因保守区域分别设计一对引物,通过双重PCR检测融合菌株部分基因片段,其中3株能够同时检测到鸭源沙门氏菌的目的条带,3株仅能够检测到某一个亲本菌株目的条带,还有一株则检测不到任何的目的条带,测序结果同源性为100%。本实验还提取了融合菌株及两亲本菌株外膜蛋白,通过SDS-PAGE电泳进行外膜蛋白成分变化分析,有3株融合菌株能够同时表达鸭源沙门氏菌和鸭源大肠杆菌双亲本菌株外膜蛋白。
[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.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61
本文编号：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.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61
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