CN102168033A - 嗜酸硫化芽孢杆菌半胱氨酸脱硫酶基因及其重组蛋白 - Google Patents
嗜酸硫化芽孢杆菌半胱氨酸脱硫酶基因及其重组蛋白 Download PDFInfo
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Abstract
嗜酸硫化芽孢杆菌半胱氨酸脱硫酶基因及其重组蛋白,涉及一种基因及其重组蛋白。提供嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶的基因序列、克隆方法、重组蛋白与制备方法,以及在制备细菌生长促进剂中的应用。克隆方法为提取嗜酸硫化芽孢杆菌TPY的基因组作为模板,扩增引物;扩增出来的PCR产物回收连接到pMD18T-Simple vector进行测序;双酶切回收连接PET-His构建表达载体PET-His-sufs+进行表达。所述嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶可作为细菌生长促进剂,所述细菌如大肠杆菌、嗜酸硫化芽孢杆菌、氧化亚铁硫杆菌等。
Description
技术领域
本发明涉及一种基因及其重组蛋白,尤其是涉及一种嗜酸硫化芽孢杆菌(Sulfobacillus acidophilus)半胱氨酸脱硫酶基因序列及其重组蛋白的功能。
背景技术
半胱氨酸脱硫酶(Cysteine des ulfurase)是一个以5-磷酸吡哆醛为辅基的二聚体酶,该酶可以催化L-型半胱氨酸转变为L-型丙氨酸,脱下的硫原子生成硫烷,形成半胱氨酸的过硫化物,从而保护半胱氨酸残基。([1]Zheng.L,White.R.H.,Cash.V.L,Dean.D.R.Cysteine desulfurase activity indicates a role for NIFS in metallocluster biosynthesis.[j]Biochemistry.1994,33:4714-4720)许多研究表明,该酶在鉄硫簇以及硫胺,tRNA中的硫代核苷,生物素,脂肪酸,NAD等的合成中均有重要作用([2]H.Mihara,N.Esaki.Bacterial cysteine desulfurases:their function and mechanisms.[j]Appl Microbiol Biotechnol.2002,60:12-23)。该酶在胞内的铁平衡以及硒蛋白的合成中也有重要作用([3]Beinert.H,Holm.R.H,Mu¨nck.E.Iron-sulfur clusters:Nature′s modular,multipurpose structures.[j]Science,1997,277:653-659)。半胱氨酸脱硫酶在硫代谢中的作用机理目前还不清楚,但是其在含有硫或硒蛋白的生物合成中都有重要作用。
铁硫簇蛋白是一类在生物当中普遍存在的蛋白,且参与了胞内许多重要代谢途径,如:DNA修复,转录调控,核苷酸和氨基酸合成,以及能量代谢([4]Zheng.L,White.R.H,Cash.V.L,Jack.R.F,Dean.D.R.Cysteine desulfurase activity indicates a role for NIFS in metallocluster biosynthesis.[j]Proc.Natl.Acad.Sci.USA,1993,90:2754-2758)。尽管有很多研究是有关Fe-S蛋白活性,但是对在体内Fe-S的组装以及如何***到蛋白中去的研究还为数不多。在大肠杆菌中,参与这一过程的两个独立***分别是:由IscSUA-hscBA-fdx基因簇编码的ISC和由SufABCDSE操纵子编码的SUF***([5]ToKumoto.U et,al.A Suf operon requirement for Fe-S cluster assembly during iron starvation in Escherichia coli.[j]Proc.Natl.Acad.Sci.USA,2004,136:199-201)。IscSUA-hscBA-fdx是参与铁硫簇合成的主要基因簇([6]Tokomoto.U et al,Transfer of Sulfur from IscS to IscU during Fe/S Cluster Assembly.[j]Biochem(ToKyo),2001,131:63-65)。该基因簇非常保守,可编码6个蛋白质,其中IscS,IscU和IscA是铁硫簇合成的主要蛋白质,HscA,HscB是一对热激关联蛋白,与IscU的活性修饰有关([7]Silberg.j.j et al,Crystal Structure of IscA,an Iron-sulfur Cluster Assembly Protein from Escherichia coli.[j]Biolchem,2004,279:53924-53926);而Fdx是一种铁氧化蛋白,在铁硫簇的组装中起着电子供体的作用([8]Takag.I.S,Sasado.T,Tamiy.A.G,et al.An efficient expression vector for transgenic medaka construction.[J]Mol Mar Biol Bioteehnol,1994,3:192-199)。研究表明,Suf***可能与铁硫簇的损伤的修复有关([9]Kokata Y et al.Reconstitution of secretase activity.[j]Biochemistry,2001,40:11007-11010)。
发明内容
本发明的目的之一在于提供嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶的基因序列。
本发明的目的之二在于提供嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶的克隆方法。
本发明的目的之三在于提供嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶的重组蛋白及其制备方法。
本发明的目的之四在于提供嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶在制备细菌生长促进剂中的应用。
本发明所述嗜酸硫化芽孢杆菌TPY(Sulfobacillus acidophilus TPY)已于2010年08月16日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M2010203,中国典型培养物保藏中心的地址为中国,武汉,武汉大学。
所述嗜酸硫化芽孢杆菌TPY(Sulfobacillus acidophilus TPY)是从太平洋热液区样品中分离纯化得到的一株中等嗜热嗜酸菌(参见本申请人的中国发明专利申请200610135418.9)。
本发明所述嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶的基因序列为:
ATGACACGGACTTTAGTGGATATCCGGCAGGATTTTCCCATTTTGACGCGGGAAATTCACGGTCATCGACTCGTATATCTTGACAGTGCCGCCACCTCCC AAAAGCCGGAGTCGGTCATTCAAGCCATTGACCACTATTATCGACACTCGAATGCCAATGTGCTCCGGAGTGTGCATACGCTCGCGGAGGAGGCGACC A C GCTCTATGAAGACGCGCGGCGCAAGGTGGCCCGCTTCATTGGCGCCAAAACCTCGCAAGAAGTGATTTTTACTCGCGGGACCACCGAGAGTTTAAA CG TC GTCGCGCGAGGCTGGGGAGATAAATTTGTCCAGCCGGGCGACGAAATTGTCGTGTCACCGATGGAGCATCATTCGAATTTAATTCCTTGGCAGC AAT TGG CGCAACGCCGGCAAGCCGTTTTACGCTTTGTGGAATTAAACGGCGACGGCACGATTGCCTTGGACGAGGTCCGTCGGGTGATTAACGCCAAA ACCA AAAT CGTGGCCCTATCCCGCATTTCCAATGTTCTCGGGACCATTAACCCGATTGCGGAAATCAGTGCTTTAGCCCATCAACAGGGTGCGGTGAT GGTGG TGGAC GGCGCGCAGTCGGTGCCGCACGAACCGACGGACGTCAAACGTCTGGGAGCGGATTTTTTGGCCTTTTCCGGGCACAAAATGTTAGGGC CGACCG GCATCG GTGTCTTATGGGGACGTCGCGAGTTGTTAGACGCGATGGATCCCGTGCTTTTTGGCGGGGAAATGATTGCCTATGTCGATCGGGAA AGGGCGA CGTGGGC CGAATTGCCGGCCAAATTGGAGGGCGGCACGCCAAACATCGCCGGAGCCATTGGGTTAGGGGCGGCCGTCGATTATTTAACCGC CATTGGCA TGGACGTC GTCGCCGCGCACGGGCAAGCCTTAGGCGAATTGGCCTACCAACGGCTAAAAGCCGTCGGCGGCGTGACCGTTTATGGCCCCC CCGCACCTC GCGGTGCCC TGGTGGCATTTAACCTGGAAGGTATCCATCCCCATGACGTGGCCCAAGTGTTTGACTCCAATGGCGTGGCCATCCGAGCC GGGCATCATT GCGCACAACC GCTGTTGGCGTGGTTAGGCGTGGGATCTACCGCCCGTGCCAGTTTCTATGTCTATAATACCGAGGCTGACATCGATAC
本发明所述嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶的克隆方法的具体步骤如下:
1)提取嗜酸硫化芽孢杆菌TPY的基因组作为模板,扩增引物为:
P1:5′-CA GAATTC ATGACACGGACTTTAGTGG-3′;
P2:5′-TT AAGCTT TCAGCGAAAGTACCTCTGC-3′;
2)扩增出来的PCR产物回收连接到pMD18T-Simple vector(可由日本TaKaRa公司提供)进行测序;
3)双酶切回收连接PET-His构建表达载体PET-His-sufs+进行表达。
本发明所述嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶的重组蛋白的序列如下:
MTRTLVDIRQDFPILTREIHGHRLVYLDSAATSQKPESVIQAIDHYYRHSNANVLRSVHTLAEEATTLYEDARRKVARFIGAKTSQEVIFTRGTTESLNVVARGWGDKFVQPGDEIVVSPMEHHSNLIPWQQLAQRRQAVLRFVELNGDGTIALDEVRRVINAKTKIVALSRISNVLGTINPIAEISALAHQQGAVMVVDGAQSVPHEPTDVKRLGADFLAFSGHKMLGPTGIGVLWGRRELLDAMDPVLFGGEMIAYVDRETATWAELPAKLEGGTPNIAGAIGLGAAVDYLTAIGMDVVAAHGQALGELAYQRLKAVGGVTVYGPPAPRGALVAFNLEGIHPHDVAQVFDSNGVAIRAGHHCAQPLLAWLGVGSTARASFYVYNTEADIDTLVEVIGETQRYFR*。
本发明所述嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶的重组蛋白的制备方法如下:
将构建好的表达载体PET-His-sufs+转入表达菌株BL21(DE3)进行表达。首先将BL21(DE3)菌株接入LB培养基,在LB培养基中加入浓度为100μg/ml的氨苄,再放入37℃摇床进行培养,培养到600nm处吸光值OD600为0.5时,加入0.1mM的IPTG;接着放入37℃摇床进行诱导表达6h后,即得嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶的重组蛋白。
将诱导表达6h后培养物5000g离心收菌,由磷酸盐缓冲液(PBS)洗涤一遍以除去残留的LB培养基;接着重悬在磷酸盐缓冲液中超声破碎10min,8000g离心得上清液和沉淀,各取上清液和沉淀100μl于EP管中,各加入2×蛋白沉降缓冲液(购自上海生工公司)100μl,煮10min,SDS胶电泳分析。
本发明所述嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶可作为细菌生长促进剂,所述细菌如大肠杆菌、嗜酸硫化芽孢杆菌或氧化亚铁硫杆菌等。
本发明的突出优点和技术效果如下:
所述嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶的重组蛋白的主要功能是促进铁硫簇的合成,在细菌的三羧酸循环中很多酶都是由铁硫簇组装而成的。由于嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶可以促进铁硫簇的组装,可以增加参与细菌三羧酸的循环的由铁硫簇组装的一些酶的活性,从而促进三羧酸的循环,促进细菌的生长。在一些恶劣的环境中,细菌可以通过增加自己的数目而对抗外界的恶劣的条件。比如在硫浓度很高的情况下大肠杆菌是很难存活的,而在培养基中加入足够的该蛋白大肠杆菌即可生长(已试验中得到证实,参见以下附图)。在一些工程菌的使用上,常常会因为细菌生长速度慢,而无法利用或者是增加成本,使生产周期加长。如一些浸矿菌至今不能应用到生产当中,其中一个很主要的阻力就是细菌生长速度慢,导致细菌浓度低,使浸矿周期长,浸矿速度慢。嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶的重组蛋白可以促进细菌的生长,可以明显缩短细菌浸矿周期,加快浸矿速度,从而促进浸矿菌在贫矿浸出工程中的应用。
附图说明
图1为嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶的基因在大肠杆菌E.coli BL21(DE3)表达的重组蛋白的电泳图。在图1中,泳道1为蛋白分子量大小,泳道2为没有转入SufS基因的全菌蛋白,泳道3为转入SufS基因的全菌蛋白,泳道4为为转入SufS基因的上清,泳道5为转入SufS基因的沉淀;箭头处为目标条带。
图2为嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶在大肠杆菌E.coli BL21(DE3)中过表达菌群及空载菌群的生长曲线。在图2中,横坐标为时间time/min,纵坐标为600nm吸光值(OD600);◆为BL21/pet-his-sufs+,■为BL21/pet-his。
图3为嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶在大肠杆菌E.coli BL21(DE3)中过表达菌及空载菌在LB平板上的菌落大小。在图3中,稀释度为10-6,培养时间为24h;a为菌落直径2~3mm,b为菌落直径1~1.5mm。
图4为大肠杆菌E.coli BL21(DE3)在LB培养基中的生长曲线。在图4中,横坐标为时间time/min,纵坐标为600nm吸光值(OD600);◆E.coli BL21(DE3)在LB培养基中的生长曲线,■E.coli BL21(DE3)在加有520ng/ml的actin蛋白的LB培养基中的生长曲线,▲E.coli BL21(DE3)在加有520ng/ml的SufS蛋白的LB培养基中的生长曲线。
图5不同量的吸收液和标准液产生的H2S的浓度在670nm处的吸光值绘制的标准曲线。在图5中,横坐标为H2S的浓度μg,纵坐标为670nm吸光值(OD670)。
具体实施方式
以下实施例将结合附图对本发明作进一步的说明。
1.嗜酸硫化芽孢杆菌TPY的基因组的提取。
1)取100ml的已经培养至稳定期的细菌培养基(此时沉淀最少),于10,000rpm下离心10min收集细菌;
2)用pH=2的去离子水洗涤细菌,直至没有沉淀,去除培养基中盐离子;
3)用500μL的TE(pH8.0)重悬,加入30μL的溶菌酶(50μg/μL)(由于该菌是革兰氏阳性菌破壁比较难),混匀,37℃水浴10min;
4)加入30μL 10%的SDS溶液和10μl RNase(20μg/μL)混匀,55℃水浴10min,再加入3μL蛋白酶K(20μg/μL),55℃水浴10min,直至菌液变澄清;
5)加入150μL 5mol/L的NaCl,充分混匀,再加入80μL的CTAB/NaCl溶液,混匀,于65℃水浴10min;
6)加入等体积的酚/氯仿/异戊醇(25∶24∶1),混匀,12,000rpm离心10min,上清液转移入新管中;如果对DNA质量要求比较高可以多抽提几次;
7)加入0.6倍体积的异丙醇,轻轻混匀,-20℃放置2~3h,直到DNA沉淀下来,12,000rpm离心10min;
8)弃上清,用70%的乙醇洗两次,晾干;
9)沉淀溶解于30~60μL水中,-20℃储存备用。
2.PCR反应***包括:
DNA模板 80~100ng
正向引物及反向引物 各1μM
10×PCR缓冲液 2.5μL
25mM MgCl2 2μL
2.5mM dNTP 2μL
Taq DNA聚合酶 1U
无菌水 补至25μL。
PCR程序:
在PCR反应***中加入所需组分按以下程序进行PCR扩增。
94℃ 5min
94℃ 1min
67℃ 1min
72℃ 1.5min 循环30次
72℃ 7min
4℃ 停止。
扩增出来的PCR产物回收连接到pMD18T-Simple vector(TaKaRa)进行测序。利用大肠杆菌E.coli BL21(DE3)作为替代宿主,将含SufS基因的质粒PET-SufS+转入E.coli BL21(DE3)中使其过表达。
3.嗜酸硫化芽孢杆菌半胱氨酸脱硫酶的重组表达
1)对照菌(即只转PET-His载体的E.coli BL21(DE3))和重组菌(转质粒PET-SufS+的E.coli BL21(DE3)分别挑取单菌落接入5ml含氨苄青霉素(100μg/ml)的LB培养基中,37℃摇床培养过夜。
2)取50μl过夜培养物接入到5ml含氨苄青霉素(100μg/ml)的LB培养基中,37℃摇床培养至对数期(OD600为0.5)。
3)在培养基中加入0.1mM的IPTG,继续在37℃摇床中培养6h。
4)取1ml的培养物于EP管中5000g离心10min,沉淀重悬于100μl磷酸盐缓冲液(PBS)中,加入2×SDS凝胶加样缓冲液(购自上海生工公司)100μl,100℃煮沸5min。
5)上述样品上样于12%SDS聚丙烯酰胺凝胶。
6)考马斯亮蓝染色(参见图1)。
4、嗜酸硫化芽孢杆菌半胱氨酸脱硫酶的纯化
该重组蛋白在上清当中,因此我们按照上清His-tag融合蛋白在上清的纯化方法纯化该蛋白。
1)100ml诱导菌液,离心(5000g,10min)取沉淀,用10ml bufferA洗涤1遍。
2)用10ml bufferA重悬超声破碎10min(破碎功率60%,5s,5s)。
3)超声破碎的菌液在18000g,4℃离心20min。
4)Ni Sepharose 4Fast Flow(invitrogen)介质用蒸馏水洗涤两次除去酒精,加入BufferA平衡10min。
5)菌裂解液上柱,在4℃轻摇4h。
6)收集流出液在重复上柱2次。
7)用buffer B洗涤介质,共4~5次(每次使介质悬浮起来)。
8)用buffer C洗涤介质,共4~5次(每次使介质悬浮起来)。
9)加入1ml的buffer D,浸泡15min,收集。共收集5管。
5、生长曲线的测定
首先得到含有PET-SufS+质粒的BL21及含有PET-His质粒的BL21相同浓度的菌液作为种子。每组3瓶250ml的锥形瓶装有100ml LB培养基,按1∶100的比例接种浓度相同的含有PET-SufS+质粒的BL21和含有PET-His质粒的BL21菌液。放入37℃,180转摇床培养,在不同的时间段取样用核酸蛋白测定仪(型号smartspe)测OD。测3次取平均值,以时间做横坐标,OD为纵坐标绘制生长曲线(参见图2和4)。
同样用得到的具有相同OD的含有PET-SufS+质粒的BL21及含有PET-His质粒的BL21菌液稀释相同的稀释度,每个稀释度涂3个相同的板(参见图3)。
6、嗜酸硫化芽孢杆菌半胱氨酸脱硫酶的酶活的测定
由于该酶可催化L-半胱氨酸脱硫形成丙氨酸,脱下的硫离子在酸性条件下在三氯化铁存在的情况下与对氨基二甲基苯胺生成亚甲基蓝,在670nm处有吸收峰,其颜色深浅与硫离子的含量成比例进行比色定量。
1)在测酶活前我们首先要测的是H2S的标准曲线,根据标准曲线计算硫离子的浓度。所需试剂有:
吸收液:取5g硫酸锌溶于500ml水中,另取6g氢氧化钠溶于300ml水中,将两种溶液混合,再取70g硫酸铵加入溶液中,搅拌溶解后加50g甘油,加水稀释至1L。
对氨基二甲基苯胺溶液:取25ml硫酸,加入15ml水中,冷却后取6g对氨基二甲基苯胺盐酸盐,溶于上述溶液中,贮于冰箱中。使用时,去取2.5ml贮备液,用1+1硫酸稀释至100ml。氯化铁溶液:称取50g三氯化铁(FeCl3·6H2O)溶于水中,稀释至50ml。若有沉淀需过滤。标准溶液:称取0.71g硫化钠晶体(Na2S·9H2O)溶于新煮沸冷却后的水中,稀释至100ml。标定后用新煮沸冷却后的水稀释成每ml含5ug的标准使用液。
标定方法:取20.00ml 10.0100N的标准溶液置于200ml碘量瓶中,准确加入10.00ml硫化钠标准溶液中,再加新煮沸冷却后的水80ml,立即加入5ml冰醋酸,混匀后在暗处放置2-3min;用0.0100N硫代硫酸钠标准溶液滴定至淡黄色,加入1ml新配置的0.5%淀粉呈蓝色,用少量水冲洗瓶内壁,在继续滴定至蓝色刚好消失为终点(此时有硫生成使溶液微浑浊,要特别注意终点颜色的突变)。记录所用硫代硫酸钠标准溶液的体积V(ml),同时,另取10ml水,做空白滴定,其操作步骤同上。记录空白滴定所用硫代硫酸钠标准溶液的体积V0ml。
硫化氢的浓度按以下公式计算:
硫化氢(H2S,mg/ml)=(V-V0)N·17/10
式中:
V0——滴定空白时,消耗硫代硫酸钠的ml数;
V——滴定硫化氢贮备液时,消耗硫代硫酸钠溶液的ml数;
N——硫代硫酸钠标准溶液的当量浓度;
17——硫化氢的当量。
绘制硫化氢的浓度标准曲线(参见图5)的数据参见表1。
表1
酶活的表示方法为产生1nmol的硫离子为1个酶活单位。测得OD670的数据如表2所示。
表2
通过标准曲线的计算的嗜酸芽孢杆菌的半胱氨酸脱硫酶的酶活为95u/μL,阴性对照为0.84u/μL。
以上各管分别加入1ml对氨基二甲基苯胺使用液,加盖混匀(不要振摇),放置1min,再加入1~2滴三氯化铁溶液,摇匀,放置30min后排除Fe3+的颜色。用2cm比色皿,于波长670nm处,以水为参比,测定吸光度绘制标准曲线。
2)酶活测定步骤:
(1)在厌氧条件下取1ml含有50mmol/l Tris-HCl(pH=8.0),10mmol/l MgCl2,1.0mmol/lL-Cysteine,5mmol/l DTT,10um/l pyridoxal 5’-5phosphate。加入2μl的酶。
(2)反应20min后,加入100μl溶于1.2mol/l的盐酸中的30mmol/l FeCl3以终止反应。
(3)加入100μl溶于7.2mol/l的盐酸中的20mmol/l N,N-dimethy-p-phenediamine dihydrochloride以产生甲基蓝,在黑暗条件下反应30min。
(4)670nm下测OD。
(5)重复3次。
Claims (6)
1.嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶的基因序列,其特征在于所述嗜酸硫化芽孢杆菌TPY(Sulfobacillus acidophilus TPY)已于2010年08月16日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M2010203;
所述嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶的基因序列为:
ATGACACGGACTTTAGTGGATATCCGGCAGGATTTTCCCATTTTGACGCGGGAAATTCACGGTCATCGACTCGTATATCTTGACAGTGCCGCCACCTCCCAAAAGCCGGAGTCGGTCATTCAAGCCATTGACCACTATTATCGACACTCGAATGCCAATGTGCTCCGGAGTGTGCATACGCTCGCGGAGGAGGCGACCACGCTCTATGAAGACGCGCGGCGCAAGGTGGCCCGCTTCATTGGCGCCAAAACCTCGCAAGAAGTGATTTTTACTCGCGGGACCACCGAGAGTTTAAACGTCGTCGCGCGAGGCTGGGGAGATAAATTTGTCCAGCCGGGCGACGAAATTGTCGTGTCACCGATGGAGCATCATTCGAATTTAATTCCTTGGCAGCAATTGGCGCAACGCCGGCAAGCCGTTTTACGCTTTGTGGAATTAAACGGCGACGGCACGATTGCCTTGGACGAGGTCCGTCGGGTGATTAACGCCAAAACCAAAATCGTGGCCCTATCCCGCATTTCCAATGTTCTCGGGACCATTAACCCGATTGCGGAAATCAGTGCTTTAGCCCATCAACAGGGTGCGGTGATGGTGGTGGACGGCGCGCAGTCGGTGCCGCACGAACCGACGGACGTCAAACGTCTGGGAGCGGATTTTTTGGCCTTTTCCGGGCACAAAATGTTAGGGCCGACCGGCATCGGTGTCTTATGGGGACGTCGCGAGTTGTTAGACGCGATGGATCCCGTGCTTTTTGGCGGGGAAATGATTGCCTATGTCGATCGGGAAACGGCGACGTGGGCCGAATTGCCGGCCAAATTGGAGGGCGGCACGCCAAACATCGCCGGAGCCATTGGGTTAGGGGCGGCCGTCGATTATTTAACCGCCATTGGCATGGACGTCGTCGCCGCGCACGGGCAAGCCTTAGGCGAATTGGCCTACCAACGGCTAAAAGCCGTCGGCGGCGTGACCGTTTATGGCCCCCCCGCACCTCGCGGTGCCCTGGTGGCATTTAACCTGGAAGGTATCCATCCCCATGACGTGGCCCAAGTGTTTGACTCCAATGGCGTGGCCATCCGAGCCGGGCATCATTGCGCACAACCGCTGTTGGCGTGGTTAGGCGTGGGATCTACCGCCCGTGCCAGTTTCTATGTCTATAATACCGAGGCTGACATCGATACCTTGGTGGAAGTCATCGGAGAAACGCAGAGGTACTTTCGCTGA。
2.嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶的克隆方法,其特征在于具体步骤如下:
1)提取嗜酸硫化芽孢杆菌TPY的基因组作为模板,扩增引物为:
P1:5′-CA GAATTC ATGACACGGACTTTAGTGG-3′;
P2:5′-TT AAGCTT TCAGCGAAAGTACCTCTGC-3′;
2)扩增出来的PCR产物回收连接到pMD18T-Simple vector(可由日本TaKaRa公司提供)进行测序;
3)双酶切回收连接PET-His构建表达载体PET-His-sufs+进行表达。
3.嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶的重组蛋白,其特征在于其序列如下:
MTRTLVDIRQDFPILTREIHGHRLVYLDSAATSQKPESVIQAIDHYYRHSNANVLRSVHTLAEEATTLYEDARRKVARFIGAKTSQEVIFTRGTTESLNVVARGWGDKFVQPGDEIVVSPMEHHSNLIPWQQLAQRRQAVLRFVELNGDGTIALDEVRRVINAKTKIVALSRISNVLGTINPIAEISALAHQQGAVMVVDGAQSVPHEPTDVKRLGADFLAFSGHKMLGPTGIGVLWGRRELLDAMDPVLFGGEMIAYVDRETATWAELPAKLEGGTPNIAGAIGLGAAVDYLTAIGMDVVAAHGQALGELAYQRLKAVGGVTVYGPPAPRGALVAFNLEGIHPHDVAQVFDSNGVAIRAGHHCAQPLLAWLGVGSTARASFYVYNTEADIDTLVEVIGETQRYFR*。
4.如权利要求3所述的嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶的重组蛋白的制备方法,其特征在于具体步骤如下:
将构建好的表达载体PET-His-sufs+转入表达菌株BL21(DE3)进行表达。首先将BL21(DE3)菌株接入LB培养基,在LB培养基中加入浓度为100μg/ml的氨苄,再放入37℃摇床进行培养,培养到600nm处吸光值OD600为0.5时,加入0.1mM的IPTG;接着放入37℃摇床进行诱导表达6h后,即得嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶的重组蛋白。
5.嗜酸硫化芽孢杆菌TPY半胱氨酸脱硫酶在制备细菌生长促进剂中的应用。
6.如权利要求5所述的应用,其特征在于所述细菌为大肠杆菌、嗜酸硫化芽孢杆菌或氧化亚铁硫杆菌。
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CN105154430A (zh) * | 2015-07-28 | 2015-12-16 | 福建师范大学 | 一种提取革兰氏阳性菌基因组的试剂盒 |
CN106085978A (zh) * | 2016-06-13 | 2016-11-09 | 国家***第三海洋研究所 | Nadph依赖型双辅基苯酚羟化酶还原酶及其制备方法 |
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CN103396964B (zh) * | 2013-08-01 | 2015-04-29 | 中南大学 | 一种浸出硫化矿复合菌群及其复配和应用方法 |
CN105154430A (zh) * | 2015-07-28 | 2015-12-16 | 福建师范大学 | 一种提取革兰氏阳性菌基因组的试剂盒 |
CN106085978A (zh) * | 2016-06-13 | 2016-11-09 | 国家***第三海洋研究所 | Nadph依赖型双辅基苯酚羟化酶还原酶及其制备方法 |
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