CN114606203B - 一种利用低温等离子体诱导溶源性噬菌体方法 - Google Patents
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Abstract
本发明公开了一种利用低温等离子诱导激活溶源性噬菌体的方法。本发明的方法包括:在含有溶原性噬菌体的细菌培养液中加入表面活性剂和诱导缓冲液,然后置于等离子体放电装置中,调节放电电压及放电功率,进行放电处理;放电处理结束后,移入孵育液中孵育培养;孵育培养结束后,依次经过破胞处理、离心和过滤,获得含有子代噬菌体的滤液。本发明采用低温等离子体技术,通过放电使细菌DNA脱稳,高效快速地诱导整合在细菌基因组中的前噬菌体。
Description
技术领域
本发明涉及一种利用低温等离子体诱导溶源性噬菌体方法,具体涉及利用高压脉冲等离子体放电技术高效快速的诱导溶源性噬菌体,获得大量子代噬菌体的方法。属于环保技术领域。
背景技术
噬菌体是感染细菌的特异性病毒,同时也是地球上数量最多、种类最多的生物实体,据估计生物圈中噬菌体的数量高达1031,由噬菌体侵染裂解导致的细菌死亡率可占总体死亡率的20-40%。因此,噬菌体对生物地球化学循环有相当大的影响。噬菌体的生命史包括裂解周期和溶源周期。进入裂解周期的噬菌体,在侵染宿主后,会利用宿主物质大量合成组装子代噬菌体,并裂解宿主细胞释放子代病毒。一些溶源性噬菌体在侵染宿主后会将自己的DNA整合到宿主菌的基因组中,形成前噬菌体(prophage)进入溶源周期,而携带前噬菌体基因的宿主细胞则被称为溶源菌。溶源周期的前噬菌体会保持休眠状态与宿主DNA一起进行复制,但在一定的外界因素影响下,整合的噬菌体DNA又可从宿主细菌的基因上脱离(这个过程称为激活),重新进入裂解周期。
目前已有大量关于利用裂解噬菌体进行噬菌体治疗的研究和应用,例如耐药细菌的控制和灭活,以及生物膜的清除。但由于裂解噬菌体的宿主特异性,相关研究和应用的特点一般是需要从外源特定的环境介质中筛选特异噬菌体,再通过富集和浓缩后投加到目标菌群体系中。然而特异性噬菌体的筛选通常比较繁琐,同时细菌会发展形成噬菌体抗性,导致裂解噬菌体治疗策略失效。而整合到细菌基因组上的前噬菌体,本身就存在于宿主细菌体内,并且其在某些外界诱导剂的刺激作用下,可能会发生生命周期转换。即:环境刺激会对宿主细菌的DNA产生损伤,触发细菌的SOS反应,诱导前噬菌体激活,导致前噬菌体重新进入裂解周期。这可作为一种潜在的“由内而外”消杀细菌的新型噬菌体治疗策略。目前关于前噬菌体的诱导激活,普遍使用的是丝裂霉素C。但丝裂霉素C除了具有致癌、致畸等风险外,对前噬菌体的诱导也存在耗时长,具有偏好性等缺点。其他化学试剂的添加,如重金属离子、抗生素、纳米量子点也可诱导溶源性噬菌体激活,但是这些化学试剂的添加会导致化学试剂残留,对环境造成二次污染。
低温等离子体技术作为一种新型高级氧化技术,集臭氧氧化、微波辐射、紫外辐射及热解等效应于一体,无选择性、无需添加化学药剂且可以在常温常压下实现操作。目前,低温等离子体技术已成功地应用于环境污染控制、纳米材料合成、生物医学和食品加工等领域。在低温等离子体处理过程中,大量的活性氧(ROS)包括羟基自由基(·OH)、原子氧(O)、过氧化氢(H2O2)、单线态氧(1O2)和臭氧(O3)被产生。这些活性氧可以通过多种方式破坏细菌的结构和功能,包括EPS氧化、细胞泄漏、DNA损伤、脂质过氧化、蛋白质变性和干扰细胞代谢等。因此可以推断出,等离子体造成的细菌DNA损伤可促发细菌的SOS反应,导致溶源性噬菌体的诱导。目前还没有关于利用低温等离子体诱导溶源性噬菌体的相关研究和报道。
发明内容
本发明解决的技术问题是:目前诱导溶源性噬菌体的方法存在步骤繁琐、耗时长、诱导效率低及二次污染风险等问题。
为了解决上述技术问题,本发明提供了一种利用低温等离子诱导溶源性噬菌体的方法,包括:在含有溶原性噬菌体的细菌培养液中加入表面活性剂和诱导缓冲液,然后置于等离子体放电装置中,调节放电电压及放电功率,进行放电处理;放电处理结束后,移入孵育液中孵育培养;孵育培养结束后,依次经过破胞处理、离心和过滤,获得含有子代噬菌体的滤液。
优选地,所述的细菌培养液中含有溶原性噬菌体的细菌浓度为102-108CFU/mL。
优选地,所述表面活性剂的加入量为0.1-40g/L,具体包括吐温0.1-40g/L、焦磷酸钠0.1-40g/L和柠檬酸钾0.1-40g/L。
优选地,所述诱导缓冲液的加入量为0.1-100mol/L的Na2HPO4或KH2PO4,以及0.1-100mmol/L CaCl2和0.1-50mmol/L MgCl2。
优选地,所述放电处理的放电电压为15-150V,放电功率为10W-80W,放电时间为1-200min。
优选地,所述的孵育液为0.1-10倍浓缩的培养基。
更优选地,所述的孵育液包括蛋白胨5-15g/L、酵母提取物2-6g/L、氯化钠5-15g/L、葡萄糖2-6g/L和苹果酸钙3-9g/L。
优选地,所述孵育培养的温度为30~40℃,时间为0.5-12h。
优选地,所述破胞处理的方法为:加入以体积比例计算的氯仿0.1-10%作为破胞剂,静置5-30min;所述离心的条件为:200-15000rpm离心3-30min;所述过滤为:采用0.2-0.45μm的滤膜过滤。
本发明的原理如下:
本发明利用低温等离子体放电过程中产生脉冲电压进行放电,放电过程中产生大量的活性物质,如羟基自由基(·OH)、氧自由基(·O)、氢自由基(·H)、臭氧(O3)、过氧化氢(H2O2)等。这些活性物质会对生物膜内的细菌DNA造成氧化损伤,触发细菌的SOS反应,从而激活前噬菌体进入裂解状态,大量组装合成子代噬菌体,裂解宿主细菌,释放子代噬菌体。此外,放电过程还会产生紫外光、冲击波等,集合了化学氧化、光化学和电化学,进一步加强作用效果。
本发明与现有技术相比,具有如下有益效果:
(1)本发明实验方法简单,操作简便,制作及运行费用较低,溶源性噬菌体诱导效果好;
(2)本发明在产生等离子体时,采用脉冲放电的方式,低温下即可产生等离子体,温度条件易满足。同时所需功率小,系统能耗低;
(3)本发明将低温等离子体技术与噬菌体技术相结合,利用低温等离子体产生的多种自由基高效、快速激活溶源性噬菌体,有效避免了常规诱导方法耗时长、诱导效率低的问题,并且无二次污染产生。
具体实施方式
为使本发明更简单易懂,兹以优选实施例,作详细说明如下。
实施例1
一种利用低温等离子体诱导溶源性噬菌体的方法,具体步骤为:
(1)选用含有前噬菌体lambda(λ)的大肠杆菌(E.coli(λ+)),采用BPY琼脂斜面培养基(含有牛肉提取物、蛋白胨、酵母提取物、葡萄糖、氯化钠、琼脂、蒸馏水、pH=7.0)在30℃下活化培养24h后,取15mL(约105CFU/mL)细菌悬浮液置于石英大圆盘反应器中,进行等离子体放电反应。
(2)调节等离子体放电电压和电流,放电的频率为8.8kHz,放电电压为50V;放电功率为40W,一次放电的时间为20min。
(3)取相同浓度的不含前噬菌体lambda的大肠杆菌液(E.coli(λ-))置于大圆盘中,放电频率、放电电压、放电电流均相同,作为实验对照。
(4)分别取相同的(E.coli(λ+))和(E.coli(λ-)),不进行放电处理作为空白对照。
(5)在所有组别中加入表面活性剂为2g/L的柠檬酸钾和焦磷酸钠,诱导缓冲液为0.1mol/L的Na2HPO4和KH2PO4,以及5mmol/L的CaCl2和MgCl2。
(6)放电结束后,在实验组、实验对照组、空白对照组中加入等体积2倍浓缩的培养基(包括蛋白胨10g/L、酵母提取物4g/L、氯化钠10g/L、葡萄糖4g/L和苹果酸钙6g/L),35℃下进行孵育5h。
(7)孵育结束后,菌液加入体积比为1.5%氯仿进行破胞,在8000r/min的参数下离心10min,取上清液过0.22μm的滤膜,所得滤液即为噬菌体裂解液。
(8)通过双层琼脂平板法测定噬菌体诱导数量,测得放电组的噬菌体浓度最大值为6.8×105PFU/mL,空白组检测不到噬菌体颗粒。。
实施例2
一种利用低温等离子体诱导溶源性噬菌体的方法,具体步骤为:
(1)选用含有前噬菌体lambda(λ)的大肠杆菌(E.coli(λ+)),采用BPY琼脂斜面培养基(含有牛肉提取物、蛋白胨、酵母提取物、葡萄糖、氯化钠、琼脂、蒸馏水、pH=7.0)在30℃下活化培养24h后,取15mL(106CFU/mL)细菌悬浮液置于石英大圆盘反应器中,进行等离子体放电反应。
(2)调节等离子体放电电压和电流,放电的频率为8.8kHz,放电电压为60V;放电功率为30W,一次放电的时间为30min。
(3)取相同浓度的不含前噬菌体lambda的大肠杆菌液(E.coli(λ-))置于大圆盘中,放电频率、放电电压、放电电流均相同,作为实验对照。
(4)分别取相同的(E.coli(λ+))和(E.coli(λ-)),不进行放电处理作为空白对照。
(5)在所有组别中加入表面活性剂为5g/L的柠檬酸钾和焦磷酸钠,诱导缓冲液为0.5mol/L的Na2HPO4和KH2PO4,以及2mmol/L的CaCl2和MgCl2。
(6)放电结束后,在实验组、实验对照组、空白对照组中加入等体积2倍浓缩的培养基(包括蛋白胨10g/L、酵母提取物4g/L、氯化钠10g/L、葡萄糖4g/L和苹果酸钙6g/L),35℃下进行孵育5h。
(7)孵育结束后,菌液加入体积比为1.5%氯仿进行破胞,在8000r/min的参数下离心15min,取上清液过0.22μm的滤膜,所得滤液即为噬菌体裂解液。
(8)通过双层琼脂平板法测定噬菌体诱导数量,测得放电组的噬菌体浓度最大值为5.2×105PFU/mL,空白组检测不到噬菌体颗粒。
实施例3
一种利用低温等离子体诱导溶源性噬菌体的方法,具体步骤为:
(1)选用含有前噬菌体lambda(λ)的大肠杆菌(E.coli(λ+)),相同浓度的不含前噬菌体lambda(λ)的大肠杆菌液(E.coli(λ-))1:1混合均匀后,取15mL(105CFU/mL)细菌悬浮液置于石英大圆盘反应器中,进行等离子体放电反应。
(2)调节等离子体放电电压和电流,放电的频率为8.8kHz,放电电压为50V;放电功率为40W,一次放电的时间为20min。
(3)取相同体积和相同浓度的E.coli(λ+)和E.coli(λ-)的混合液,不进行放电处理作为空白对照。
(4)在所有组别中加入表面活性剂为5g/L的柠檬酸钾和焦磷酸钠,诱导缓冲液为0.5mol/L的Na2HPO4和KH2PO4,以及10mmol/L的CaCl2和MgCl2。
(5)放电结束后,在实验组、空白对照组中加入等体积3倍浓缩的培养基(包括蛋白胨15g/L、酵母提取物6g/L、氯化钠15g/L、葡萄糖6g/L和苹果酸钙9g/L),35℃下进行孵育5h。
(6)孵育结束后,菌液加入体积比为2%氯仿进行破胞,在1000r/min的参数下离心15min,取上清液过0.22μm的滤膜,所得滤液即为噬菌体裂解液。
(7)通过双层琼脂平板法测定噬菌体诱导数量,测得放电组的噬菌体浓度最大值为1.02×106PFU/mL,空白组检测不到噬菌体颗粒。
Claims (4)
1.一种利用低温等离子诱导溶源性噬菌体的方法,其特征在于,包括:在含有溶原性噬菌体的细菌培养液中加入表面活性剂和诱导缓冲液,然后置于等离子体放电装置中,调节放电电压及放电功率,进行放电处理;放电处理结束后,移入孵育液中孵育培养;孵育培养结束后,依次经过破胞处理、离心和过滤,获得含有子代噬菌体的滤液;
所述诱导缓冲液的加入量为0.1-100 mol/L 的 Na2HPO4或KH2PO4,以及0.1-100 mmol/LCaCl2和0.1-50 mmol/L MgCl2;
所述放电处理的放电电压为50-60V,放电功率为30W-40W,放电时间为20-30min;
所述的孵育液包括蛋白胨 5-15 g/L、酵母提取物 2-6 g/L、氯化钠5-15g/L、葡萄糖2-6g/L和苹果酸钙3-9g/L。
2. 如权利要求1所述的利用低温等离子诱导溶源性噬菌体的方法,其特征在于,所述的细菌培养液中含有溶原性噬菌体的细菌浓度为102-108 CFU /mL。
3. 如权利要求1所述的利用低温等离子诱导溶源性噬菌体的方法,其特征在于,所述孵育培养的温度为30~40℃,时间为0.5-12 h。
4. 如权利要求1所述的利用低温等离子诱导溶源性噬菌体的方法,其特征在于,所述破胞处理的方法为:加入以体积比例计算的氯仿0.1-10%作为破胞剂,静置5-30 min;所述离心的条件为:200-15000rpm离心3-30min;所述过滤为:采用0.2-0.45μm的滤膜过滤。
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