CN114763544A - 一种铜绿假单胞菌高产蛋白菌株的生物诱变方法 - Google Patents
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Abstract
一种铜绿假单胞菌高产蛋白菌株的生物诱变方法,属于微生物遗传育种技术领域。为了解决物理诱变和化学诱变方法存在的问题,获得高产蛋白的铜绿假单胞菌,本发明选取铜绿假单胞菌典型菌落,经肉汤增菌培养基、血液琼脂培养基和营养琼脂培养基37℃多次传代培养,获得高产蛋白菌株。本发明提供的生物诱变方法获得的铜绿假单胞菌高产蛋白菌株制备的蛋白可用于制备防治铜绿假单胞菌感染的药物或疫苗。
Description
技术领域
本发明属于微生物遗传育种技术领域,具体涉及一种铜绿假单胞菌高产蛋白菌株的生物诱变方法。
背景技术
铜绿假单胞菌(Pseudomonas aeruginosa)又称绿脓杆菌,在自然界中广泛存在,是一种机会致病菌,在机体免疫系统功能低下或受损时引发感染。铜绿假单胞菌已成为医院内感染的重要病原菌之一,亦是战伤感染的常见致病菌,还可引起禽畜感染及败血性死亡等,对人体健康和养殖业造成了较大的危害和经济损失。
铜绿假单胞菌代谢产生的蛋白大多具有免疫原性,可刺激机体产生抗体,激发机体免疫系统功能。因此,对已筛选到的具有较高免疫效果的铜绿假单胞菌进行诱变,获得高产蛋白菌株,用于制备预防和治疗铜绿假单胞菌感染的药物或疫苗,是十分有意义的。
菌种诱变方法主要有物理诱变和化学诱变,常用的物理诱变方法有紫外照射、电离辐射、离子注入、激光辐射、航天育种等;化学方法常采用烷化剂、碱基类似物和无机化合物等进行诱变。对于物理诱变方法,紫外照射过的细胞易受光修复作用而造成突变失败,同时对菌液的适宜浓度要求较高;电离辐射有一定局限性,操作要求过高,且有一定的危险性;离子注入和激光辐射实验室条件难以达到,应用较少;航天育种方法技术含量高、成本高,一般难以实现。化学诱变方法采用的试剂,有的诱变效果一般,有的负诱变率很高,往往不易得到好的突变体。
发明内容
本发明为了解决物理诱变和化学诱变方法存在的缺陷,获得高产蛋白的铜绿假单胞菌,提供了一种铜绿假单胞菌高产蛋白菌株的生物诱变方法,所述生物诱变方法是将铜绿假单胞菌经肉汤增菌培养基、血液琼脂培养基和营养琼脂培养基多次传代培养进行诱变,获得高产蛋白菌株。
进一步地限定,所述铜绿假单胞菌为铜绿假单胞菌(Pseudomonas aeruginosa)PA-393、铜绿假单胞菌(Pseudomonas aeruginosa)PA-017和铜绿假单胞菌(Pseudomonasaeruginosa)PA-189中的任一一种,这三株菌均于2015年8月24日保藏在位于北京市朝阳区北辰西路1号院3号的中国微生物菌种保藏管理委员会普通微生物中心,其中,菌株PA-393的保藏编号为CGMCC NO.11267,菌株PA-017的保藏编号为CGMCC NO.11268,菌株PA-189的保藏编号为CGMCC NO.11269。
进一步地限定,所述的生物诱变方法的具体操作步骤如下:
步骤一、无菌操作条件下,取铜绿假单胞菌营养琼脂培养物,用接种环挑取一环接入肉汤增菌培养基中,37℃培养箱内静止培养24h,获得呈菌膜生长的肉汤增菌培养基的培养物;
步骤二、无菌操作条件下,将步骤一获得的培养物的菌膜接种于肉汤增菌培养基中,37℃培养箱内静止培养24h,按照本步骤所述方法传代培养2~3次;
步骤三、无菌操作条件下,将步骤二末次培养物的菌膜接种于肉汤增菌培养基中,漩涡振荡器振荡,充分混匀后,用接种环挑取一环以分区划线接种方式接种到血液琼脂平板培养基上,37℃培养箱内静止培养18h;
步骤四、无菌操作条件下,取步骤三获得的血液琼脂平板培养基培养物,用接种环挑取外形小、乳头状且表面有凹的菌落,以分区划线接种方式接种到营养琼脂斜面培养基上,37℃培养箱内静止培养18h;
步骤五、无菌操作条件下,取步骤四获得的营养琼脂斜面培养基培养物,用接种环挑取一环菌体,以分区划线接种方式接种到血液琼脂平板培养基上,37℃培养箱内静止培养48~72h;
步骤六、无菌操作条件下,取步骤五获得的血液琼脂平板培养基培养物,用接种环挑取外形大、扁平、表面呈山脉状且周边向周围扩展的菌落,接种到营养琼脂斜面培养基上,37℃培养箱内静止培养18h,即得高产蛋白菌株。
本发明还提供了所述生物诱变方法获得的铜绿假单胞菌高产蛋白菌株制备的蛋白在制备预防或/和治疗铜绿假单胞菌感染药物中的应用。
本发明还提供了所述生物诱变方法获得的铜绿假单胞菌高产蛋白菌株制备的蛋白在制备铜绿假单胞菌疫苗中的应用。
有益效果
本发明提供的生物诱变方法与物理诱变和化学诱变方法相比,操作简单,受过微生物学培训的技术人员均可在配备有超净工作台的实验室内完成;成本低,仅需要上述几种培养基即可完成;诱变效果好,本发明提供的诱变方法属于定向诱变,很容易获得高产蛋白的突变体;安全性高,不存在辐射和试剂毒害。
利用本发明提供的生物诱变方法获得的诱变菌株与出发菌株相比,蛋白产量明显提高,菌株PA-393经诱变后液体发酵蛋白含量由52.3%提升为86.1%,菌株PA-017经诱变后液体发酵蛋白含量由39.3%提升为84.4%,菌株PA-189经诱变后液体发酵蛋白含量由40.6%提升为82.6%。
附图说明
图1.菌株PA-393诱变前后对比图(血液琼脂平板培养基),其中,图A为原始出发菌株,图B为诱变后的菌株;
图2.菌株PA-017诱变前后对比图(血液琼脂平板培养基),其中,图A为原始出发菌株,图B为诱变后的菌株;
图3.菌株PA-189诱变前后对比图(血液琼脂平板培养基)其中,图A为原始出发菌株,图B为诱变后的菌株。
具体实施方式
以下结合具体实施例和附图,对本发明作进一步的详细说明。
下述实施例中所使用的实验方法,如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。下述实施例中涉及的培养基组成如下:
1、肉汤增菌培养基:
每1000mL培养基含:蛋白胨10.0g,牛肉膏粉3.0g,氯化钠5.0g,校正PH值7.4±0.2。
2、血液琼脂平板培养基:
每1000mL培养基含:酪蛋白胰酶消化物10.0g,心胰酶消化物3.0g,玉米淀粉1.0g,琼脂14.0g,肉胃酶消化物5.0g,酵母浸出粉5.0g,氯化钠5.0g,蒸馏水1000mL,无菌脱纤维羊血60mL,校正PH值至7.2~7.4。
3、营养琼脂斜面培养基:
每1000mL培养基含:蛋白胨10.0g,牛肉膏粉3.0g,氯化钠5.0g,琼脂15.0g,校正PH值7.3±0.2。
4、产蛋白培养基:谷氨酸钠20.0g,葡萄糖7.5g,MgSO4·7H2O 0.2g,Ca(NO3)20.01g,FeSO4·7H2O 0.00005g,KH2PO40.252 g,Na2HPO4·12H2O5.63g,溶于1000mL蒸馏水中,校正pH至7.2~7.4,115℃25min高压蒸汽灭菌。
实施例1
铜绿假单胞菌菌株PA-393、菌株PA-017和菌株PA-189鉴定
⑴菌落形态特征:
将菌株PA-393、菌株PA-017和菌株PA-189分别接种于血液琼脂平板培养基和营养琼脂平板培养基上,37℃培养18h,三种菌株两种培养基上的菌落特征均为光滑微隆起、边缘整齐、灰色、表面湿润、不透明、直径约1.5~2.5mm,三种菌株在血液琼脂平板培养基上均出现溶血环,均可使营养琼脂平板培养基变为黄绿色,镜检均为革兰氏阴性短杆状。
⑵生化鉴定:菌株PA-393、菌株PA-017和菌株PA-189氧化酶实验阳性,MR试验和VP试验阴性,甘露醇试验阴性,能分解葡萄糖,产酸不产气,能液化明胶和利用枸橼酸盐,不产硫化氢,因此鉴定证实菌株PA-393、菌株PA-017和菌株PA-189为铜绿假单胞菌,鉴定结果如表1所示。
表1.PA-393株、PA-017株、PA-189株主要生化特征
注:“+”:阳性;“-”:阴性。
(一)菌株准备:
分别将PA-393、PA-017、PA-189菌株冻干管无菌开启,接入肉汤增菌培养基中,37℃静置培养22h。用无菌加样枪吸取30uL,均匀涂布接种于血液琼脂平板培养基中,37℃培养24h。用接种环挑取平板上溶血圈大的典型单菌落,在血液琼脂平板上进行分区划线接种,37℃培养22h。选取平板上溶血圈大、典型的单菌落接种于营养琼脂斜面培养基上,37℃培养24h。镜检,确定为铜绿假单胞菌。
(二)生物诱变:
步骤一、无菌操作条件下,取铜绿假单胞菌菌株PA-393、PA-017和PA-189中的任意一株菌的营养琼脂培养物,用接种环挑取一环接入肉汤增菌培养基中,37℃培养箱内静止培养24h,获得呈菌膜生长的肉汤增菌培养基的培养物;
步骤二、无菌操作条件下,将步骤一获得的培养物的菌膜接种于肉汤增菌培养基中,37℃培养箱内静止培养24h,按照本步骤所述方法传代培养2~3次;
步骤三、无菌操作条件下,将步骤二末次培养物的菌膜接种于肉汤增菌培养基中,漩涡振荡器振荡,充分混匀后,用接种环挑取一环以分区划线接种方式接种到血液琼脂平板培养基上,37℃培养箱内静止培养18h;
步骤四、无菌操作条件下,取步骤三获得的血液琼脂平板培养基培养物,用接种环挑取外形小、乳头状且表面有凹的菌落,以分区划线接种方式接种到营养琼脂斜面培养基上,37℃培养箱内静止培养18h;
步骤五、无菌操作条件下,取步骤四获得的营养琼脂斜面培养基培养物,用接种环挑取一环菌体,以分区划线接种方式接种到血液琼脂平板培养基上,37℃培养箱内静止培养48~72h;
步骤六、无菌操作条件下,取步骤五获得的血液琼脂平板培养基培养物,用接种环挑取外形大、扁平、表面呈山脉状且周边向周围扩展的菌落,接种到营养琼脂斜面培养基上,37℃培养箱内静止培养18h,即得高产蛋白菌株。
将由菌株PA-393诱变获得的高产蛋白菌株命名为PA-393-YB,将由菌株PA-017诱变获得的高产蛋白菌株命名为PA-017-YB,将由菌株PA-189诱变获得的高产蛋白菌株命名为PA-189-YB。
(三)诱变菌株鉴定
⑴菌落形态特征:
将菌株PA-393-YB、菌株PA-017-YB和菌株PA-189-YB分别接种于血液琼脂平板培养基和营养琼脂平板培养基上,37℃条件下培养18h,三种菌株在两种培养基上的菌落特征均为光滑微隆起、边缘整齐、灰色、表面湿润、不透明、直径约1.5~2.5mm,三种菌株在血液琼脂平板培养基上均出现溶血环,均可使营养琼脂平板培养基变为黄绿色,镜检均为革兰氏阴性短杆状。
⑵生化鉴定:菌株PA-393-YB、菌株PA-017-YB、菌株PA-189-YB氧化酶实验阳性,MR试验和VP试验阴性,甘露醇试验阴性,能分解葡萄糖,产酸不产气,能液化明胶和利用枸橼酸盐,不产硫化氢,因此鉴定证实PA-393-YB、PA-017-YB、PA-189-YB株为铜绿假单胞菌,如表2所示。
表2.PA-393-YB株、PA-017-YB株、PA-189-YB株主要生化特征
注:“+”:阳性;“-”:阴性。
菌株PA-393、菌株PA-017和菌株PA-189诱变前后对比图(血液琼脂平板培养基)分别如图1、图2和图3所示。
(四)原始出发菌株和诱变菌株液体发酵蛋白产量测定
1.利用产蛋白培养基液体发酵制备蛋白:
⑴取PA-393、PA-017、PA-189菌株和PA-393-YB、PA-017-YB、PA-189-YB菌株,分别接种于营养琼脂斜面培养基上,37℃培养18h,然后用产蛋白培养基将菌体洗下,将菌液标定为3.0×108CFU/mL(细菌浊度计TA-2XJ,北京天安联合科技有限公司),接入装有1000mL产蛋白培养基的三角瓶中,接种量为10%(v/v),旋转摇床(175转/min)37℃振荡培养24h。
⑵取步骤⑴获得的培养物,于4℃,10000rpm/min的条件下离心30min,分别收集沉淀和上清液,沉淀烘干,称重后加入上清液中混匀,校正pH至8.5,37℃甲苯重层自溶48h。暗视野镜检自溶完全后,将自溶液校正pH至7.2,G5砂芯漏斗抽滤,得到棕色透明液体,用50%ZnCl2沉淀蛋白(3.2mL50%ZnCl2/100mL抽滤液),4℃,4000rpm/min离心20min,弃掉上清,沉淀用适量20%Na2HPO4·7H2O溶液溶解,37℃静置2h,4℃冰箱过夜保存。
⑶取步骤⑵获得的溶液,4℃,4000rpm离心20min,收集上清液,校正pH至7.0~7.2,装入透析袋(截留分子量:8000~14000)流水透析48h后,电扇吹风浓缩至体积50~80mL。
⑷取步骤⑶获得的浓缩液,校正pH至7.0~7.2,冷丙酮沉淀(浓缩液体积:冷丙酮体积=1:2),4℃,4000rpm/min离心20min,弃掉上清,沉淀加少量ddH2O溶解,电扇吹风浓缩,真空冷冻干燥,获得蛋白。
2.蛋白含量测定:
蛋白含量测定方法选取考马斯亮蓝法(Bradford法),具体操作步骤如下:
⑴试剂配制:
①标准蛋白溶液:精确称取酪蛋白25mg,加dd H2O溶解并定容至100mL,吸取上述溶液40mL,用dd H2O稀释至100mL,即为100ug/mL的标准蛋白溶液。
②染料溶液:称取0.1g考马斯亮蓝G-250溶于50mL 95%的酒精,再加入85%的浓磷酸100mL,用dd H2O稀释至1000mL,混匀备用。
⑵操作步骤:
①按表3分别向各支试管内加入各种试剂,充分混匀,5min后在595nm波长处以0号管调零,测定各管吸光度值(A)。以吸光度值为纵坐标,蛋白质浓度为横坐标绘制标准曲线。
表3.标准曲线绘制
②样品测定:分别取1mL各样品溶液(约含25~250ug蛋白质),加入染料溶液5mL混匀,5min后测定其595nm吸光度值,对照标准曲线求得蛋白质浓度。测定结果如表4所示。
表4.原始出发菌株和诱变菌株液体发酵蛋白产量测定
结果表明,铜绿假单胞菌原始出发菌株经过生物诱变后,产蛋白能力大幅提高,与原始出发菌株相比,差异显著。
虽然本发明已以较佳的实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可以做各种改动和修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (5)
1.一种铜绿假单胞菌高产蛋白菌株的生物诱变方法,其特征在于,所述生物诱变方法是将铜绿假单胞菌经肉汤增菌培养基、血液琼脂培养基和营养琼脂培养基多次传代培养进行诱变,获得高产蛋白菌株。
2.根据权利要求1所述的生物诱变方法,其特征在于,所述铜绿假单胞菌为铜绿假单胞菌(Pseudomonas aeruginosa)PA-393、铜绿假单胞菌(Pseudomonas aeruginosa)PA-017和铜绿假单胞菌(Pseudomonas aeruginosa)PA-189中的任一一种,其中,菌株PA-393的保藏编号为CGMCC NO.11267,菌株PA-017的保藏编号为CGMCC NO.11268,菌株PA-189的保藏编号为CGMCC NO.11269。
3.根据权利要求2所述的生物诱变方法,其特征在于,具体操作步骤如下:
步骤一、无菌操作条件下,取铜绿假单胞菌营养琼脂培养物,用接种环挑取一环接入肉汤增菌培养基中,37℃培养箱内静止培养24h,获得呈菌膜生长的肉汤增菌培养基的培养物;
步骤二、无菌操作条件下,将步骤一获得的培养物的菌膜接种于肉汤增菌培养基中,37℃培养箱内静止培养24h,按照本步骤所述方法传代培养2~3次;
步骤三、无菌操作条件下,将步骤二末次培养物的菌膜接种于肉汤增菌培养基中,漩涡振荡器振荡,充分混匀后,用接种环挑取一环以分区划线接种方式接种到血液琼脂平板培养基上,37℃培养箱内静止培养18h;
步骤四、无菌操作条件下,取步骤三获得的血液琼脂平板培养基培养物,用接种环挑取外形小、乳头状且表面有凹的菌落,以分区划线接种方式接种到营养琼脂斜面培养基上,37℃培养箱内静止培养18h;
步骤五、无菌操作条件下,取步骤四获得的营养琼脂斜面培养基培养物,用接种环挑取一环菌体,以分区划线接种方式接种到血液琼脂平板培养基上,37℃培养箱内静止培养48~72h;
步骤六、无菌操作条件下,取步骤五获得的血液琼脂平板培养基培养物,用接种环挑取外形大、扁平、表面呈山脉状且周边向周围扩展的菌落,接种到营养琼脂斜面培养基上,37℃培养箱内静止培养18h,即得高产蛋白菌株。
4.权利要求1所述生物诱变方法获得的铜绿假单胞菌高产蛋白菌株制备的蛋白在制备预防或/和治疗铜绿假单胞菌感染药物中的应用。
5.权利要求1所述生物诱变方法获得的铜绿假单胞菌高产蛋白菌株制备的蛋白在制备铜绿假单胞菌疫苗中的应用。
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