爱媛类芽孢杆菌南京分离株的鉴定及其体内外的抑菌活性
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摘要
类芽孢杆菌能分泌多种胞外抗菌物质,对多种致病菌有抑制活性,能显著提高动物的抗病性,随着科学研究的深入,类芽孢杆菌抗菌特性的应用将会得到重视。
采用双层琼脂平板法,从30份水样和2份猪粪样品中分离出12株具有抑菌活性的细菌,通过抗菌谱,筛选出一株分离自健康猪粪的类芽孢杆菌,名为NJ2008。通过菌落、菌体形态观察、生理生化反应及16S rDNA序列分析,同时结合用微生物自动鉴定系统验证,鉴定这株菌属于爱媛类芽孢杆菌属(Paenibacillus ehimensis)。此菌细胞杆状,革兰氏染色成阴性,菌体中具有中生的椭园形芽孢,芽孢囊膨大；在普通琼脂平板上菌落乳白色,半透明,边缘光滑,呈白蜡状；在肉汤培养基中出现混浊且形成薄菌膜,最适生长温度为37℃,最佳生长酸碱条件pH6。
用双层琼脂平板法检测爱媛类芽孢杆菌,抗菌谱表明其对肠道致病菌有明显体外抑菌活性,能够抑制大肠杆菌和鼠伤寒沙门菌的生长,也能够抑制水产动物病害的不动杆菌,迟缓爱德华菌和嗜水气单胞菌.除此之外,对革兰氏阳性细菌,猪链球菌2型、猪链球菌9型和马链球菌兽疫亚种也有抑制作用。
鉴于爱媛类芽孢杆菌NJ2008体外能够抑制鼠伤寒沙门菌,以人工感染的小鼠鼠伤寒沙门菌病为模型,分析爱媛类芽孢杆菌NJ2008对小鼠的急性毒性,并评估其预防和治疗小鼠感染沙门菌的效果.爱媛类芽孢杆菌NJ2008分别以107cfu／mL、108cfu／mL、109cfu／mL给小鼠灌胃,试验组和空白对照组小鼠均健康,证明NJ2008对小鼠无毒性。以109cfu／mL爱媛类芽孢杆菌NJ2008预防人工感染鼠伤寒沙门菌小鼠,小鼠存活率为8／10(80%),空白对照组存活率仅为4／10(40%),试验组和空白对照组的差异显著,显示爱媛类芽孢杆菌NJ2008对小鼠感染鼠伤寒沙门菌有良好的预防效果。分别用106cfu／mL,107cfu／mL,108cfu／mL和109cfu／mL爱媛类芽孢杆菌NJ2008治疗人工感染鼠伤寒沙门菌小鼠,小鼠存活率分别为4／10(40%),4／10(40%),4／10(40%)和6／10(60%),空白对照组的存活率为4／10(40%),试验组和空白对照组无差异,治疗效果不佳。以上结果表明,爱媛类芽孢杆菌NJ2008能够抑制致病菌,可以作为一种微生物制剂用于预防动物的细菌疾病。
通过PCR扩增,从质粒pFPV25.1中获取绿色荧光蛋白基因片段,克隆入pP43NMK的多克隆区,构建成pP43NMK-GFP,通过限制性核酸内切酶KpnⅠ与HindIII对所建质粒进行分析,并转化大肠杆菌,观察绿色荧光蛋白表达,证实成功构建了pP43NMK-GFP.该质粒可用于下一步转化爱媛类芽孢杆菌NJ2008,在动物体内定位爱媛类芽孢杆菌NJ2008。
A strain (NJ2008) was isolated from the intestinal tract of healthy swine. Through study on its morphological, physiological and biochemical characteristics as well as 16S rDNA sequence homology comparison the strain NJ2008 was identified as Paenibacillus ehimensis. Antagonistic activity of NJ2008 was tested against Escherichia coli(7), Edwardsiella tarda(3), Acinetobacter baumannii(1), Salmonella typhimurium(1), Aeromonas hydrophila(1), Streptococcus equi subsp.zooepidemicus(1), Streptococcus suis serotype 2(1) and Streptococcus suis serotype 9(1). The Results showed that NJ2008 was found to have strong antagonistic activity against Escherichia coli. The best optimal condition for growth is pH 6.0 and 37℃.
In vitro, Paenibacillus ehimensis NJ2008 could lyse Salmonella typhimurium. The safty test was carried out through drenching ICR mice with 107 cfu/mL,108 cfu/mL and 109 cell/mL Paenibacillus ehimensis NJ2008. All the mice of test group and control group were healthy. This Result showed Paenibacillus ehimensis NJ2008 was safty to mice. The mice in test group first were protected by the 109 cell/mL Paenibacillus ehimensis NJ2008 then infected by the Salmonella typhimurium were 80%survival. Apparently, it is higher than the rate of control group. The mice infected with Salmonella typhimurium artificially were cured by 106 cfu/mL,107 cfu/mL,108 cfu/mL and 109 cell/mL Paenibacillus ehimensis NJ2008. The survival rates were 40%,40%,40%and 60%, respectively. There were not different from the survival rate of control group. However, the Result showed that Paenibacillus ehimensis NJ2008 was effective to prevent Salmonellosis.
The vector pP43NMK lantern was digested with Kpn I and HindⅢ, the encoding region of green fluorescent protein tag was inserted into the pP43NMK. The recombinant was confirmed by restriction enzyme map and transformed into TOP 10 to ensure the expression of green fluorescent protein. The Results of the restriction enzyme digestion confirmed the recombinant vector was constructed successfully. Furthermore it can express green fluorescent protein in TOP 10. The vector pP43NMK-GFP was constructed successfully. It is convenient to transfer it into the isolate strain Paenibacillus ehimensis NJ2008. Paenibacillus ehimensis NJ2008 with the plasmid pP43NMK-GFP can show its trace in vivo. It also laid foundation for making the vector as a reporter gene plasmid in vivo.
采用双层琼脂平板法,从30份水样和2份猪粪样品中分离出12株具有抑菌活性的细菌,通过抗菌谱,筛选出一株分离自健康猪粪的类芽孢杆菌,名为NJ2008。通过菌落、菌体形态观察、生理生化反应及16S rDNA序列分析,同时结合用微生物自动鉴定系统验证,鉴定这株菌属于爱媛类芽孢杆菌属(Paenibacillus ehimensis)。此菌细胞杆状,革兰氏染色成阴性,菌体中具有中生的椭园形芽孢,芽孢囊膨大；在普通琼脂平板上菌落乳白色,半透明,边缘光滑,呈白蜡状；在肉汤培养基中出现混浊且形成薄菌膜,最适生长温度为37℃,最佳生长酸碱条件pH6。
用双层琼脂平板法检测爱媛类芽孢杆菌,抗菌谱表明其对肠道致病菌有明显体外抑菌活性,能够抑制大肠杆菌和鼠伤寒沙门菌的生长,也能够抑制水产动物病害的不动杆菌,迟缓爱德华菌和嗜水气单胞菌.除此之外,对革兰氏阳性细菌,猪链球菌2型、猪链球菌9型和马链球菌兽疫亚种也有抑制作用。
鉴于爱媛类芽孢杆菌NJ2008体外能够抑制鼠伤寒沙门菌,以人工感染的小鼠鼠伤寒沙门菌病为模型,分析爱媛类芽孢杆菌NJ2008对小鼠的急性毒性,并评估其预防和治疗小鼠感染沙门菌的效果.爱媛类芽孢杆菌NJ2008分别以107cfu／mL、108cfu／mL、109cfu／mL给小鼠灌胃,试验组和空白对照组小鼠均健康,证明NJ2008对小鼠无毒性。以109cfu／mL爱媛类芽孢杆菌NJ2008预防人工感染鼠伤寒沙门菌小鼠,小鼠存活率为8／10(80%),空白对照组存活率仅为4／10(40%),试验组和空白对照组的差异显著,显示爱媛类芽孢杆菌NJ2008对小鼠感染鼠伤寒沙门菌有良好的预防效果。分别用106cfu／mL,107cfu／mL,108cfu／mL和109cfu／mL爱媛类芽孢杆菌NJ2008治疗人工感染鼠伤寒沙门菌小鼠,小鼠存活率分别为4／10(40%),4／10(40%),4／10(40%)和6／10(60%),空白对照组的存活率为4／10(40%),试验组和空白对照组无差异,治疗效果不佳。以上结果表明,爱媛类芽孢杆菌NJ2008能够抑制致病菌,可以作为一种微生物制剂用于预防动物的细菌疾病。
通过PCR扩增,从质粒pFPV25.1中获取绿色荧光蛋白基因片段,克隆入pP43NMK的多克隆区,构建成pP43NMK-GFP,通过限制性核酸内切酶KpnⅠ与HindIII对所建质粒进行分析,并转化大肠杆菌,观察绿色荧光蛋白表达,证实成功构建了pP43NMK-GFP.该质粒可用于下一步转化爱媛类芽孢杆菌NJ2008,在动物体内定位爱媛类芽孢杆菌NJ2008。
A strain (NJ2008) was isolated from the intestinal tract of healthy swine. Through study on its morphological, physiological and biochemical characteristics as well as 16S rDNA sequence homology comparison the strain NJ2008 was identified as Paenibacillus ehimensis. Antagonistic activity of NJ2008 was tested against Escherichia coli(7), Edwardsiella tarda(3), Acinetobacter baumannii(1), Salmonella typhimurium(1), Aeromonas hydrophila(1), Streptococcus equi subsp.zooepidemicus(1), Streptococcus suis serotype 2(1) and Streptococcus suis serotype 9(1). The Results showed that NJ2008 was found to have strong antagonistic activity against Escherichia coli. The best optimal condition for growth is pH 6.0 and 37℃.
In vitro, Paenibacillus ehimensis NJ2008 could lyse Salmonella typhimurium. The safty test was carried out through drenching ICR mice with 107 cfu/mL,108 cfu/mL and 109 cell/mL Paenibacillus ehimensis NJ2008. All the mice of test group and control group were healthy. This Result showed Paenibacillus ehimensis NJ2008 was safty to mice. The mice in test group first were protected by the 109 cell/mL Paenibacillus ehimensis NJ2008 then infected by the Salmonella typhimurium were 80%survival. Apparently, it is higher than the rate of control group. The mice infected with Salmonella typhimurium artificially were cured by 106 cfu/mL,107 cfu/mL,108 cfu/mL and 109 cell/mL Paenibacillus ehimensis NJ2008. The survival rates were 40%,40%,40%and 60%, respectively. There were not different from the survival rate of control group. However, the Result showed that Paenibacillus ehimensis NJ2008 was effective to prevent Salmonellosis.
The vector pP43NMK lantern was digested with Kpn I and HindⅢ, the encoding region of green fluorescent protein tag was inserted into the pP43NMK. The recombinant was confirmed by restriction enzyme map and transformed into TOP 10 to ensure the expression of green fluorescent protein. The Results of the restriction enzyme digestion confirmed the recombinant vector was constructed successfully. Furthermore it can express green fluorescent protein in TOP 10. The vector pP43NMK-GFP was constructed successfully. It is convenient to transfer it into the isolate strain Paenibacillus ehimensis NJ2008. Paenibacillus ehimensis NJ2008 with the plasmid pP43NMK-GFP can show its trace in vivo. It also laid foundation for making the vector as a reporter gene plasmid in vivo.
引文
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