CN113430146A - 高效表达石杉碱甲的苏云金芽孢杆菌hw1株及其应用 - Google Patents
高效表达石杉碱甲的苏云金芽孢杆菌hw1株及其应用 Download PDFInfo
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Abstract
本发明公开了一种高效表达石杉碱甲的苏云金芽孢杆菌HW1株及其应用,该菌株为从野生蛇足石杉(Huperzia serrata石杉科,石杉属)内生细菌中分离获得,属于苏云金芽孢杆菌(Bacillus thuringiensis)HW1株,其氨基序列如SEQ ID NO:1所示。本发明苏云金芽孢杆菌HW1株,作为治疗阿尔茨海默病的生物菌株,将其优良的产石杉碱甲高效表达基因转入其他模式菌株中,通过基因改造获得更多的优良性状,此外还可通过诱变育种达到菌株的进一步优化。对其研究将会为生物合成工业化生产石杉碱甲,保护植物资源、解决临床一线用药需求、降低治疗阿尔茨海默病医药费用有着巨大意义。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种高效表达石杉碱甲的苏云金芽孢杆菌菌株,本发明还涉及该菌株的应用。
背景技术
目前,乙酰胆碱酯酶抑制剂类药物在阿尔茨海默病药物市场上处于主导地位。石杉碱甲为从民间草药蛇足石杉中分离获得的新型石松类单体生物碱,是一种强效的可逆性的胆碱酯酶抑制剂,具有多靶点作用,对乙酰胆碱酯酶的抑制作用为毒扁豆碱的3倍、加兰他敏的30倍,而且外周不良反应低,是治疗阿尔茨海默病临床优选药物之一。
源于天然植物的药物开发历史悠久且富有生命力,植物提取获得石杉碱甲是目前最简便的一种方式,但植物提取获得石杉碱甲已面临植物资源匮乏瓶颈;化学合成生产石杉碱甲仍是研发重要途径之一,但合成步骤繁琐、代价昂贵,很难获得纯光学活性的合成物。
因此利用从野生蛇足石杉内生细菌中筛选分离具有产石杉碱甲能力的菌株对于治疗阿尔茨海默病和保护植物资源意义重大。虽然目前国内外已有对产石杉碱甲菌株进行开发和研究,但苏云金芽孢杆菌(Bacillus thuringiensis)具备产石杉碱甲功能却鲜有报道。对已获得的苏云金芽孢杆菌(Bacillus thuringiensis)HW1株可通过生物技术改造菌株为获得石杉碱甲工业化生产提供新的菌种资源。同时,通过对其菌株产石杉碱甲机理的研究,利用合成生物学使其获得更优良稳定的产石杉碱甲性状和能力。
发明内容
本发明的目的是提供一种高效表达石杉碱甲的苏云金芽孢杆菌HW1株,解决植物提取获得石杉碱甲面临植物资源匮乏瓶颈、化学合成步骤繁琐、代价昂贵,很难获得纯光学活性的合成物的问题。
本发明的另一个目的是提供一种高效表达石杉碱甲的苏云金芽孢杆菌HW1株作为治疗阿尔茨海默病的功能菌株在生物合成制药方面的应用。
本发明所采用的第一种技术方案是,一种高效表达石杉碱甲的苏云金芽孢杆菌HW1株,菌株保藏于中国普通微生物菌种保藏中心,保藏编号:CGMCC No.21398,保藏日期为2020年12月18日。
本发明所采用第一种技术方案的特点还在于,
菌株从野生蛇足石杉(Huperzia serrata)内生细菌中分离获得,属于苏云金芽孢杆菌(Bacillus thuringiensis)HW1株,其氨基酸序列如SEQ ID NO:1所示。
菌株高效表达石杉碱甲培养条件为:
(1)培养基:马铃薯300g,葡萄糖20g,蒸馏水1000ml;
(2)培养条件:25℃,220r/min;
(3)装液量:250ml锥形瓶,装液量为100ml;
(4)培养时间:3天。
菌株的实验室保存条件为:(1)菌株每次在培养基固体斜面传代后,生长时间为24h;(2)保存菌种条件为4℃,12h后再将其放置-25℃冰箱中进行保存。
本发明所采用的第二个技术方案是,一种高效表达石杉碱甲的苏云金芽孢杆菌HW1株作为治疗阿尔茨海默病的功能菌株在生物合成制药方面的应用,菌株从野生蛇足石杉(Huperzia serrata)内生细菌中分离获得,属于苏云金芽孢杆菌(Bacillusthuringiensis)HW1株,保藏编号:CGMCC No.21398。
本发明所采用的第三个技术方案是,一种高效表达石杉碱甲的苏云金芽孢杆菌HW1株的应用,通过转基因、诱变育种对细菌菌株进行基因改造,以获得表达效率更高、遗传稳定性更好的系列菌株。
本发明的有益效果是,本发明一种高效表达石杉碱甲的苏云金芽孢杆菌HW1株,作为治疗阿尔茨海默病的生物菌株,将其优良的产石杉碱甲高效表达基因转入其他模式菌株中,通过基因改造获得更多的优良性状,此外还可通过诱变育种达到菌株的进一步优化。对其研究将会为生物合成工业化生产石杉碱甲,保护植物资源、解决临床一线用药需求、降低治疗阿尔茨海默病医药费用有着巨大意义。
附图说明
图1是本发明苏云金芽孢杆菌HW1株产石杉碱甲能力的高效液相检测图;
图2是本发明石杉碱甲标准品高效液相检测图;
图3为本发明琼脂糖凝胶电泳检测本发明细菌菌株中所抽提DNA的PCR产物电泳图;
图4为本发明高效表达石杉碱甲的细菌菌株形态图。
具体实施方式
下面结合附图和具体实施方式对本发明进行详细说明。
本发明一种高效表达石杉碱甲的苏云金芽孢杆菌HW1株,该菌株为从野生蛇足石杉(Huperzia serrata石杉科,石杉属)内生细菌中分离获得,属于苏云金芽孢杆菌(Bacillus thuringiensis)HW1株,其氨基酸序列如SEQ ID NO:1所示。
该菌株已在国家知识产权局指定的保藏单位中国普通微生物菌种保藏中心保藏。
生物材料保藏信息:
名称:苏云金芽孢杆菌(Bacillus thuringiensis)HW1株
保藏编号:CGMCC No.21398
保藏单位:中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)
保藏时间:2020年12月18日
地址:北京市朝阳区北辰西路1号院3号
(一)本发明高效表达石杉碱甲菌株的分离方法如下:
将新鲜蛇足石杉植株用自来水冲洗后,先用浓度为75%的酒精进行表面消毒30s,无菌水冲洗4次,接着用浓度为10%的NaClO浸泡5min,无菌水冲洗4次,最后再用75%乙醇表面消毒30s,无菌水冲洗4次后,用无菌刀片将茎切成0.5cm大小,叶片切成0.3cm×0.3cm的小块,接种到含有15μg/ml链霉素和1mg/mL脱氧胆酸钠的固体培养基上,倒置于25℃培养箱中进行暗培养。为了检验上述表面消毒是否彻底,同时把在消毒过程中最后一次冲洗组织块的无菌水涂布在培养基上进行培养。2d后,无菌操作法挑取在茎段和叶片切口上所长出的菌丝,平板划线法转接到新鲜的固体培养基平板上,重复上述操作直到得到纯化的内生菌为止。
(二)高效表达石杉碱甲菌株发酵液制备:将上述分离得到的纯化内生菌分别接入装有100mL液体培养基的250ml锥形瓶(每个菌株接3瓶),置于摇床上,25℃、220r/min振荡培养3d,设置3个重复,内生细菌在振荡培养2d后,发酵液于10000r/min离心15min。
(三)高效表达石杉碱甲菌株产物分析测定:取发酵液离心后的上清液500ml,采用二氯甲烷和乙醚提取方法提取石杉碱甲,用0.01mol/L的盐酸溶解定容至5mL,用高效液相法测定发酵上清液产石杉碱甲的含量。高效液相(HPLC)色谱柱为Agilent Cl8柱(4.60mm×250mm,5μm);色谱条件为:流动相为磷酸盐缓冲液-乙腈(86:14);流量1.0ml/min;柱温25℃,进样量20μL;检测波长310nm,以石杉碱甲标准品作对照。
精密称取石杉碱甲标准品,用0.01mol/L盐酸溶液溶解并依次制备成2,0.2,0.02,0.002,0.0002mg/mL质量浓度,分别进样20μL。每次5次重复,以确定精密度良好。以峰面积与对应石杉碱甲标准品质量绘制标准曲线,得线性回归方程。通过线性回归方程计算分离获得产石杉碱甲菌株代谢产物含量。如图1及图2所示,为本发明产石杉碱甲能力与石杉碱甲标准品高效液相检测对照图,图1为石杉碱甲标准品高效液相色谱,图2为本发明细菌菌株高效液相色谱;结果表明石杉碱甲标准品出峰时6.678min(图1),菌株HW1发酵液提取物出峰时间6.866min(图1),二者出峰时间接近,当向菌株HW1发酵液提取物中添加标准品后HPLC出峰时间重叠,结果表明HW1发酵液提取液中存在石杉碱甲。
(四)高效表达石杉碱甲菌株分子生物学鉴定:
1.基因组DNA提取
1)取1.5mL离心管,加入200μL预处理液和三颗玻璃珠,然后加入适量紫云金杆菌样品,放入研磨仪中研磨至充分。
2)加入20μL蛋白酶(Proteinase)K溶液,混匀,37℃放置30~60min。
3)加入200μL裂解液,充分颠倒混匀,70℃放置10min。
4)加200μL无水乙醇,充分颠倒混匀,离心以去除管盖内壁液滴。
5)过吸附柱,洗涤液洗1次,漂洗液洗2次。
6)吸附柱室温放置5~10分钟,以彻底晾干吸附材料中残余的漂洗液。
7)将吸附材料转入一个新离心管,向吸附膜中间位置悬空滴加50~100μLddHW1O,室温放置5~10min,12000rpm离心2min,将溶液收集到离心管中。
2.16S扩增
1)引物序列:
27F:5’-AGAGTTTGATCCTGGCTCAG-3’;
1492R:5’-TACGGCTACCTTGTTACGACTT-3’;
2)PCR反应体系
3)PCR循环条件
3.PCR产物检测及纯化
3μL PCR产物进行1.0%的琼脂糖凝胶检测,Marker条带从上到下依次为5000bp、3000bp、2000bp、1000bp、750bp、500bp、250bp、100bp。电泳PCR产物结果如图3所示,图3中,a为菌株检测区,b为Marker对照区;图3(a)为菌株检测区,图3(b)为Marker对照区,2000bp条带浓度为60ng/3ul显示为加亮带,其余条带浓度均为30ng/3μl。结果:目的检测HW1菌株DNA扩增条带经琼脂糖凝胶电泳测定大小约为1000~2000bp,且上下无杂带。
PCR产物纯化按照磁珠纯化标准操作流程操作,利用磁珠能够吸附或者释放带电荷物质的原理,在高盐低pH溶液吸附DNA,在低盐高pH溶液释放DNA,从而达到分离提纯DNA产物的目的。
4.测序
PCR产物结果鉴定,委托北京六合华大基因科技有限公司进行测序。
根据blast结果,菌株HW1与Bacillus thuringiensis亲源关系最近。
本发明高效表达石杉碱细菌菌株具有下述特征:
1.镜下形态特征
图4为本发明高效表达石杉碱甲的细菌菌株形态图,图4(a)为光学显微镜下菌体形态图,图4(b)为菌落形态图,如图4(a)所示,该菌株G+杆菌,革兰氏染色紫色,呈椭圆形杆,排列成短链或长链,芽孢为椭圆形近中间生长,芽孢囊微膨大。
2.菌落形态特征
如图4(b)所示,该细菌菌株在固体培养基上菌落大,淡黄色至黄色、圆形或椭圆形、边缘不整齐呈滴蜡状。
3.该细菌菌株高效表达石杉碱甲的培养条件为:
(1)培养基:马铃薯300g,葡萄糖20g,蒸馏水1000ml。
(2)培养条件:25℃,220r/min。
(3)装液量:250ml锥形瓶,装液量为100ml。
(4)培养时间:3天。
本发明的HW1株为苏云金芽孢杆菌(Bacillus thuringiensis)细菌,经液体发酵培养提取代谢产物,可获得较高产量石杉碱甲。
本发明细菌菌株的16S与同属的相同种的比对结果如下:
与Bacillus thuringiensis strain FDAARGOS_796的相似度为99.86%;
与Bacillus mobilis HFBP14的相似度为99.86%;
与Bacillus paranthracis KUBOTAB9的相似度为99.86%。
该属鲜有报道具有产石杉碱甲能力。通过对其机理研究,可以进一步加深菌株产石杉碱甲中高效表达基因的研究,从而构建新的转基因生物,使其获得更高效的表达。苏云金芽孢杆菌(Bacillus thuringiensis)HW1株作为一种新的产石杉碱甲菌株,将为生物合成途径合成石杉碱甲提供新的菌种资源。
SEQUENCE LISTING
<110> 西安医学院
<120> 高效表达石杉碱甲的苏云金芽孢杆菌HW1株及其应用
<130> 2021
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1435
<212> DNA
<213> 苏云金芽孢杆菌(Bacillus thuringiensis)
<400> 1
tcaggatgaa cgctggcggc gtgcctaata catgcaagtc gagcgaatgg attgagagct 60
tgctctcaag aagttagcgg cggacgggtg agtaacacgt gggtaacctg cccataagac 120
tgggataact ccgggaaacc ggggctaata ccggataata ttttgaaccg catggttcga 180
aattgaaagg cggcttcggc tgtcacttat ggatggaccc gcgtcgcatt agctagttgg 240
tgaggtaacg gctcaccaag gcaacgatgc gtagccgacc tgagagggtg atcggccaca 300
ctgggactga gacacggccc agactcctac gggaggcagc agtagggaat cttccgcaat 360
ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa ggctttcggg tcgtaaaact 420
ctgttgttag ggaagaacaa gtgctagttg aataagctgg caccttgacg gtacctaacc 480
agaaagccac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttat 540
ccggaattat tgggcgtaaa gcgcgcgcag gtggtttctt aagtctgatg tgaaagccca 600
cggctcaacc gtggagggtc attggaaact gggagacttg agtgcagaag aggaaagtgg 660
aattccatgt gtagcggtga aatgcgtaga gatatggagg aacaccagtg gcgaaggcga 720
ctttctggtc tgtaactgac actgaggcgc gaaagcgtgg ggagcaaaca ggattagata 780
ccctggtagt ccacgccgta aacgatgagt gctaagtgtt agagggtttc cgccctttag 840
tgctgaagtt aacgcattaa gcactccgcc tggggagtac ggccgcaagg ctgaaactca 900
aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960
aagaacctta ccaggtcttg acatcctctg aaaaccctag agatagggct tctccttcgg 1020
gagcagagtg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa 1080
gtcccgcaac gagcgcaacc cttgatctta gttgccatca ttaagttggg cactctaagg 1140
tgactgccgg tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat 1200
gacctgggct acacacgtgc tacaatggac ggtacaaaga gctgcaagac cgcgaggtgg 1260
agctaatctc ataaaaccgt tctcagttcg gattgtaggc tgcaactcgc ctacatgaag 1320
ctggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc gggccttgta 1380
cacaccgccc gtcacaccac gagagtttgt aacacccgaa gtcagtgggg taacc 1435
Claims (6)
1.一种高效表达石杉碱甲的苏云金芽孢杆菌HW1株,其特征在于,所述菌株保藏于中国普通微生物菌种保藏中心,保藏编号:CGMCC No.21398。
2.根据权利要求1所述的一种高效表达石杉碱甲的苏云金芽孢杆菌HW1株,其特征在于,所述菌株从野生蛇足石杉(Huperzia serrata)内生细菌中分离获得,属于苏云金芽孢杆菌(Bacillus thuringiensis)HW1株,其氨基酸序列如SEQ ID NO:1所示。
3.根据权利要求2所述的一种高效表达石杉碱甲的苏云金芽孢杆菌HW1株,其特征在于,所述菌株高效表达石杉碱甲培养条件为:
(1)培养基:马铃薯300g,葡萄糖20g,蒸馏水1000ml;
(2)培养条件:25℃,220r/min;
(3)装液量:250ml锥形瓶,装液量为100ml;
(4)培养时间:3天。
4.根据权利要求2所述的一种高效表达石杉碱甲的苏云金芽孢杆菌HW1株,其特征在于,所述菌株的实验室保存条件为:(1)菌株每次在培养基固体斜面传代后,生长时间为24h;(2)保存菌种条件为4℃,12h后再将其放置-25℃冰箱中进行保存。
5.一种高效表达石杉碱甲的苏云金芽孢杆菌HW1株作为治疗阿尔茨海默病的功能菌株在生物合成制药方面的应用,所述菌株从野生蛇足石杉(Huperzia serrata)内生细菌中分离获得,属于苏云金芽孢杆菌(Bacillus thuringiensis)HW1株,保藏编号:CGMCCNo.21398。
6.一种高效表达石杉碱甲的苏云金芽孢杆菌HW1株的应用,其特征在于,通过转基因、诱变育种对细菌菌株进行基因改造,以获得表达效率更高、遗传稳定性更好的系列菌株。
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