草鱼病原菌三元融合子构建及生物学特性研究
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摘要
采用细胞融合技术首次构建草鱼烂鳃病、赤皮病和肠炎病的病原菌融合子。应用多种经典检测方法对融合子的形态、构造、生理生化特性、致病性、抗原性和DNA同源性展开深入研究，以揭示融合子生物学特性的遗传规律。建立多元细胞融合技术，为研制鱼类细胞融合疫苗，创造有多种价值的益生菌等优良菌种资源提供科学依据。
应用定性和定量药物敏感性试验与诱变相结合，筛选出荧光假单细胞56-12-10菌株的遗传标记为链霉素抗性，二元融合子AM-1菌株的遗传标记为环丙沙星抗性。在这两种细菌原生质体融合试验中，选择培养基中链霉素的应用浓度为300Iu/ml，环丙沙星应用浓度为100Iu/ml。
在酶解温度37℃下，选出最适酶解时间为60min，最适溶菌酶浓度为5mg/ml。以PEG为促融剂的条件下，荧光假单细胞56-12-10菌株与二元融合子AM-1菌株的原生质体进行了融合，56-12-10菌株的原生质体形成率为81.6％，原生质体再生率19.8％，AM-1菌株的原生质体形成率为79.2％，原生质体再生率为8.2％，初筛的原生质体融合率为6‰，点种100个初筛融合子菌落在双抗培养基上连续传代10次，得到1个稳定的融合子菌落，其稳定率为1％；同时制得三者的原生质体并悬浮，先取其中58-20-9菌株、G_4菌株的原生质体进行融合，再将56-12-10菌株的原生质体与此二元融合原生质体液混匀并进行第二次融合，经单抗选择培养基上的筛选率约为3.5‰，而经过连续传代10次后，共得到两个菌株，其中一个菌株为特殊变异株，另一为稳定三元融合子。两种方法均可获得相同的稳定三元融合子，后者更为快捷。
筛选的融合子菌株菌体短杆状，近于AM-1亲本株，平均长轴4.52μm，平均短轴0.75μm，平均体积1.33μm~3，革兰氏阴性，有鞭毛，运动，巨大菌落圆形，表面光滑、湿润，半透明，边缘整齐。对融合子菌株进行酶类性状及生理生化鉴定，其介于三原始亲本菌株之间。
用融合子菌株对当年健康草鱼种进行人工感染攻毒，说明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、烂鳃病及赤皮病三种疾病的部分症状。对融合菌株进行抗原性研究，结果表明，受融合子菌株免疫保护的草鱼种注射56-12-10菌株、AM-1菌株及三原始亲本，其免疫保护率分别达64.9％、68.0％、62.5％，抗体效价最高达1：256和1：512。对融合子及其亲本的DNA含量进行测定及琼脂糖凝胶电泳分析，融合子的DNA含量明显高于任一亲本。至于基因具体整合及表达情况，有待于进一步试验。
This experiment used Aeromonas punctta f. intestinalis 58-20-9 strains,Myxococcs piscicola sp. nov G4 strains and Pesudomonas fluorescens 56-12-10 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 the sonsanguinity of DNA.it is in order to coform the technology of building trinal fusant and grope for the basic theory and feasibility of preparing fusants bacterins of fish pathogeny,which also supply for creating bacteria with many values. The main results were presented as follows:
Using the qualitative and quantitative antimicrobial susceptibility experiment to screen the genetic markers of protoplast fusion between 56-12-10 strains and AM-1 strains,they are Smr(Streptomycin resistance) and Cmr(Ciprofloxacin resistance) respectively. In this protoplast fusion experiment between two bacteria,the applying density in selective culture medium of Streptomycin and Streptomycin are 300 Iu / ml and 100 Iu / ml.
When the temperature of enzymolysis was 37C,we singled out the best enzymolysis time to be 60 minute and the best lysozyme density to be 5 mg/ml . In the condition of PEG operating as agent of accelerating fusion,AM-1 strains and 56-12-10 strains conducted protoplast fusion,the protoplast formation rates of 56-12-10 strains are 81.6% and the protoplast regenerative rates of 56-12-10 strains were 19.8%,the protoplast formation rates of AM-1 strains were 79.2% and the protoplast regenerative rates of AM-1 strains were 8.2%.Arch-screening protoplast fusion rates were 6%o.Breeding respectively 100 fusants colony of arch-screening on duplexing repel select culture medium and generating continuously 10 times,derived a steady fusant conoly.The fusants steady rates were 1%; preparated the threes' protoplast at the same time,at first let 58-20-9 strains and G4 strains conduct protoplast fusion,then mixed it with 56-12-10 strains and finished the second fusion. Arch-screening protoplast fusion rates were 3.5%o.After
breeded on single repel select culture medium one by one,derived two strains.one is nomal,the other is aberrance.it can gain steady trinal fusion through the two methods,but the latter is more shortcut.
The screened fusants are short bacilliform,they are similar to AM-1 strains.their average macro-axis are 4.52um,average brachy-axis are 0.75um,average volume are 1.33um3.Gram female,flagellum,motion.The colour of giga-colony which was cultured 30 days is roundness,wetness and lranslucency,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 among three parental strains.
Using fusant strains to infect manually to health grass carps which were born in just that year.After observing two weeks,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 as bacterial enteritis disease, Bac terial gill rot disease and Red-skin disea.Studied on antigenicity of fusant strains,the results showed that after the grass carps which were immunized by fusanl strains infecting AM-lstrains,56-12-10 strains and three parental strains,their survival rates run to 64.9%, 68 % and 62.5%,valency of antibody upmost to 1:256 and 1:512 on the fourth week.Under the same experiment condition,we abstract the DNAof these five fusants and conduct with
analysis of agrose,the result revealed that the DNA content of binary fusants and trinal fusants are more than any parental strains.As to the gene's concrete integration and its express circumstance,awaiting the further trial.
应用定性和定量药物敏感性试验与诱变相结合，筛选出荧光假单细胞56-12-10菌株的遗传标记为链霉素抗性，二元融合子AM-1菌株的遗传标记为环丙沙星抗性。在这两种细菌原生质体融合试验中，选择培养基中链霉素的应用浓度为300Iu/ml，环丙沙星应用浓度为100Iu/ml。
在酶解温度37℃下，选出最适酶解时间为60min，最适溶菌酶浓度为5mg/ml。以PEG为促融剂的条件下，荧光假单细胞56-12-10菌株与二元融合子AM-1菌株的原生质体进行了融合，56-12-10菌株的原生质体形成率为81.6％，原生质体再生率19.8％，AM-1菌株的原生质体形成率为79.2％，原生质体再生率为8.2％，初筛的原生质体融合率为6‰，点种100个初筛融合子菌落在双抗培养基上连续传代10次，得到1个稳定的融合子菌落，其稳定率为1％；同时制得三者的原生质体并悬浮，先取其中58-20-9菌株、G_4菌株的原生质体进行融合，再将56-12-10菌株的原生质体与此二元融合原生质体液混匀并进行第二次融合，经单抗选择培养基上的筛选率约为3.5‰，而经过连续传代10次后，共得到两个菌株，其中一个菌株为特殊变异株，另一为稳定三元融合子。两种方法均可获得相同的稳定三元融合子，后者更为快捷。
筛选的融合子菌株菌体短杆状，近于AM-1亲本株，平均长轴4.52μm，平均短轴0.75μm，平均体积1.33μm~3，革兰氏阴性，有鞭毛，运动，巨大菌落圆形，表面光滑、湿润，半透明，边缘整齐。对融合子菌株进行酶类性状及生理生化鉴定，其介于三原始亲本菌株之间。
用融合子菌株对当年健康草鱼种进行人工感染攻毒，说明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、烂鳃病及赤皮病三种疾病的部分症状。对融合菌株进行抗原性研究，结果表明，受融合子菌株免疫保护的草鱼种注射56-12-10菌株、AM-1菌株及三原始亲本，其免疫保护率分别达64.9％、68.0％、62.5％，抗体效价最高达1：256和1：512。对融合子及其亲本的DNA含量进行测定及琼脂糖凝胶电泳分析，融合子的DNA含量明显高于任一亲本。至于基因具体整合及表达情况，有待于进一步试验。
This experiment used Aeromonas punctta f. intestinalis 58-20-9 strains,Myxococcs piscicola sp. nov G4 strains and Pesudomonas fluorescens 56-12-10 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 the sonsanguinity of DNA.it is in order to coform the technology of building trinal fusant and grope for the basic theory and feasibility of preparing fusants bacterins of fish pathogeny,which also supply for creating bacteria with many values. The main results were presented as follows:
Using the qualitative and quantitative antimicrobial susceptibility experiment to screen the genetic markers of protoplast fusion between 56-12-10 strains and AM-1 strains,they are Smr(Streptomycin resistance) and Cmr(Ciprofloxacin resistance) respectively. In this protoplast fusion experiment between two bacteria,the applying density in selective culture medium of Streptomycin and Streptomycin are 300 Iu / ml and 100 Iu / ml.
When the temperature of enzymolysis was 37C,we singled out the best enzymolysis time to be 60 minute and the best lysozyme density to be 5 mg/ml . In the condition of PEG operating as agent of accelerating fusion,AM-1 strains and 56-12-10 strains conducted protoplast fusion,the protoplast formation rates of 56-12-10 strains are 81.6% and the protoplast regenerative rates of 56-12-10 strains were 19.8%,the protoplast formation rates of AM-1 strains were 79.2% and the protoplast regenerative rates of AM-1 strains were 8.2%.Arch-screening protoplast fusion rates were 6%o.Breeding respectively 100 fusants colony of arch-screening on duplexing repel select culture medium and generating continuously 10 times,derived a steady fusant conoly.The fusants steady rates were 1%; preparated the threes' protoplast at the same time,at first let 58-20-9 strains and G4 strains conduct protoplast fusion,then mixed it with 56-12-10 strains and finished the second fusion. Arch-screening protoplast fusion rates were 3.5%o.After
breeded on single repel select culture medium one by one,derived two strains.one is nomal,the other is aberrance.it can gain steady trinal fusion through the two methods,but the latter is more shortcut.
The screened fusants are short bacilliform,they are similar to AM-1 strains.their average macro-axis are 4.52um,average brachy-axis are 0.75um,average volume are 1.33um3.Gram female,flagellum,motion.The colour of giga-colony which was cultured 30 days is roundness,wetness and lranslucency,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 among three parental strains.
Using fusant strains to infect manually to health grass carps which were born in just that year.After observing two weeks,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 as bacterial enteritis disease, Bac terial gill rot disease and Red-skin disea.Studied on antigenicity of fusant strains,the results showed that after the grass carps which were immunized by fusanl strains infecting AM-lstrains,56-12-10 strains and three parental strains,their survival rates run to 64.9%, 68 % and 62.5%,valency of antibody upmost to 1:256 and 1:512 on the fourth week.Under the same experiment condition,we abstract the DNAof these five fusants and conduct with
analysis of agrose,the result revealed that the DNA content of binary fusants and trinal fusants are more than any parental strains.As to the gene's concrete integration and its express circumstance,awaiting the further trial.
引文
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