【作者】 钟蕾；

【导师】 肖克宇；

【作者基本信息】 湖南农业大学 ， 基础兽医学， 2002， 硕士

【摘要】 本试验以肠型点状气单细胞菌58-20-9菌株和鱼害粘球菌G₄菌株作为初发菌株进行原生质体融合，构建融合子，并对其进行形态结构、生理生化、酶类性状及病原性、抗原性等方面的研究，主要结果如下： 1、融合子遗传标记的筛选：应用定性和定量药物敏感性试验，筛选出肠型点状气单细胞58-20-9菌株的遗传标记为E_m^r（红霉素抗性），鱼害粘球菌G₄菌株的遗传标记为G_m^r（庆大霉素抗性）。在这两种细菌原生质体融合试验中，选择培养基中红霉素的应用浓度为3000Iu／ml，庆大霉素应用浓度为50Iu／ml。 2、融合子的构建：在酶解温度37℃下，选出最适酶解时间为40min，最适溶菌酶浓度为4mg／ml。以PEG为促融剂的条件下，肠型点状气单菌胞58-20-9菌株与鱼害粘球菌G₄菌株的原生质体进行了融合，58-20-9菌株的原生质体形成率为54.7％，原生质体再生率18.2％，G₄菌株的原生质体形成率为50.8％，原生质体再生率为12.2％，初筛的原生质体融合率为4‰，点种500个初筛融合子菌落在双抗培养基上连续传代12次，得到1个稳定的融合子菌落，其稳定率为0.2％。 3、融合子形态结构及理化性状鉴定：筛选的融合子菌株菌体短杆状，比G₄菌株稍短，但较58-20-9菌株略粗，近圆形，平均长轴5.60μm，平均短轴0.67μm，平均体积1.34μm³，革兰氏阴性，无鞭毛，不运动，培养30天后形成的巨大菌落颜色乳白色，有光泽，中央较厚、隆起、边缘较整齐。对融合子菌株进行酶类性状及生理生化鉴定，其特性有些倾向于58-20-9菌株，有些倾向于G₄菌株。 4、病原性与抗原性研究：用融合子菌株对当年健康草鱼种进行人工感染攻毒，连续观察两周，草鱼种死亡率达95％，空白对照组成活率100％，显示融合子菌株也具有致病性，进一步剖检病死鱼，发现由融合菌株攻毒的草鱼种具有肠炎病和烂鳃病两种疾病的部分症状。用融合菌株制成灭活抗原进行抗原性研究，结果表明，受融合子菌株免疫保护的草鱼种用58-20-9菌株和G₄菌株分别攻毒，其成活率达76.6％和80.0％，免疫保护率达74.9％和75.5％，抗体效价于第四周达到峰值，分别为1：322.5和1：435.5。 总之，在本试验条件下，肠型点状气单胞菌5820习菌株与鱼害粘球菌G。菌】～ 株的原生质体能够融合并形成稳定的融合子，且融合子菌株具有两亲本的病原性 和抗原性。更多还原

【Abstract】 This experiment used Aeromonas punctta f. intestinalis 58-20-9 strains and Myxococcs piscicola sp.nov G4 strains to operate as arch-strains to conduct protoplast fusion,established fusants and studied their morphological configuration physiologic biochemical,enzyme character and pathogenicity,antigenicity and so on . The main results were presented as follows:1. Screening of fusants’ genetic markers:Using the qualitative and quantitative antimicrobial susceptibility experiment to screen the genetic markers of protoplast fusion between Aeromonas punctta f. intestinalis 58-20-9 strains and Myxococcs piscicola sp.nov 64 strains,they are Emr(Erythomycin resistance) and Gmr(Gentamicin resistance) respectively . In this protoplast fusion experiment between two bacteria,the applying density in selective culture medium of Erythomycin and Gentamicin are 3000 lu / ml and 50 lu / ml.2. Establishment of fusants:When the temperature of enzymolysis was 37C,we singled out the best enzymolysis time to be 40 minute and the best lysozyme density to be 4 mg/ml . In the condition of PEG operating as agent of accelerating fusion,Aeromonas punctta f. intestinalis 58-20-9 strains and Myxococcs piscicola sp.nov G4 strains conducted protoplast fusion,the protoplast formation rates of Aeromonas punctta f. intestinalis 58-20-9 strains are 54.7 % and the protoplast regenerative rates of 58-20-9 strains were 18.2%,the protoplast formation rates of Myxococcs piscicola sp.nov 64 strains were 50.8% and the protoplast regenerative rates of 64 strains were 12.2% . Arch-screening protoplast fusion rates were 4 . Breeding respectively 500 fusants colony of arch-screening on duplexing repel select culture medium and generating continuously 12 times,derived a steady fusant conoly . The fusants steady rates were 0.2% .3. Judging of the characters of fusants on morphological and configuration physics and chemical:The screened fusants are short rhabditiform . they are shorterthan 64 strains,but more grossness than 58-20-9 strains .They are near round .and their average macro-axis are 5.60um,average brachy-axis are 0.67um,average volume are 1.34um3 .Gram female,no flagellum,no motion .The colour of giga-colony which was cultured 30 days is scream white and have brilliancy,the colony’s center is thicker than rim .The colony is bulging and it’s rim is orderliness .The fusants’ characters of enzyne and physiologic and biochemical exist between 58-20-9 strains and 64 strains .4. Studies on pathogenicity and antigenicity:Using fusant strains to infect manually to health grass carps which were born in just that year .After observing two weeks,the mortality rates of grass carps were 95%,but the control group’s survival rates were 100%,the results showed that the fusant strains also had pathogenicity .Examined the disease and dead grass carps further,we found that the grass carps which were infected fusant strains had part symptom not only AKromonu.s punctta f. intestinalis 58-20-9 strains but also Myxococcs piscicola sp.nov G4 strains . Studied on antigenicity of fusant strains,the results showed that after the grass carps which were immunized by fusant strains infecting 58-20-9 strains and G4 strains,their survival rates run to 76.6% and 80.0%,ralative percents survival were 74.9% and 75.5%,valency of antibody upmost to 1:322.5 and 1:435.5 on the fourth week.In summary,under the present conditions,Aeromonas punctta f. intestinalis 58-20-9 strains and Myxococcs piscicola sp.nov G4 strains protoplasts could fusion and form steady fusants,the fusants also have two parents’ pathogenicity and antigenicity .更多还原