产河豚毒素微生物的研究
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摘要
1、从河豚鱼的卵巢中分离出35株野生菌,其中15株菌株遗传性状稳定。用小鼠生物检测法对15株菌株的发酵产物进行了检测,初筛选出11菌株进行复筛,结果有6株菌毒素含量均在400ng/ml,其中菌株B-333毒素含量最高为489.40ng/ml,因此选择菌株B-333作为出发菌株,并命名为ZY-23。
2、通过对ZY-23菌株的形态特征、培养特征、生理生化特征进行了研究与分析。结果表明,ZY-23菌株具有沙雷氏菌的典型的形态特征和生理生化特征。该菌株最适生长温度26℃,最适pH7.0,最适Nacl浓度2%。
3、通过提取ZY-23菌株的基因组DNA,利用16S rRNA基因通用引物,进行扩增、转化感受态大肠杆菌,将阳性克隆测序,获得ZY-23菌株的16S rRNA基因序列,构建进化树,证实ZY-23菌株与Serratia sp.Tp5有99%的同源性。
4、通过小鼠生物法测定发酵离心液和破壁后的菌体细胞上清液,结果表明发酵离心液中含有毒性成分,破壁后的细胞上清液不含毒性成分,ZY-23产生的毒素为胞外产物。采用荧光分光光度计对发酵离心液进行测定,结果在423nm处标准品与ZY-23提取物都有相同的发射峰,初步证实发酵液含有河豚毒素。
5、通过研究确定了高效液相检测法测定TTX的条件:流动相乙腈:水15:85,流速0.5ml/min,检测波长191nm,柱温23℃,进样量:20ul。HPLC检测结果显示,ZY-23菌株的发酵产物中提取得到的毒性成份保留时间与标准品基本一致。初步证实菌株ZY-23的发酵产物为河豚毒素或河豚毒素衍生物。
The experiment reserched bacteria producing tetrodotoxin and 15 strain was discovered and isolated from the ovary of fish. Mouse bioassay method was used in order to analyze the production of these strain. Strain B-333 having the most toxin content was studied and named ZY-23.
The characteristics of morphology,culture,physiochemist of ZY-23 strain has been reserched. The results indicated that strain ZY-23 had the typical characteristic of morphology,physiochemist of Serratia .The optimum culture conditions of strain ZY-23 were temperature 26℃,pH7.0,salinity 2%.
The 16S rRNA gene of ZY-23 was cloned, amplified, liked with pMD-18 T vector, then transferred competent Escherichia coli and the masc clones were sequenced. Thel6S rRNA gene sequence was submitted to Blastn to construct cladogram in order to understand the genetic background of this strain. The result showed that the 16S rDNA of ZY-23 was highly homology to that of Serratia sp.Tp5.
Mouse bioassay method was used in order to analyze fermentation broth and cell supernatant. The result showed fermentation broth produced toxin and cell supernatant did not produce toxin. The toxin was extracellular products. The result of fluorescence spectrophotometry proved strain ZY-23 would produce tetrodotoxin.
Determining the content of TTX by HPLC,the determination conditions were zorbax sb-aq chromatographic column, column temperature 23℃, detection wave length191nm, Acetonitrile: water 15:85,flow rate 0.5ml/min. The result of HPLC showed that the retion time of the production of strain ZY-23 is similar with standard substance. The result showed that the production is tetrodotoxin or its ramification.
2、通过对ZY-23菌株的形态特征、培养特征、生理生化特征进行了研究与分析。结果表明,ZY-23菌株具有沙雷氏菌的典型的形态特征和生理生化特征。该菌株最适生长温度26℃,最适pH7.0,最适Nacl浓度2%。
3、通过提取ZY-23菌株的基因组DNA,利用16S rRNA基因通用引物,进行扩增、转化感受态大肠杆菌,将阳性克隆测序,获得ZY-23菌株的16S rRNA基因序列,构建进化树,证实ZY-23菌株与Serratia sp.Tp5有99%的同源性。
4、通过小鼠生物法测定发酵离心液和破壁后的菌体细胞上清液,结果表明发酵离心液中含有毒性成分,破壁后的细胞上清液不含毒性成分,ZY-23产生的毒素为胞外产物。采用荧光分光光度计对发酵离心液进行测定,结果在423nm处标准品与ZY-23提取物都有相同的发射峰,初步证实发酵液含有河豚毒素。
5、通过研究确定了高效液相检测法测定TTX的条件:流动相乙腈:水15:85,流速0.5ml/min,检测波长191nm,柱温23℃,进样量:20ul。HPLC检测结果显示,ZY-23菌株的发酵产物中提取得到的毒性成份保留时间与标准品基本一致。初步证实菌株ZY-23的发酵产物为河豚毒素或河豚毒素衍生物。
The experiment reserched bacteria producing tetrodotoxin and 15 strain was discovered and isolated from the ovary of fish. Mouse bioassay method was used in order to analyze the production of these strain. Strain B-333 having the most toxin content was studied and named ZY-23.
The characteristics of morphology,culture,physiochemist of ZY-23 strain has been reserched. The results indicated that strain ZY-23 had the typical characteristic of morphology,physiochemist of Serratia .The optimum culture conditions of strain ZY-23 were temperature 26℃,pH7.0,salinity 2%.
The 16S rRNA gene of ZY-23 was cloned, amplified, liked with pMD-18 T vector, then transferred competent Escherichia coli and the masc clones were sequenced. Thel6S rRNA gene sequence was submitted to Blastn to construct cladogram in order to understand the genetic background of this strain. The result showed that the 16S rDNA of ZY-23 was highly homology to that of Serratia sp.Tp5.
Mouse bioassay method was used in order to analyze fermentation broth and cell supernatant. The result showed fermentation broth produced toxin and cell supernatant did not produce toxin. The toxin was extracellular products. The result of fluorescence spectrophotometry proved strain ZY-23 would produce tetrodotoxin.
Determining the content of TTX by HPLC,the determination conditions were zorbax sb-aq chromatographic column, column temperature 23℃, detection wave length191nm, Acetonitrile: water 15:85,flow rate 0.5ml/min. The result of HPLC showed that the retion time of the production of strain ZY-23 is similar with standard substance. The result showed that the production is tetrodotoxin or its ramification.
引文
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