CN116640782A - 锑结合蛋白AntC在锑污染修复中的应用 - Google Patents
锑结合蛋白AntC在锑污染修复中的应用 Download PDFInfo
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Abstract
本发明属于基因工程及微生物技术领域,公开了锑结合蛋白AntC在锑污染修复中的应用,本发明首次发现AntC可以与Sb(III)结合,以三个半胱氨酸为金属结合位点,通过半胱氨酸的巯基与Sb(III)紧密结合因此该蛋白可用于Sb(III)的污染处理。表达AntC的基因工程菌可用于Sb(III)的污染处理,将AntC和GlpF进行联合表达,可提高Sb(III)的处理能力。申请人同时提供了一种高效的处理锑污染的基因工程菌,保藏编号为CCTCC NO:M 20222051,该菌株成本低,效率高,更为关键的是环境友好,不会造成二次污染,在锑污染水体及土壤修复中具有较好的应用前景。
Description
技术领域
本发明属于基因工程及微生物技术领域,涉及锑结合蛋白AntC在锑污染修复中的应用,本发明提供的锑结合蛋白AntC可用于制备具有锑吸附能力的基因工程菌,该工程菌可用于处理含锑废水或锑污染土壤。
背景技术
锑是一种广泛分布于自然界中类金属,主要以亚锑酸盐[Sb(III)]和锑酸盐[Sb(V)]的形式存在,其中Sb(III)的毒性远大于Sb(V)。锑在工业上具有广泛用途,被大量应用于合金和半导体等行业上。作为一种剧毒的类金属,长期的锑暴露可能会引起呼吸道疾病,肺损伤,心血管疾病,胃肠道疾病等众多疾病,甚至诱导癌变。人为活动如矿石的开采或燃料的燃烧及工业生产大大增加了锑造成的环境污染。在锑污染地区的土壤、水体和沉积物中的锑含量都远大于正常的背景浓度。因此,寻求一种经济可靠修复锑污染废水及锑污染土壤的方法成为人们关注的焦点。
含锑废水的处理方法主要有吸附法、离子交换、膜处理技术等,但这些方法成本高、操作复杂、易产生二次污染。微生物吸附法作为一种安全、经济、环境友好、避免二次污染的除锑方法与技术,备受学者的青睐和关注。此外,微生物功能菌剂在锑污染土壤的修复中也具有广泛应用。特定微生物菌株可以降低土壤中锑的生物有效性,并提高土壤酶活,促进土壤健康。在厌氧还原的条件下,异化锑酸盐还原地杆菌Geobacter sp.SVR可将Sb(V)还原与乙酸盐氧化相耦合,形成Sb2O3沉淀从而高效去除污染水体中的锑。然而,地杆菌作为专性厌氧菌,其对Sb(V)的还原去除仅发生在厌氧条件下,且需要一定浓度乙酸盐作为电子供体,极大的限制其对锑污染修复的应用。此外,硫酸盐还原菌也被广泛应用在废水中Sb(V)的去除。在厌氧条件下,硫酸盐被还原为S2-,同时Sb(V)被还原为Sb(III),两者形成Sb2S3沉淀而去除。但是这类微生物的作用效力受底物(硫酸盐)的影响,当环境中可用硫酸盐浓度低或不存在时则无锑去除效果。目前,大部分微生物修复锑污染主要针对Sb(V),针对毒性更大的Sb(III)的修复技术研究较少。因此,急需开发构建出不受厌氧条件或硫酸盐等物质限制的高效去除并修复Sb(III)污染的菌株或技术。
发明内容
本发明的第一个目的在于提供锑结合蛋白AntC在锑污染修复中的应用,所述的锑结合蛋白为SEQ ID NO.2所示。本发明首次发现该蛋白是可与锑离子结合的蛋白
本发明的另一个目的在于提供了可用于锑污染修复的工程菌,申请人将AntC及GlpF蛋白构建进入表达载体制备成基因工程菌,该基因工程菌可用于处理锑污染的体系。
本发明的最后一个目的在于提供了上述基因工程菌在锑污染修复中的应用。
为了达到上述目的,本发明采取以下技术措施:
申请人对锑抗性细菌C.testosteroni JL40的iTRAQ差异蛋白质组学分析得到AntC可以与Sb(III)结合,所述的AntC蛋白为SEQ ID NO.2所示,编码其的多核苷酸优选为SEQ ID NO.1所示。
本申请的保护内容包括:表达AntC蛋白的基因工程菌在吸附Sb(III)中的应用,所述的应用包括,上述基因工程菌用于处理锑(III)污染的体系,例如土壤,水体等。
以上所述的应用中,优选的,所述的基因工程菌为可同时表达AntC蛋白和GlpF蛋白的基因工程菌,所述的GlpF蛋白为SEQ ID NO.3所示。
以上所述的基因工程菌,具体为大肠杆菌(Escherichia coli)DH5α(pUC-Lac-G/Trp-C),该基因工程菌已于2022年12月23日送至中国典型培养物保藏中心(CCTCC)保藏,其保藏编号为CCTCC NO:M20222051,分类命名:大肠杆菌(Escherichia coli)DH5α(pUC-Lac-G/Trp-C),地址:中国武汉武汉大学。
与现有技术相比,本发明具有以下优点:
(1)本发明首次发现AntC可以与Sb(III)结合,以三个半胱氨酸为金属结合位点,通过半胱氨酸的巯基与Sb(III)紧密结合因此该蛋白可用于Sb(III)的污染处理。
(2)利用AntC蛋白的特点,申请人将其与Sb(III)摄取蛋白GlpF的在生物载体中共表达,获得的基因工程株可以与Sb(III)结合将其固定在细胞内
(3)本发明的基因工程菌能降低锑污染土壤中锑的生物有效性,具有开发成为新型的重金属钝化菌剂的应用潜力。
(4)本发明的基因工程菌能高效处理剧毒Sb(III)污染废水,且不受厌氧条件限制,无需添加高浓度硫酸盐等辅助物质。
附图说明
图1为载体pUC-Lac-G/Trp-C示意图;
利用同源重组的方式将pUC19载体的氨苄青霉素(Amp)的抗性基因替换为安普霉素(Apr);将原始载体Lac启动子的operator敲除掉,达到组成型表达GlpF蛋白的目的;在安普霉素抗性基因后面加入色氨酸(Trp)启动子,从而高效高表达AntC蛋白。
图2为AntC蛋白与Sb(III)结合位点示意图;
其中A图是Swiss-model预测的AntC的空间(https://swissmodel.expasy.org);B图是根据胞内结合测定了AntC与Sb(III)的结合能力,其三个半胱氨酸形成Sb(III)结合位点。
图3为土壤模拟探究表达AntC的大肠杆菌对锑污染土壤的钝化效果;
其中Control是无菌株施加的对照组;DH5α(pUC18)是施加原始大肠杆菌的对照组;DH5α(pUC18-AntC)是施加表达AntC的大肠杆菌的实验组。
图4为模拟废水实验探究表达AntC的大肠杆菌对锑污染水体的修复效果;
其中Control是无菌株施加的对照组;Vector是施加原始大肠杆菌的对照组;GlpF是施加表达GlpF的大肠杆菌的实验组;AntC是施加表达AntC的大肠杆菌的实验组;GlpF+AntC是施加共表达AntC与GlpF的大肠杆菌的实验组。
具体实施方式
下面结合具体实施例对本发明做进一步详细说明。实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。下述实施例中所用方法如无特别说明均为常规方法。
实施例1:
AntC蛋白具有与Sb(III)的结合能力:
本发明提供的AntC蛋白是通过对锑抗性细菌C.testosteroni JL40的iTRAQ差异蛋白质组学分析得到。C.testosteroni JL40分离自湖南省冷水江市锡矿山锑矿中,具有很强的Sb(III)抗性及Sb(III)氧化能力。JL40对Sb(III)的最低抑制浓度为5mM,该菌株能在72h内完全氧化100μM Sb(III)生成Sb(V)。该菌基因组已测序,在GenBank数据库中基因组序列注册号为AWOR00000000。AntC蛋白的编码基因antC从菌株JL40基因组通过PCR扩增得到。编码AntC蛋白的基因为SEQ ID NO.1所示,AntC蛋白为SEQ ID NO.2所示。
AntC蛋白与Sb(III)结合位点测定
具体步骤如下所述:
(1)为测定AntC蛋白的Sb(III)结合位点,将AntC蛋白的30,34,36号位点处的半胱氨酸分别单独或全部突变为丙氨酸,然后连接到pUC18载体上,构建得到重组突变体载体分别命名为:pUC18-30C,pUC18-34C,pUC18-36C,pUC18-3C。其中pUC18-30C指得是AntC蛋白的30号位点处的半胱氨酸突变为丙氨酸后,连接到pUC18载体;pUC18-34C,pUC18-36C以此类推;pUC18-3C指得是AntC蛋白的30,34,36号位点处的半胱氨酸均突变为丙氨酸后,连接到pUC18载体。将AntC蛋白连接到pUC18载体上得到pUC18-AntC,作为野生型对照组。
将AntC野生型及上述重组突变体载体转化至大肠杆菌W3110的arsRBC基因敲除株AW3110细胞中,特用于锑的结合检测,敲除了arsRBC基因的W3110在本申请中称为AW3110,得到菌株AW3110(pUC18-AntC),AW3110(pUC18-30C)和AW3110(pUC18-34C),AW3110(pUC18-36C),AW3110(pUC18-3C),用于AntC蛋白及半胱氨酸突变体Sb(III)结合能力测定。
(2)将菌株AW3110(pUC18),AW3110(pUC18-AntC),AW3110(pUC18-30C)和AW3110(pUC18-34C),AW3110(pUC18-36C),AW3110(pUC18-3C)新鲜菌液分别按1%的接种量接种于100mL LB液体培养基中,在37℃摇床培养16h,然后于4℃下6000rpm离心5min收集菌体,用0.85%的生理盐水洗涤2次,重悬于50mL 0.85%的氯化钠溶液中,调整OD600值为3.0左右。
(3)吸取5mL重悬后的菌液于10mL离心管中,加入终浓度为100μM Sb(III)溶液后于28℃摇床震荡培养,每个菌株作三重复。30min后取出1mL溶液于6000rpm离心5min,去除上清后菌体沉淀用1mL 0.85%的氯化钠溶液洗涤2次后,加入400μL硝酸消解2h。利用原子荧光光谱仪(HG-AFS)对消解后的菌体进行锑浓度的检测。
(4)将所测试的结果数据用SPSS Statistics 24.0统计软件处理,以单位质量菌体吸附的锑含量纵坐标,菌体处理的时间为横坐标,用GraphPad Prism 8软件进行图表绘制。
(5)通过分析AntC的半胱氨酸突变株与野生株的Sb(III)结合能力差异探究其Sb(III)的结合位点。结果发现,野生型AntC对Sb(III)具有很强的结合能力,而突变第30,34,36号位点处的半胱氨酸后,显著降低了其Sb(III)的结合能力;将该3个半胱氨酸全部突变后,其Sb(III)的结合能力进一步降低,说明锑结合蛋白AntC的第30,34,36号位点处的半胱氨酸参与形成其Sb(III)结合位点(图2)。
实施例2:土壤模拟探究表达AntC的大肠杆菌对锑污染土壤的钝化效果
具体步骤如下所述:
(1)以菌株JL40的基因组为模板进行PCR扩增得到antC基因的DNA序列,然后连接到pUC18载体上,构建得到载体pUC18-AntC。将载体pUC18及pUC18-AntC分别转化至大肠杆菌DH5α细胞中,得到菌株DH5α(pUC18)和DH5α(pUC18-AntC)。
(2)准备15个150mL大小带盖玻璃瓶,每个瓶子称入50g风干土,在每盒中加入100mL去离子水,并加入终浓度50mg/kg的Sb(III),于室温平衡48h。实验一共3个处理,每个处理设5重复。
(3)平衡结束后,向土壤悬液中喷洒DH5α(pUC18)及DH5α(pUC18-AntC)菌液,接种量约1.0×108cfu/g(土)。Control组是无菌株施加的对照组;DH5α(pUC18)组是施加原始大肠杆菌的对照组;DH5α(pUC18-AntC)组是施加表达AntC的大肠杆菌的实验组。
然后置于室温(25℃~28℃)孵育,期间保持土壤的含水率恒定,并于第4、8、12天取样。取样方法为,将土样悬液充分混匀后吸取5mL土壤悬液,并根据Walter(2001)年发表文章中的方法利用0.05mol/L(NH4)2SO4及0.05mol/L NH4H2PO4对土壤可利用态锑进行提取;此外,额外吸取5份5mL土壤悬液离心去除上清,烘干土壤后用于计算干湿比。最后使用原子荧光光谱仪(HG-AFS)(北京吉天仪器有限公司)检测提取液中锑含量并计算土壤可利用态锑的浓度。
(4)将所测试的结果数据用SPSS Statistics 24.0统计软件处理,以土壤单位重量可利用态锑含量纵坐标,时间为横坐标,GraphPad Prism 8软件进行图表绘制。
(5)通过比较基因工程菌与对照处理组的土壤可利用态锑含量可发现,施加表达AntC的基因工程菌显著性降低了土壤可利用态锑浓度。锑的生物有效性在施加菌株后的第4、8、12天分别降低了13.9%,30.3%,25.5%,而施加野生株的大肠杆菌对其生物有效性无显著影响。说明锑结合蛋白AntC在底盘细胞中表达后可有效将锑固定,降低土壤中生物可利用态锑的含量,该工程菌株在土壤锑污染修复中具有很好的应用潜力(图3)。
实施例3:
表达载体pUC-Lac-G/Trp-C及基因工程菌株的构建
首先利用同源重组的方式将pUC19载体的Lac启动子的operator敲除掉,达到组成型表达GlpF蛋白的目的;此外在Apr抗性基因后面加入Trp启动子,从而高效高表达AntC蛋白。
(1)以大肠杆菌Escherichia coli K12的基因组为模板,以表1中GlpF-F/R引物进行PCR扩增,选择Mix(green)DNA Polymerase高保真酶(擎科生物)进行,PCR条件为预变性98℃,3min;扩增阶段30个循环,按照98℃,10s,52℃,10s,72℃,10s进行;终延伸72℃,10min,获得GlpF的基因片段;
(2)以pUC19质粒为模板,以表1中的引物p19-F/R进行反向PCR扩增,选择Mix(green)DNA Polymerase高保真酶(擎科生物)进行,PCR条件为预变性98℃,3min;扩增阶段30个循环,按照98℃,10s,50℃,10s,72℃,10s进行;终延伸72℃,10min,将载体质粒线性化;
(3)将步骤(1)得到的基因片段GlpF与步骤(2)得到的载体pUC19线性化的PCR产物分别纯化并通过同源重组的方法进行连接,连接体系为:2×Buffer 5μl,线性化载体2μl,GlpF的基因片段3μl。体系配制完成后,于50℃反应30min。待反应完成后,立即将反应管置于冰水浴中冷却5min,然后转化至Escherichia coli DH5α感受态细胞中。挑取转化成功的转化子DH5α(pUC-Lac-GlpF),并提取其质粒pUC-Lac-GlpF进行测序验证。
(4)以质粒pUC19及pUC-Lac-GlpF为模板,以表1中的引物p19Trp-F/R进行反向PCR扩增,将两个质粒分别线性化;同时用表1中的引物AntC-F/R以菌株JL40的基因组为模板进行PCR扩增得到AntC的DNA序列;最后以Trp启动子的DNA及AntC的DNA为模板,用引物Trp-F及AntC-R进行PCR扩增,以重叠延伸PCR的方式扩增得到Trp-AntC的DNA片段;
(5)将扩增得到Trp-AntC的DNA片段与线性化的载体pUC19连接,然后转化至Escherichia coli DH5α感受态细胞中,得到菌株DH5α(pUC-Trp-AntC);
将扩增得到Trp-AntC的DNA片段与pUC-Lac-GlpF通过同源重组的方法进行连接后,再与线性化的载体pUC19连接,然后转化至Escherichia coli DH5α感受态细胞中,得到菌株DH5α(pUC-Lac-G/Trp-C);
提取菌株DH5α(pUC-Lac-G/Trp-C)的质粒pUC-Lac-G/Trp-C,通过测序验证,共表达GlpF与AntC的载体构建成功(图1),制备出的基因工程菌已于2022年12月23日送至中国典型培养物保藏中心(CCTCC)保藏,其保藏编号为CCTCC NO:M20222051,分类命名:大肠杆菌(Escherichia coli)DH5α(pUC-Lac-G/Trp-C),地址:中国武汉武汉大学。
表1
Primer | Primer sequence(5'-3') |
GlpF-F | CCGGCTCGTATGTTGTGTGGACACAGGAAACAGCTatgagtcaaacatcaacct |
GlpF-R | CGTTGTAAAACGACGGCCAGTGttacagcgaagctttttgttc |
p19-F | CACTGGCCGTCGTTTTACAACGTC |
p19-R | TCCACACAACATACGAGCCGG |
p19Trp-F | CTGTCAGACCAAGTTTACTCA |
p19Trp-R | TCAGCCAATCGACTGGCGAGCG |
Trp-F | GCTCGCCAGTCGATTGGCTGACGCCAAGCTTCCGGCAAAT |
AntC-F | ATGGCTAGCAAAGGAGAAGAAATGGAAAAGAAAAACAGCG |
AntC-R | TGAGTAAACTTGGTCTGACAGTCATGCCTTCCCGTTGTC |
实施例4:模拟废水实验探究表达AntC的基因工程菌对锑污染水体的修复效果
具体步骤如下所述:
(1)将菌株DH5α(pUC19),DH5α(pUC-Lac-GlpF),DH5α(pUC-Trp-AntC)和DH5α(pUC-Lac-G/Trp-C)新鲜菌液分别按1%的接种量接种于100mL LB液体培养基中,在37℃摇床培养16h,然后于4℃下6000rpm离心5min收集菌体,用0.85%的生理盐水洗涤2次,重悬于50mL0.85%的氯化钠溶液中,调整OD600值为3.0左右。
(2)吸取5mL重悬后的菌液于10mL离心管中,加入终浓度为20μM Sb(III)溶液后于28℃摇床震荡培养,每个菌株作三重复。每隔2h取出1mL溶液于6000rpm离心5min,转移上清并对其锑浓度进行测定,菌体沉淀用1mL 0.85%的氯化钠溶液洗涤2次后,加入400μL硝酸消解2h。利用原子荧光光谱仪(HG-AFS)对锑溶液上清及消解后的菌体进行锑浓度的检测。
(3)将所测试的结果数据用SPSS Statistics 24.0统计软件处理,以溶液上清及菌体吸附的锑含量纵坐标,菌体处理的时间为横坐标,用GraphPad Prism 8软件进行图表绘制。
(4)通过比较基因工程菌与空载对照菌株对模拟废水中锑去除效果可发现,单独表达GlpF的大肠杆菌、单独表达AntC的大肠杆菌、共表达GlpF与AntC的大肠杆菌在4小时后,菌株对锑的吸附量相比对照分别提高了938%,999%,1166%,同时对模拟废水中的锑去除率分别为74.7%,85.8%,90.9%。共表达GlpF与AntC的大肠杆菌处理4小时后,模拟废水溶液中的锑浓度降低至国家工业废水排放标准(0.3mg/kg)以下。说明锑结合蛋白AntC在工程菌中高效表达后可有效将锑固定在细胞内,共表达GlpF能进一步提高细胞对锑的摄取及积累量,该工程菌株在锑污染废水中具有很好的应用前景(图4)。
Claims (7)
1. 表达AntC蛋白的基因工程菌在吸附Sb(III)中的应用,所述的AntC蛋白为SEQ IDNO.2所示。
2.根据权利要求1所述的应用,其特征在于:表达AntC蛋白的基因工程菌在Sb(III)污染修复中的应用。
3.根据权利要求2所述的应用,所述的污染对象为水或土壤。
4. 同时表达AntC蛋白和GlpF蛋白的基因工程菌在吸附Sb(III)中的应用,所述的AntC蛋白为SEQ ID NO.2所示,所述的GlpF蛋白为SEQ ID NO.3所示。
5.根据权利要求4所述的应用,其特征在于:同时表达AntC蛋白和GlpF蛋白的基因工程菌在Sb(III)污染修复中的应用。
6.根据权利要求5所述的应用,所述的污染对象为水或土壤。
7. 根据权利要求4所述的应用,所述的基因工程菌的保藏编号为CCTCC NO:M20222051。
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