鸭源巴氏杆菌与鸭源沙门氏菌融合菌株的制备及鉴定

发布时间：2018-06-21 08:30

本文选题：鸭源巴氏杆菌 + 鸭源沙门氏菌　；参考：《四川农业大学》2015年硕士论文

【摘要】：细菌原生质体融合是一个随机的过程,通过标记双亲本菌株来筛选阳性融合子。本试验利用了亲本菌株在培养特性及耐药性两方面存在的差异,标记亲本菌株对融合菌株进行筛选。通过药敏纸片试验和最低抑菌浓度试验(MIC)筛选出两亲本菌株鸭源沙门氏菌(S6)和鸭源巴氏杆菌(CBa),确定亲本菌株药物标记,药敏试验结果表明鸭源沙门氏菌(S6)对壮观霉素敏感,抑菌圈为26 mm,鸭源巴氏杆菌(CBa)对壮观霉素耐药,抑菌圈为0mm。通过测定最低抑菌浓度(MIC),确定32 μg/ml的壮观霉素作为选择培养基的抗生素浓度。壮观霉素应用浓度试验结果表明,在含32μg/ml壮观霉素的麦康凯培养基中S6完全不能生长,在含32μg/ml的新霉素的血琼脂培养基中CBa能良好生长。筛选出的亲本菌株通过紫外分光光度计测量OD600值绘制CBa和S6的生长曲线,确定原生质体制备时间：CBa为12-14 h,S6为14-16 h。在溶菌酶的作用下制备原生质体,确定制备CBa原生质体的最佳溶菌酶浓度为3 g/L,可得到95.25%的原生质体制备率以及33.52%的再生率；制备S6原生质体的最佳溶菌酶浓度为2.5g/L,可得到95.32%的原生质体制备率以及32.19%的再生率。通过聚乙二醇(PEG)法进行原生质体融合,利用抗药性结合培养条件对融合子进行筛选,再对融合菌株进行培养特性、菌落形态、菌体形态、血清学试验、稳定性检测等初步鉴定最终得到5株能够在32μg/ml壮观霉素的麦康凯平板上稳定遗传的融合菌株。所得融合子进行血清学试验均能凝集双亲菌株阳性血清,菌体形态为中等大小两端顿圆的革兰氏阴性杆菌,能够在麦康凯培养基长出圆形淡黄色菌落,生化试验结果表明氧化酶为阳性,吲哚、枸橼酸钠、蔗糖、乳糖、侧金盏花、硫化氢、氰化钾均为阴性。根据CBa的外膜蛋白H (OmpH)基因和S6的外膜蛋白A (OmpA)基因的保守区域设计两对引物。利用双重PCR法检测5株融合菌株,结果表明3株能够同时检测到双亲本的基因,其余2株则只能检测到单一亲本基因,双重PCR产物测序结果表明与亲本菌株同源性为100%。
[Abstract]:Bacterial protoplast fusion is a random process. In this experiment, we used the differences in culture characteristics and drug resistance of parent strains to screen the fusion strains. Two parent strains, Salmonella duck-origin S6) and Pasteurella duckelis CBaA, were screened by drug sensitive disk test and minimal inhibitory concentration test (MICM). The drug markers of parent strains were determined. The results of drug sensitivity test showed that Salmonella duck-derived Salmonella S6) was sensitive to spectinomycin. The antimicrobial circle was 26 mm, the resistance of CBato to spectinomycin was 0 mm. The antibiotic concentration of 32 渭 g/ml spectinomycin as the selective medium was determined by the determination of the minimum inhibitory concentration (MIC). The results of spectinomycin concentration test showed that S6 could not grow completely in wheat Kang Kai medium containing 32 渭 g/ml spectinomycin, but it could grow well in blood Agar medium containing 32 渭 g/ml neomycin. The growth curves of CBa and S6 were plotted by measuring OD600 value by UV spectrophotometer, and the protoplast preparation time was determined to be 12-14 hS6 and 14-16 h. Protoplasts were prepared under the action of lysozyme. The optimum concentration of lysozyme was 3 g / L to obtain 95.25% protoplast preparation rate and 33.52% regeneration rate. The optimum concentration of lysozyme for preparation of S6 protoplast was 2.5 g / L, and the protoplast preparation rate and regeneration rate were 95.32% and 32.19% respectively. The protoplast fusion was carried out by polyethylene glycol (PEG) method. The fusants were screened by the combination of drug resistance and culture conditions. The culture characteristics, colony morphology, mycelial morphology, serological tests of the fusion strains were carried out. Finally, five fusion strains with stable inheritance on the wheat Kang Kai plate with 32 渭 g/ml spectinomycin were obtained. In serological tests, the fusion could agglutinate the positive sera of the parent strains, and the bacteria were of medium size and round Gram-negative bacilli, and could grow round yellowish colonies in wheat Kang Kai medium. The results of biochemical tests showed that oxidase was positive, indole, sodium citrate, sucrose, lactose, lateral calendula, hydrogen sulfide and potassium cyanide were all negative. Two pairs of primers were designed according to the conserved region of the outer membrane protein (H) gene of CBa and the outer membrane protein A (OmpA) gene of S6. The double PCR method was used to detect 5 fusion strains. The results showed that 3 strains could detect parental genes at the same time, the other 2 strains could only detect single parent genes. The sequencing of double PCR products showed that the homology was 100% with parent strain.
【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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