CN114934044A - 一种重组大肠杆菌在养护铅酸蓄电池中的应用 - Google Patents
一种重组大肠杆菌在养护铅酸蓄电池中的应用 Download PDFInfo
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Abstract
本发明属于基因工程应用领域,涉及一种编码ST肽的基因、含有该基因的重组载体pET30α‑X1以及包含上述重组载体的大肠杆菌和其在延缓铅酸蓄电池老化以及修复老化铅酸蓄电池领域的应用。本发明通过基因工程技术利用重组大肠杆菌表达具有特殊结构的多肽链,将多肽物质纯化后加入到铅酸蓄电池电解液中,利用其能够吸附在海绵状铅的表面的功能,增大电极的表面积,防止铅电极的钝化,同时抑制硫酸铅形成大结晶,使得铅离子在充电过程中能够有效的还原成极板上的活性物质而延长铅酸蓄电池的寿命。适用于对铅酸蓄电池的养护。
Description
技术领域
本发明属于基因工程应用领域,具体公开了一种编码ST肽的基因、含有该基因的重组载体pET30α-X1以及包含上述重组载体的大肠杆菌和其在延缓铅酸蓄电池老化以及修复老化铅酸蓄电池方向的应用。
背景技术
铅酸蓄电池(Lead-acid battery),是由铅、二氧化铅和30%的硫酸溶液组成的一种二次电池,阀控式密闭铅酸蓄电池是目前应用最为广泛的铅酸蓄电池。由于铅酸蓄电池的价格便宜、可靠性高、所用的原材料容易得到、放电电流大等优点,使得其广泛运用于交通、通信、后备电源等领域。铅酸蓄电池目前主要的问题是其不可逆硫酸盐化导致电池的寿命短,需要经常维护修理。由于铅酸蓄电池的老化而导致其提前失效报废,不仅会污染环境,而且造成了极大的浪费。
铅酸蓄电池虽然经过了160多年的不断改进创新,但在长时间的使用后还是会出现容量下降甚至失效等问题。其中主要的失效原因有:板栅氧化腐蚀、正极板活性物质脱落、不可逆硫酸盐化等,这些失效原因通常会相互作用相互影响,但其中最主要的原因是不可逆硫酸盐化。
发明内容
为解决背景技术中列举的技术问题,本发明提供了一种重组大肠杆菌,该重组大肠杆菌能够合成ST肽,吸附在海绵状铅的表面,增大电极的表面积,来抑制铅酸蓄电池在充放电中形成大的的硫酸铅晶体,使得铅离子在充电过程中能够有效的还原成极板上的活性物质,从而修复和延缓铅酸蓄电池的衰老。具体技术方案如下:
本发明提供一种编码ST肽链的基因,所述基因的核苷酸序列如SEQ ID NO:1所示。
本发明还提供了一种重组载体pET30α-X1,所述重组载体pET30α-X1以质粒pET30α为原始载体,携带有上述方案所述编码ST肽的基因;
所述质粒pET30α的核苷酸序列如SEQ ID NO:2所示。
优选的,所述编码ST肽的基因的插入质粒pET30α中的位点为NdeI和XhoI。
本发明还提供了一种包含上述方案所述重组载体pET30α-X1的重组大肠杆菌。
本发明还提供了上述方案所述重组大肠杆菌在延缓铅酸蓄电池的衰老中的应用。
本发明还提供了上述方案所述重组大肠杆菌在修复老化铅酸蓄电池中的应用。
本发明的有益效果:本发明提供了一种编码ST肽的基因,该基因根据大肠杆菌((Escherichia coli)对简并密码子的使用偏嗜性对ST成熟多肽编码序列进行密码子优化得到,与其他编码ST肽的基因相比大肠杆菌(宿主菌)对本发明的基因使用频率更高;本发明还提供了一种重组载体pET30α-X1以及一种包含重组载体pET30α-X1的重组大肠杆菌。本发明通过基因工程技术利用重组大肠杆菌表达具有特殊结构的多肽链,将多肽物质纯化后加入到铅酸蓄电池电解液中,利用其能够吸附在海绵状铅的表面的功能,增大电极的表面积,防止铅电极的钝化,同时抑制硫酸铅形成大结晶,使得铅离子在充电过程中能够有效的还原成极板上的活性物质而延长铅酸蓄电池的寿命。经检测,所述重组大肠杆菌分泌得到的ST肽浓度达到95.919μg/ml。本发明的技术方案为延缓铅酸蓄电池老化以及修复老化铅酸蓄电池提供了一种新型的解决思路和应用方向,有利于延长铅酸蓄电池的寿命,降低蓄电池报废的数量以及电池报废产生的费用,减少蓄电池报废带来的污染。
附图说明
图1为重组载体pET30α-X1的质谱图;
图2为实施例2中BL21-pET30α-X1重组子菌液PCR验证电泳图;
图3为实施例3中X1重组多肽在大肠杆菌的胞内表达鉴定结果;
图4为实施例5中不同的浓度的ST多肽对电池负极板上形成的硫酸铅晶大小影响的结果。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面结合附图及具体实施例对本发明进行描述。显然,所描述的实施例仅仅是本发明的一部分实施例,而不是全部的实施例。基于该实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
本发明提供了一种编码ST肽的基因,所述基因的核苷酸序列如SEQ ID NO:1所示。
在不改变其氨基酸的前提下,根据大肠杆菌(Escherichia coli)对简并密码子的使用偏嗜性对ST成熟多肽编码序列进行密码子优化(从编码同一种氨基酸的简并密码子中选择一个宿主菌使用频率高的序列,U→T),送武汉擎科创新生物技术有限公司全基因合成获得目的基因(编码ST肽的基因)。本发明中,所述编码ST肽的基因与其他编码ST肽的基因相比宿主菌使用频率更高。
实施案例2
一种重组载体pET30α-X1,所述重组载体pET30α-X1以质粒pET30α为原始载体,携带有上述方案所述编码ST肽的基因;所述质粒pET30α的核苷酸序列如SEQ ID NO:2所示;所述编码ST肽的基因的插入质粒pET30α中的位点为NdeI和XhoI;所述重组载体pET30α-X1的质谱图参见图1。
本发明中,所述重组载体pET30α-X1的制备方法优选的包括以下步骤:编码ST肽的基因和质粒pET30α双酶切,回收酶切产物,酶切产物酶连,得到重组载体pET30α-X1。
本发明中,所述编码ST肽的基因和质粒pET30α双酶切体系参见表1;所述酶切的温度优选为35~40℃,更优选为37℃;所述酶切的时间优选为4~8h,更优选为6h。
表1编码ST肽的基因和质粒pET30α双酶切体系
本发明中,所述回收酶切产物的试剂盒优选为PCR清洁回收试剂盒,购自于OMEGA公司;所述回收酶切产物后优选的还包括除去反应体系中的酶等物质,琼脂糖凝胶电泳验证成功后待连。
本发明中,所述酶切产物(目的片段与载体片段)酶连的体系参见表2;所述酶连的温度优选为15~18℃,更优选为16℃;所述酶连的时间优选为8~12h,更优选为9~10h。
表2目的片段与载体片段酶连体系
将得到的重组载体pET30α-X1电转化大肠杆菌感受态细胞,37℃静置复苏后涂布含有氨苄青霉素的LB平皿,于37℃恒温倒置培养过夜后筛选、验证、获得阳性重组子。验证结果参见图2,M为Trans 2K Plus DNA Marker,1为NdeI/XhoI双酶切产物电泳图。重组质粒,经NcoI/KpnI双酶切后产物预期大小分别为5236bp和203bp。由琼脂糖凝胶核酸电泳结果图可知,质粒双切结果与预期一致,条带大小相符。2-7分别为从平皿上挑取的10个转化子。pET30α空载PCR产物大小为188bp,pET30α-X1重组质粒PCR产物大小为192bp。由琼脂糖凝胶核酸电泳结果图可知,条带单一明亮,10个泳道的PCR产物片段大小一致,将PCR产物送测序,测序结果与预期相符,进一步说明初步获得了正确的pET30α-X1阳性克隆。
实施案例3
划线并挑取实施例2中阳性克隆重组大肠杆菌(BL21-pET30α-X1),转接至5mL含Kan的LB培养基中,37℃180r/min震荡培养24小时。其中预期优化的ST肽的氨基酸序列如SEQ ID NO:3所示。以BL21及携带空载pET30α的重组菌BL21-pET30α为阴性对照,将菌体培养至相同OD。收集各菌菌液1mL,超生波破碎细胞后10000×g离心30min,取其破碎细胞上清液作为待检样品进行Western blot鉴定。鉴定结果参见图3,其中M为蛋白Marker,泳道1-2分别为受体菌(BL21)和质粒载体转化子(BL21-pET30α)泳道3-4为重组菌(BL21-pET30α-X1)。
由WB结果可知,空菌和空载均未检测到目的蛋白,只有重组菌检测到蛋白大小与预期15kDa相符,说明重组目的蛋白可在大肠杆菌Escherichia coli中成功分泌表达。
实施例4重组目的蛋白的分离纯化
活化培养实施例2中重组大肠杆菌培养至24小时收集菌液,压力破碎细胞,4℃、10000r/min离心30min,上清液过0.22μm滤膜除杂除菌后,进行镍柱亲和层析。上样前,先用20%乙醇、超纯水冲洗镍柱,再用Buffer A平衡镍柱。柱平衡后,将离心、过滤处理后的样品以1mL/min的流速通过衡流泵加载到Ni柱上。挂柱完成后,依次用含不同浓度咪唑的BufferA/Buffer B混合液开始梯度洗脱目的产物。根据洗脱过程实时出峰情况,当流出液的紫外吸光度开始明显上升时收集流出液,至流出液的紫外吸光度下降至最低值时停止收液体。将收集的各管样品进行12%SDS-PAGE检测分析。根据电泳结果,选择相应收集管中的洗脱蛋白在4℃环境下透析除去咪唑,通过蛋白标曲测定浓度。
实施例5实施例4获得的重组目的蛋白的按照不同浓度加入铅酸电池组中,经过长时间充放电后将电池的负极极板拆下置于扫描电子显微镜下观察其围观结构,由此判断重组蛋白对硫酸铅晶体形成大小的影响。结果参见图4,由图4中(1)-(3)为分别加入0.1%、0.2%、0.5%的重组蛋白,(4)-(6)分别为加入0.1%、0.2%、0.5%的混合试剂,电池的负极板上形成的硫酸铅晶体有显著的差异。通过比较发现1号、2号电池负极板上的硫酸铅晶体要小于其他电池。3号电池负极板上形成了较大的硫酸铅晶体,同时一些细小的晶体附着在大晶体的表面。在4号、5号、6号中大硫酸铅晶体比较多。在电池充电的时候难以溶解,所以电池的容量相对于其他的电池更低。说明重组目的蛋白能够有效延缓铅酸蓄电池老化。
实施例6挑选6只同型号的老旧铅酸蓄电池。将老旧蓄电池充满电后,进行一次核容测试,并记录初始状态。将6只铅酸蓄电池串联为一组,在保留1只为空白对照样品外,将其余5只蓄电池分别添加不同剂量的基于重组大肠杆菌生物技术的助剂。再次将老旧蓄电池充满电,并持续浮充20天后。进行二次核容测试,并记录修复后状态。测试结果如表3所示。
表3基于重组大肠杆菌生物技术的助剂修复效果测试
测试项目 | 蓄电池1 | 蓄电池2 | 蓄电池3 | 蓄电池4 | 蓄电池5 | 蓄电池6 |
一次核容 | 244Ah | 66.06Ah | 360.8Ah | 234.3Ah | 269.4Ah | 354.5Ah |
添加剂量 | 空白对照 | 80ml | 64ml | 32ml | 16ml | 8ml |
二次核容 | 225.3Ah | 134.7Ah | 393.9Ah | 271.2Ah | 294.7Ah | 374Ah |
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 国网电力科学研究院武汉南瑞有限责任公司
<120> 一种重组大肠杆菌在养护铅酸蓄电池中的应用
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caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa 1800
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cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa 2220
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gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg 2340
cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat 2400
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tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt 2520
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cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc 2820
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cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 3240
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gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 3600
cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 3660
taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 3720
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caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg 5100
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tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg 5220
ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa 5280
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Gly Ser Glu Ser Thr Thr Glu Asp Gln Ala Asp Asp Gln Thr Ser Ser
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Lys Leu
65
Claims (6)
1.一种编码ST肽的基因,所述基因的核苷酸序列如SEQ ID NO:1所示。
2.一种重组载体pET30α-X1,所述重组载体pET30α-X1以质粒pET30α为原始载体,携带有权利要求1所述编码ST肽的基因;
所述质粒pET30α的核苷酸序列如SEQ ID NO:2所示。
3.根据权利要求2所述的重组载体pET30α-X1,其特征在于,所述编码ST肽的基因的插入质粒pET30α中的位点为NdeI和XhoI。
4.一种包含权利要求2或3所述重组载体pET30α-X1的重组大肠杆菌。
5.权利要求4所述重组大肠杆菌在延缓铅酸蓄电池老化中的应用。
6.权利要求4所述重组大肠杆菌在修复老化铅酸蓄电池中的应用。
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