CN115089733A - 用于治疗高赖氨酸血症的组合物及应用 - Google Patents
用于治疗高赖氨酸血症的组合物及应用 Download PDFInfo
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Abstract
本发明提供一种用于治疗高赖氨酸血症的组合物及应用。该组合物的活性成分为代谢赖氨酸的工程菌I和II;菌I是以大肠杆菌为出发菌株,通过敲除ldcC1和ldcC2基因,并在大肠杆菌中表达来自酿酒酵母菌BY4741的LKR和SDR基因构建得到的;菌II是以大肠杆菌为出发菌株,通过敲除ldcC1和ldcC2基因,并在大肠杆菌中表达来自酿酒酵母菌BY4741的lys5和lys2基因构建得到的。本发明以两种工程菌作为宿主菌株,通过两种菌株的复合、包被,从而为利用益生菌组治疗基因缺陷型疾病(如高赖氨酸血症)奠定基础。
Description
技术领域
本发明属于生物医药领域,具体地说,涉及一种用于治疗高赖氨酸血症的组合物及应用。
背景技术
高赖氨酸血症(Hyperlysinemia),是一种罕见的常染色体隐性遗传性的代谢紊乱疾病,由于分解赖氨酸的酶缺乏引起,其特征是血液中赖氨酸的浓度异常增高。高赖氨酸血症确诊年龄多集中在5-10岁。高赖氨酸血症可分为Ⅰ型和II型。通常Ⅰ型患者的临床症状并不明显,仅仅是血液中赖氨酸浓度偏高;而II型患者血液中除了赖氨酸浓度升高,还伴有酵母氨酸浓度增高,患者会表现出严重的神经损伤和发育迟缓,多数患者在成年之前便死亡。目前高赖氨酸血症的致病基因已经明确,即赖氨酸降解途径的关键酶:双功能酶α-氨基半醛合酶(α-aminoadipic semialdehyde synthase,AASS)。AASS基因发生基因突变会造成高赖氨酸血症,该疾病目前无特效药,临床只能通过给病人食用不含赖氨酸的食物来缓解病症。
大肠杆菌Nissle 1917(EcN),是一种肠道益生菌,该益生菌具有多方面的优良性能,如改善肠道微生物群落,抑制病原菌生长,不含肠毒素等对人体致病的有害因子,对肿瘤具有高靶向性。研究发现EcN对常见胃肠道疾病有良好疗效,被广泛用于预防传染性腹泻、溃疡性结肠炎等炎性肠疾病,防止新生儿消化道内病原菌定殖等被认为是一种安全菌株。近年来,EcN表达外源基因作为疾病诊治载体的研究越来越多,其作为微观生活的“机器人工厂”,能特异性定植于人体,长期使用具有安全性和遗传可追踪性,可用于实体诊断的临床研究中,具有作为运送载体对疾病进行治疗的潜在应用价值。
目前,利用合生生物学构建工程菌株治疗高赖氨酸血症尚未进行人类临床研究。
发明内容
本发明的目的是提供一种用于治疗高赖氨酸血症的组合物及应用。
为了实现本发明目的,第一方面,本发明提供一种用于治疗高赖氨酸血症的组合物,其活性成分为代谢赖氨酸的工程菌I和II。
其中,工程菌I是以大肠杆菌为出发菌株,通过敲除ldcC1和ldcC2基因,并在大肠杆菌中表达来自酿酒酵母菌BY4741的LKR和SDR基因构建得到的。
优选地,所述大肠杆菌为Nissle 1917。
ldcC1基因为:
a1)SEQ ID NO:1所示的核苷酸序列;
b1)SEQ ID NO:1所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;
c1)在严格条件下与SEQ ID NO:1所示序列杂交且表达相同功能蛋白质的核苷酸序列,所述严格条件为在含0.1%sds的0.1×sspe或含0.1%sds的0.1×ssc溶液中,在65℃下杂交,并用该溶液洗膜;或
d1)与a1)、b1)或c1)的核苷酸序列具有90%以上同源性且表达相同功能蛋白质的核苷酸序列;
ldcC2基因为:
a2)SEQ ID NO:2所示的核苷酸序列;
b2)SEQ ID NO:2所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;
c2)在严格条件下与SEQ ID NO:2所示序列杂交且表达相同功能蛋白质的核苷酸序列,所述严格条件为在含0.1%SDS的0.1×SSPE或含0.1%SDS的0.1×SSC溶液中,在65℃下杂交,并用该溶液洗膜;或
d2)与a2)、b2)或c2)的核苷酸序列具有90%以上同源性且表达相同功能蛋白质的核苷酸序列;
LKR基因为:
a3)SEQ ID NO:3所示的核苷酸序列;
b3)SEQ ID NO:3所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;
c3)在严格条件下与SEQ ID NO:3所示序列杂交且表达相同功能蛋白质的核苷酸序列,所述严格条件为在含0.1%SDS的0.1×SSPE或含0.1%SDS的0.1×SSC溶液中,在65℃下杂交,并用该溶液洗膜;或
d3)与a3)、b3)或c3)的核苷酸序列具有90%以上同源性且表达相同功能蛋白质的核苷酸序列;
SDR基因为:
a4)SEQ ID NO:4所示的核苷酸序列;
b4)SEQ ID NO:4所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;
c4)在严格条件下与SEQ ID NO:4所示序列杂交且表达相同功能蛋白质的核苷酸序列,所述严格条件为在含0.1%SDS的0.1×SSPE或含0.1%SDS的0.1×SSC溶液中,在65℃下杂交,并用该溶液洗膜;或
d4)与a4)、b4)或c4)的核苷酸序列具有90%以上同源性且表达相同功能蛋白质的核苷酸序列;
工程菌II是以大肠杆菌为出发菌株,通过敲除ldcC1和ldcC2基因,并在大肠杆菌中表达来自酿酒酵母菌BY4741的lys5和lys2基因构建得到的。
优选地,所述大肠杆菌为Nissle 1917。
lys5基因为:
a5)SEQ ID NO:5所示的核苷酸序列;
b5)SEQ ID NO:5所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;
c5)在严格条件下与SEQ ID NO:5所示序列杂交且表达相同功能蛋白质的核苷酸序列,所述严格条件为在含0.1%SDS的0.1×SSPE或含0.1%SDS的0.1×SSC溶液中,在65℃下杂交,并用该溶液洗膜;或
d5)与a5)、b5)或c5)的核苷酸序列具有90%以上同源性且表达相同功能蛋白质的核苷酸序列;
lys2基因为:
a6)SEQ ID NO:6所示的核苷酸序列;
b6)SEQ ID NO:6所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;
c6)在严格条件下与SEQ ID NO:6所示序列杂交且表达相同功能蛋白质的核苷酸序列,所述严格条件为在含0.1%SDS的0.1×SSPE或含0.1%SDS的0.1×SSC溶液中,在65℃下杂交,并用该溶液洗膜;或
d6)与a6)、b6)或c6)的核苷酸序列具有90%以上同源性且表达相同功能蛋白质的核苷酸序列。
进一步地,工程菌I的构建方法包括:
分别构建靶向ldcC1和ldcC2基因的CRISPR-Cas9系统,共同导入大肠杆菌得到重组菌,然后将来自酿酒酵母菌BY4741的LKR和SDR基因通过质粒导入所述重组菌中,即得代谢赖氨酸的工程菌;其中,CRISPR-Cas9系统中靶向ldcC1基因的gRNA的核苷酸序列为:5′-CTGTTTAAATATGTTCGTGA-3′,靶向ldcC2基因的gRNA的核苷酸序列为:5′-CAATATGCGTATTCAGGATC-3′。
进一步地,工程菌II的构建方法包括:
分别构建靶向ldcC1和ldcC2基因的CRISPR-Cas9系统,共同导入大肠杆菌得到重组菌,然后将来自酿酒酵母菌BY4741的lys5和lys2基因通过质粒导入所述重组菌中,即得代谢赖氨酸的工程菌;其中,CRISPR-Cas9系统中靶向ldcC1基因的gRNA的核苷酸序列为:5′-CTGTTTAAATATGTTCGTGA-3′,靶向ldcC2基因的gRNA的核苷酸序列为:5′-CAATATGCGTATTCAGGATC-3′。
所述组合物中工程菌I和II的活菌数比为1:1~1:4,优选1:1、1:2、1:3或1:4,更优选1:2。
第二方面,本发明提供一种复合菌剂的制备方法,将代谢赖氨酸的工程菌I和II按一定比例混合后发酵培养,发酵液经离心,菌体沉淀用去离子水重悬清洗2~3次,然后将菌体沉淀经海藻酸钠包被形成微球,即得。
进一步地,代谢赖氨酸的工程菌I和II按OD600比为1:1~1:4混合,优选1:1、1:2、1:3或1:4,更优选1:2。
进一步地,将两种菌体沉淀混合后,取0.1~2g混合物,向其中加入2%海藻酸钠溶液0.1~5mL,搅拌均匀得到海藻酸钠微生物胶体;将1mL胶体匀速滴入1~4%氯化钙溶液中,在30~37℃、转速100~700r/min条件下交联2~15min。
第三方面,本发明提供按照所述方法制备的复合菌剂。
第四方面,本发明提供所述组合物或所述复合菌剂在制备用于改善或治疗基因缺陷型赖氨酸紊乱疾病的药物或保健品中的应用。
所述疾病包括但不限于高赖氨酸血症。
本发明通过改造肠道微生物中的共生细菌,实现对肠道菌群状态的靶向调控,可以有效改善宿主赖氨酸水平的健康状态。该方法可塑性较强,可调控的靶标范围广、调控针对性强,副作用少,成为生物医学开发的重要方法。
本发明以两种EcN工程菌作为宿主菌株,通过两种菌株的复合、包被,从而为利用益生菌组治疗基因缺陷型疾病(如高赖氨酸血症)提供新思路。
附图说明
图1为本发明较佳实施例中pTrc99a质粒、LKR、SDR基因电泳图;M:DL5000Maker;1:pTrc99a质粒;2:LKR基因;3:SDR基因。
图2为本发明较佳实施例中pTLS质粒图谱。
图3为本发明较佳实施例中lys5基因电泳图。
图4为本发明较佳实施例中lys2-1、lys2基因电泳图。
图5为本发明较佳实施例中pK18mobSacB质粒及酶切电泳图。
图6为本发明较佳实施例中pK25质粒图谱。
图7为本发明较佳实施例中菌发酵pTLS和pK25比例分别为1:1、1:2、1:3、1:4的生长及代谢情况比较;(A)混菌发酵菌体生长速率情况比较;(B)混菌发酵菌体耗糖情况比较;(C)混菌发酵菌体消耗赖氨酸情况比较。
图8为本发明较佳实施例中海藻酸钠微球的形态与性能分析;(A)海藻酸钠微球形态;(B)扫描电子显微镜下的海藻酸钠微球形态;(C)海藻酸钠微球的生长情况;(D)海藻酸钠微球的代谢情况。
图9为本发明较佳实施例中小鼠血浆赖氨酸水平。
图10为本发明较佳实施例中小鼠血浆酵母氨酸代谢水平。
*、**和****分别表示5%、1%和0.01%水平上的显著性。
具体实施方式
本发明提供一种混合益生菌剂及其包被方法和应用。
本发明采用如下技术方案:
本发明提供由两种合成益生菌组成的复合菌剂,将菌1和菌2分别发酵培养后,按照一定比例将菌1和菌2进行混合,用海藻酸钠对复合菌剂进行包被,形成微球。采用固定比例的复合菌剂用于快速利用酵母氨酸途径代谢赖氨酸,从而实现赖氨酸紊乱疾病如高赖氨酸血症的治疗。
本发明采用混合益生菌治疗赖氨酸代谢疾病的方法,该方法在益生菌中构建了一条外源表达的用于赖氨酸代谢的酵母氨酸通路。该通路的特点是,通过基因工程改造,赖氨酸在酵母氨酸合成酶的催化下转化为酵母氨酸,并继续在己二酸半醛合成酶的催化下转化为己二酸半醛,并阻断赖氨酸生成戊二胺的途径,促进赖氨酸向己二酸的转化。
本方法可用于赖氨酸代谢相关疾病,如高赖氨酸血症的治疗、改善或相关保健食品中的应用。
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例均按照常规实验条件,如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001),或按照制造厂商说明书建议的条件。
以下实施例中涉及的菌株信息见表1,质粒信息见表2,引物信息见表3。
表1
菌株 | 描述 | 来源 |
E.coli DH5α | / | TaKaRa大连宝生物公司 |
E.coli Nissle 1917(EcN) | 编号DM6601 | 购自德国微生物菌种中心 |
E.coli Nissle 1917ΔldcC1(EcNO) | 缺失ldcC1基因 | 本发明构建 |
E.coli Nissle 1917ΔldcC1 ΔldcC2(EcNT) | 缺失ldcC1,ldcC2基因 | 本发明构建 |
表2
实施例1代谢赖氨酸的工程菌的构建方法
1、LKR、SDR酶基因的获得
以pTrc99a质粒为模板,pTrc99a-XhoI-F和pTrc99a-EcorI-R为上下游引物,获得带有XhoI、EcoRI双酶切位点的pTrc99a载体片段,以酿酒酵母菌BY4741(购自中国普通微生物菌种保藏中心,编号bio-82064)为模板,分别以LKR-EcoRI-F和LKR-XhoI-R,SDR-XhoI-R和SDR-NdeI-F为上下游引物PCR扩增LKR、SDR基因,获得带有带有XhoI、EcoRI双酶切位点的LKR基因片段和带有XhoI、NdeI双酶切位点的SDR基因片段。LKR、SDR基因和pTrc99a载体片段大小为:LKR 1119bp,SDR 1399bp,pTrc99a 4176bp。琼脂糖凝胶电泳见图1。
2、宿主菌株EcN中ldcC1和ldcC2基因的敲除
采用CRISPR-Cas9技术敲除大肠杆菌Nissle 1917中的ldcC1和ldcC2基因。双质粒系统由pREDCas和pGRB质粒组成(表2),用于基因组编辑。pREDCas9质粒主要用于构建Cas9的结构表达和CRISPR介导的大肠杆菌基因组编辑质粒凝固系统的诱导表达。利用pGRB质粒在大肠杆菌中表达gRNA。
为了构建gRNA质粒,利用一套引物对表达gRNA的pGRB质粒进行PCR扩增,引导Cas9靶向到目的基因。运用SnapGene 4.3.6对待敲除的基因进行设计,在敲除基因的PAM位点后选20bp的gRNA序列加入引物中(靶向ldcC1基因的gRNA的核苷酸序列为:5′-CTGTTTAAATATGTTCGTGA-3′,靶向ldcC2基因的gRNA的核苷酸序列为:5′-CAATATGCGTATTCAGGATC-3′)。pGRB质粒模板,高速高保真PCR酶PrimeSTAR Max DNAPolymerase为限制酶,相应上下游引物进行PCR扩增反应,PCR反应体系见表4。
表4 PCR扩增反应体系
试剂 | 体积(μL) |
pGRB | 1 |
PrimeSTAR Max Premix(2×) | 25 |
gRNA-up(10mM) | 2 |
gRNA-down(10mM) | 2 |
灭菌水 | 20 |
总体系 | 50 |
将上述试剂在PCR管中充分混匀,置于PCR仪中进行反应,PCR反应条件为:98℃,10min热变性;98℃,10s,55℃,15s,72℃,1min,循环32次;72℃,10min;4℃保存。
将回收的gRNA片段与T4 PNK进行非放射性磷酸化,在冰上的微离心管中进行以下反应:反应体系为DNA 7μL,T4 PNK反应缓冲液(10×)1μL,ATP(10mM)1μL,T4 PNK 0.2μL。37℃孵育30分钟,然后加入T4连接酶0.5μL,4℃过夜进行连接反应。将获得的gRNA质粒转化到DH5α中进行复制,并使用引物check-sgRNA-R/check-sgRNA-F进行测序验证。通过测序证实了gRNA质粒构建成功,得到ldcC1-gRNA和ldcC2-gRNA。
为了生成供体DNA(donor DNA),可以通过三步PCR获得donor基因。以ldcC1基因为例:以大肠杆菌Nissle 1917基因组为模板,在ldcC1基因的上游和下游扩增引物ldcC1-up-R/ldcC1-up-F和ldcC1-down-R/ldcC1-down-F。利用引物ldcC1-up-R/ldcC1-down-R对两个片段进行重叠扩增,获得ldcC1 donor DNA。以同样的方法获得ldcC2 donor DNA。将获得的供体DNA进一步连接到载体上,用于保存和测序。Blunting Kination反应:在微量离心管中配制以下反应液:donor DNA 2μL,10×Blunting Kination Buffer 1μL,BluntingKination Enzyme Mix 0.5μL,ddH2O 6.5μL。37℃反应10分钟。0℃热处理5分钟。连接反应:上述体系2.5μL,对照质粒(pUC118/Hind III/BAP)0.5μL,Ligation Solution I 3μL。16℃反应1小时。全量反应液转化50μL的DH5α细胞,在含有Amp、IPTG、X-Gal的平板培养基上进行蓝白斑筛选获得相应的转化子。
3、pTrc99a-LKR-SDR(pTLS)重组质粒的构建
采用双酶切连接的方式将基因片段与载体质粒进行连接。首先使用XhoI、EcoRI双酶切LKR和pTrc99a质粒,T4连接酶进行连接,得到pTrc99a-LKR重组质粒。为了后期能够表征重组质粒表达蛋白情况,将该重组质粒引入红色荧光蛋白(RFP)基因,以pK18mobSacB为模板,pBb-XbaI-F和pBb-EcoRI-R为上下游引物进行PCR扩增,获得引入XbaI、EcoRI酶切位点的RFP基因,以LKR-EcoRI-F和LKR-XbaI-R为上下游引物,PCR扩增pTrc99a-LKR片段,得到引入XbaI、EcoRI酶切位点pTrc99a-LKR片段,将RFP基因和pTrc99a-LKR片段进行XbaI,EcoRI双酶切,T4连接酶进行连接,得到pTrc99a-LKR-RFP重组质粒。
以pBb-SDR-NdeI-R和pTrc99a-XhoI-F为上下游引物扩增pTrc99a-LKR-RFP重组片段,得到引入XhoI、NdeI双酶切的载体片段,将SDR基因和pTrc99a-LKR-RFP片段进行XhoI、NdeI双酶切,T4连接酶进行连接,得到pTrc99a-LKR-RFP-SDR重组质粒。将该重组进行菌落PCR阳性筛选后送测序。经测序结果证明:得到重组质粒pTrc99a-LKR-RFP-SDR,将该重组质粒命名为pTLS(图2)。
4、pK18-LYS5-LYS2(pK25)重组质粒的构建
为了从酿酒酵母菌BY4741上获得lys5,lys2基因,首先利用TaKaRa提基因组试剂盒对酿酒酵母菌BY4741进行基因组提取。以BY4741基因组为模板,为将lys5基因加入EcoRⅠ与XbaⅠ酶切位点为后续酶切连接反应做准备,设计引物lys5-EcoRI-F和lys5-XbaI-R为上下游引物进行PCR扩增反应,琼脂糖凝胶电泳结果如图3所示,得到引入EcoRⅠ与XbaⅠ酶切位点的lys5基因片段,其大小约为863bp。
lys2基因的获得,因其引物特异性较差,因此采用巢氏PCR的方法进行扩增。首先以BY4741基因组为模板,以lys2-up和lys2-down为上下游引物,进行第一轮PCR,PCR扩增产物进行琼脂糖凝胶电泳,胶回收得到基因片段为lys2-1,其大小约为4179bp。为引入双酶切位点,以lys2-XbaI-F和lys2-HindIII-R为上下游引物,lys2-1为模板,进行第二轮PCR扩增反应。琼脂糖凝胶电泳结果如图4所示,得到引入XbaI与HindIII酶切位点的lys2基因片段,其大小约为4203bp。
为了构建重组质粒,选择pK18mobSacB质粒为载体,其质粒大小为5721bp。EcoRI和XbaI双酶切质粒得到片段大小为5694bp和27bp,pK18mobSacB质粒的提取以及酶切验证琼脂糖凝胶电泳图如图5所示。
lys5基因与pK18mobSacB载体的连接,采用双酶切连接的方法,将lys5基因片段与pK18mobSacB载体分别进行EcoRI和XbaI双酶切反应,T4连接酶进行连接反应,将连接反应产物转化至E.coli DH5α感受态细胞中,将转化成功的菌落进行阳性克隆筛选后送测序。经测序结果证明:得到重组质粒pK18-LYS5。将lys2基因与pK18-LYS5重组质粒使用XbaI和HindIII双酶切,lys2酶切片段长度为4203bp,pK18-LYS5载体片段的长度为6515bp和24bp,其中24bp远离凝胶。酶切产物经T4连接酶连接反应后转化至E.coli DH5α感受态细胞中,得到的转化子经过菌落PCR反应阳性筛选后送测序,进一步验证重组质粒是否构建成功。经测序结果表明,得到重组质粒pK18-LYS5-LYS2,将该重组质粒命名为pK25(图6)。
为了构建重组菌株,制备了EcN感受态细胞,将pREDCas9转化到该细胞中,得到EcN(pREDCas9)。然后制备含有pREDCas9的大肠杆菌Nissle 1917感受态细胞。在50μL该细胞中加入100ng供体DNA和100ng gRNA质粒。使用电穿孔仪在1.80kV的0.1cm试管中进行电穿孔,然后立即在2mL LB中悬浮,30℃复苏3h,涂布于含大观霉素(50μg/mL)和氨苄西林(100μg/mL)的LB平板上,30℃孵育过夜。利用菌落聚合酶链反应和DNA测序技术对其进行鉴定。将正确的克隆接种到含有大观霉素和10%L-阿拉伯糖的LB上,培养过夜以丢失gRNA。通过对大观霉素(50mg/L)的敏感性,证实了已敲除ldcC1基因获得E.coli Nissle 1917ΔldcC1(EcNO)。采用上述相同的方法,进行ldcC2基因编辑,获得E.coli Nissle 1917 ΔldcC1 ΔldcC2(EcNT)。
将重组质粒pTLS、pK25转化改造菌株EcNT中,分别获得工程菌株EcNT(pTLS)和EcNT(pK25)。
实施例2复合菌剂的制备和赖氨酸代谢效果验证
1、摇瓶发酵获得菌种
将两种工程菌株在LB液体培养基中培养过夜,得到种子菌株。发酵培养基(10g/L葡萄糖、1g/L酵母膏、5g/L KH2PO4、5g/L(NH4)2SO4、0.7g/L KCl、0.003g/L FeSO4·7H2O、0.003g/L MnSO4、1g/L MgSO4、25g/L赖氨酸)在1%接种量,37℃,200rpm进行发酵培养。为了诱导质粒基因的表达,将0.2mol/L IPTG诱导剂加入100mL发酵培养基中。初始OD600为0.1,在不同的时间间隔收集样品,离心发酵菌液,收集上清,以测量菌株的生长并代谢物绘制出具有代表性的发酵谱图。
2、分析方法
生物菌体量的测定,利用紫外分光光度计(GE Health-care)在600nm处测量吸光度,测定菌体吸光度。
发酵液中的葡萄糖浓度通过葡萄糖测定试剂盒(葡萄糖氧化酶法)检测。试剂条件:温度37℃,波长505nm,反应时间10min,样本用量10μL,试剂用量1000μL,测量光径1.0cm。测定方法:终点法。用空白管调“零”点测定各管的吸光度。取样本10μL加入到1000μL试剂中,于37℃金属浴上加热反应10min后立即使用1.0cm光径比色杯检测OD505nm处的吸光度。计算方法如下:
葡萄糖(mmol/L)=样品管吸光度(A)/标准管吸光度(A)×标准液浓度
发酵液中的赖氨酸的含量通过高效液相色谱仪检测。样品前处理混匀后过HPLC专用膜待检测。色谱柱分析条件,采用Diamonsil AAA5μm(4.6mm×250)色谱柱和紫外检测器(360nm)的高效液相色谱(HPLC)分析赖氨酸。流动相A为0.02mol/L Na2HPO4和0.02mol/LNaH2PO4水溶液,流动相B为甲醇:乙腈=10:90(V:V)。柱温保持在45℃,流速为1mL/min。采用二元梯度洗脱的方法进行分析。
3.野生型和宿主改造型菌株生长和代谢情况比较
赖氨酸代谢途径中重组菌pTLS和pK25中的基因分别参与了赖氨酸代谢的一级和次级代谢反应。进行pTLS和pK25的比例(OD600比)1:1、1:2、1:3、1:4之间混菌发酵的比较,pTLS与pK25不同比例混菌比较,各比例生长速率和葡萄糖代谢水平相似,赖氨酸代谢水平pTLS:pK25为1:2代谢效果最好(图7)。
实施例3复合菌剂的包被
1、菌株的活化
将工程菌株pTLS、pK25分别接种于LB培养基中,在37℃,200rpm条件下置于恒温摇床中过夜培养。以初始OD600=0.1的接种量将经过富集培养后的菌株接种到发酵培养基中,37℃,200rpm条件下置于恒温摇床中培养6h,活化后的菌液(pTLS和pK25的活菌数比为1:2)置于4℃冰箱中保存。
2、微球的制备
(1)收集活化后的菌液到无菌的2mL离心管中,加入去离子水重悬清洗3次,收集菌体沉淀。
取约0.2g菌体沉淀,向其中加入2%海藻酸钠溶液1mL,搅拌均匀得到海藻酸钠微生物胶体。
(2)将氯化钙溶液置于磁力搅拌器上进行搅拌。用1mL无菌针管吸取海藻酸钠微生物胶体1mL并以均匀的速度滴入4%氯化钙溶液中,选择速度700r/min,交联时间15min,交联温度30℃,以确保微球制剂稳定。
(3)取上述发酵液用高速冷冻离心机在4℃,5000r/min条件下离心10min,灭菌水洗涤3次去掉氯化钙后重悬在灭菌水中,4℃储存。
对照组中使用的对照微球,省略步骤(1)直接将2%海藻酸钠胶体溶液滴入4%氯化钙溶液中,收集和储存与微生物的微球相同。
采用海藻酸钠法成功制备了微生物微球,使用扫描电子显微镜观察微球的形貌,如图8所示,观察到直径约1mm的粗糙球体。对实验制备的海藻酸钠微球的溶出与代谢能力进行发酵评价,结果显示,微生物微球在发酵培养基中处理24h,葡萄糖代谢旺盛且培养基中几乎没有菌液溶出,微球在24h时间内无崩解。
实施例4合成微生物组的体外效果检测
1、实验动物及分组
20只鼠龄4周龄SPF级C57BL/6小鼠(清洁级,雌雄各半),由济南朋悦实验动物繁育有限公司提供,实验鼠标准饲料由济南朋悦实验动物繁育有限公司提供。饲养级别为无特定病原体(SPF)级,昼夜时间等长;入室后适应性喂养2周再进行实验;雌雄分笼,每笼5只小鼠,共4笼;整个过程中动物饲养条件保持一致。共设2组,每组10只小鼠;第一组为模型对照组,赖氨酸喂养但不予药物干预;第二组为模型药物组,赖氨酸喂养且给予药物干预。
2、菌株的培养和富集
EcN(pTLS)菌株EcN(pK25)菌株混合和接种在50mL LB液体培养基中,37℃,200rpm过夜培养。次日将菌体全量离心,5000rpm,10min收集菌体。用灭菌超纯水洗涤2次,并重悬于灭菌水中配置成益生菌给药菌液,给予给药组小鼠食用。
3、动物实验
C57BL/6小鼠雌雄分笼(共4笼,每笼5只),标号、称重,适应性喂养2周后开始实验。实验过程给水始终为40g/L赖氨酸水溶液喂养。对照组无益生菌喂养,模型组给予益生菌(即实施例3的复合菌剂)喂养。喂养采用隔天益生菌喂养的方式,即模型组采用一天赖氨酸水溶液喂养,次日益生菌溶液喂养的方式交替进行,对照组实验过程给水始终为赖氨酸水溶液喂养。益生菌药物为保持菌株活性需每天更换药物组新鲜菌液;药物喂养约6周。
4、标本收集及取材
实验结束后,随机取出药物组和对照组小鼠3只,采集粪便以及血液样品。收集粪便样品时,用无菌镊子收集新鲜小鼠粪便3-5粒放置于无菌冻存管中,立即液氮速冻30min,然后置于-80℃保存,血液样品首先使用3.33%水合氯醛0.2-0.4mL麻醉小鼠后进行摘眼球取血并进行处死,血液收集于肝素钠抗凝管中,于离心机5000rpm,5min离心,收集上清血浆部分后置于-80℃保存。
5、指标测定
(1)小鼠体重指标:从给药第一天起记录小鼠体重,后称重5天/次,直至实验结束。取每组小鼠的体重平均值,绘制小鼠体重变化曲线,比较其差异性。
(2)小鼠血浆指标:小鼠血浆中赖氨酸和酵母氨酸含量指标送至上海美吉生物科技有限公司进行样品分析。
取结束治疗后的小鼠血浆进行LC-MS/MS赖氨酸和酵母氨酸含量分析,其结果如图9和图10所示。结果表明,与对照组相比,合成型肠道微生物药物治疗组能有效降低血浆赖氨酸,酵母氨酸水平,其代谢赖氨酸速率较对照组提高约50%,代谢酵母氨酸速率较对照组提高越30%。该结果说明来自本研究的合成肠道微生物组能有效降低血浆中赖氨酸,酵母氨酸含量,使得高赖氨酸血症患者因无法完成自身代谢而引起的副作用得到缓解。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之做一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 滨州医学院
<120> 用于治疗高赖氨酸血症的组合物及应用
<130> PI202210006
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2148
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 1
atgaacgtta ttgcaatatt gaatcacatg ggggtttatt ttaaagaaga acccatccgt 60
gaacttcatc gcgcgcttga acgtctgaac ttccagattg tttacccgaa cgaccgtgac 120
gacttattaa aactgatcga aaacaatgcg cgtctgtgcg gcgttatttt tgactgggat 180
aaatataatc tcgagctgtg cgaagaaatt agcaaaatga acgagaacct gccgttgtac 240
gcgttcgcta atacgtattc cactctcgat gtaagcctga atgacctgcg tttacagatt 300
agcttctttg aatatgcgct gggtgctgct gatgatattg ctaacaagat caagcagacc 360
actgacgaat atatcaacac tattctgcct ccgctgacca aagcactgtt taaatatgtt 420
cgtgaaggta aatatacttt ctgtactcct ggtcacatgg gtggtactgc attccagaaa 480
agcccggtag gtagcctgtt ctatgatttc tttggtccga acaccatgaa atctgatatt 540
tccatttcag tatctgaatt gggttctctg ctggatcaca gtggtccaca caaagaagca 600
gaacagtata tcgctcgcgt ctttaacgca gaccgcagct acatggtgac caacggtact 660
tccactgcga acaaaattgt tggtatgtac tctgctccgg caggcagcac cattctgatt 720
gaccgtaact gccacaaatc gctgacccac ctgatgatga tgagcgatgt tacgccaatc 780
tatttccgcc cgacccgtaa cgcttacggt attcttggtg gtatcccaca gagtgaattc 840
cagcacgcta ccattgctaa gcgcgtgaaa gaaacaccaa acgcaacctg gccggtacat 900
gctgtaatta ccaactctac ctatgatggt ctgctgtaca acaccgactt catcaagaaa 960
acactggatg tgaaatccat ccactttgac tccgcgtggg tgccttacac caacttctca 1020
ccgatttacg aaggtaaatg cggtatgagc ggtggccgtg tagaagggaa agtgatttac 1080
gaaacccagt ctactcacaa actgctggcg gcgttctctc aggcttccat gatccacgtt 1140
aaaggtgacg taaacgaaga aacctttaac gaagcctaca tgatgcacac caccacctct 1200
ccgcactacg gtatcgtggc gtccactgaa accgctgcgg cgatgatgaa aggcaatgca 1260
ggtaaacgtc tgatcaatgg ttccattgaa cgtgcgatca aattccgtaa agagatcaaa 1320
cgtctgagaa cggaatctga tggctggttc tttgatgtat ggcagccgga tcatatcgat 1380
acgactgaat gctggccgct gcgttctgac agcacatggc acggcttcaa aaacatcgat 1440
aacgagcaca tgtatcttga cccgatcaaa gtcaccctgc tgactccggg gatggaaaaa 1500
gacggcacca tgagcgactt tggtattccg gccagcatcg tggcgaaata cctcgacgaa 1560
catggcatcg ttgttgagaa aaccggtccg tataacctgc tgttcctgtt cagcatcggt 1620
atcgataaga ccaaagcact gagcctgctg cgtgctctga ctgacttcaa acgtgcgttc 1680
gacctgaacc tgcgtgtgaa aaacatgctg ccgtctctgt atcgtgaaga tcctgaattc 1740
tatgaaaaca tgcgtattca ggaactggct cagaatatcc acaaactgat tgttcaccac 1800
aatctgccgg atctgatgta tcgcgcattt gaagttctgc cgactatggt aatgactccg 1860
tatgctgcgt tccagaaaga gctgcacggt atgaccgaag aagtttacct cgacgaaatg 1920
gtcggtcgta ttaacgccaa tatgatcctt ccgtatccgc cgggagttcc tctggtaatg 1980
ccgggtgaaa tgatcaccga agaaagccgt ccggttctgg agttcctgca gatgctgtgt 2040
gaaatcggcg ctcactatcc gggctttgaa accgatattc acggtgcata ccgtcaggct 2100
gatggccgct ataccgttaa agtattgaaa gaagaaagca aaaaataa 2148
<210> 2
<211> 2142
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 2
atgaacatca ttgccattat gggaccgcat ggcgtctttt ataaagatga gcccatcaaa 60
gaactggagt cggcgctggt ggcgcaaggc tttcagatta tctggccaca aaacagcgtt 120
gatttgctga agtttatcga acataaccca cgaatttgcg gcgtgatttt tgactgggat 180
gagtacagtc tcgatttatg tagcgatatc aatcaactta atgaatatct cccgctttat 240
gccttcatca acacccactc gacgatggat gtcagcgtgc aggatatgcg gatggcgctc 300
tggttttttg aatatgcgct ggggcaggcg gaagatatcg ccattcgtat gcgtcagtac 360
accaacgaat atcttgataa cattacgccg ccgttcacga aagccttgtt tacctacgtc 420
aaagagcgga agtacacctt ttgtacgccg gggcatatgg gcggcaccgc atatcaaaaa 480
agcccggttg gctgtctgtt ttatgatttt ttcggcggga ataccctcaa ggcggacgtc 540
tctatttcgg tcaccgagct tggttcgttg ctcgaccaca ccgggccaca cctggaagcg 600
gaagagtaca tcgcgcggac gtttggcgcg gaacagagtt atatcgttac caacggaaca 660
tcgacgtcga acaaaattgt gggtatgtac gccgcgccat ccggcagtac gctgttgatc 720
gaccgcaatt gtcataaatc gctggcgcat ctgttgatga tgaacgatgt agtgccagtc 780
tggctgaaac cgacgcgtaa tgcgttgggg attctgggtg ggatcccgcg cggtgaattt 840
actcgcgaca gcatcgaaga gaaagtcgct gccaccacgc aggcacaatg gccggttcat 900
gcggtgatca ccaactccac ctatgatggc ttgctctaca acaccgactg gatcaaacag 960
acgctggatg tcccgtcgat tcacttcgat tctgcctggg tgccgtacac ccattttcat 1020
ccaatctacc agggtaaaag tggtatgagc ggcgagcgtg ttgcaggaaa agtgatcttc 1080
gaaacgcagt cgacccacaa aatgctggcg gcgttatcgc aggcgtcgct gatccacatt 1140
aaaggtgagt atgacgaaga ggcgtttaac gaagccttta tgatgcatac caccacctcg 1200
cccagttatc ccattgttgc ttctgttgag acggcggcgg cgatgttgcg tggtaatccg 1260
ggcaaacggc tgattaaccg ttcagtagaa cgagctctgc attttcgcaa agaggtccag 1320
cggctgcggg aagagtctga cggctggttt ttcgatatct ggcaaccgcc gcaggtggat 1380
gaagccgaat gctggcccgt tgcgcctggc gaacagtggc acggctttag cgatgcggat 1440
gccaatcaca tgtttctcga tccggttaaa gtcactattt tgacaccggg gatggacgag 1500
cagggcaata tgagcgagga ggggatcccg gcggcgctgg tggcaaaatt cctcgacgaa 1560
cgtgggatcg tagtagagaa aaccggccct tataacctgc tgtttctctt tagtattggc 1620
atcgataaaa ccaaagcaat gggattattg cgtgggttga cggaatttaa gcgctcttac 1680
gatctcaacc tgcggatcaa aaatatgctg cccgatctct atgcagaaga tcccgatttc 1740
taccgcaata tgcgtattca ggatctggcg caagggatcc ataagctgat tcgtaaacac 1800
gatcttcccg gtttgatgtt gcgggcattt gataccttgc cggagatgat catgacgcca 1860
catcaggcat ggcagagaca gattaaaggc gaagtagaaa ccattgcgct ggaacaactg 1920
gtcggtagag tatcggcaaa tatgatcctg ccttatccac ccggcgtacc gctgctgatg 1980
ccgggagaaa tgcttaccga agagagccgc acggtactcg attttctact gatgctttgt 2040
tccgtagggc aacattaccc cggttttgaa acggatattc acggcgcgaa acaggacgaa 2100
gacggtgttt accgcgtacg agtcctaaaa atggcaggat aa 2142
<210> 3
<211> 1122
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 3
atggctgccg tcacattaca tctaagagct gaaactaaac ccctagaggc acgtgctgcc 60
ttaacaccta ccacggttaa aaaactgata gctaagggct tcaaaatata tgtagaggac 120
agtccacaat ctactttcaa tattaacgaa tatcgtcaag caggtgccat tatagtgcct 180
gcaggttcat ggaaaaccgc tccacgcgac agaatcatta taggtttgaa ggaaatgcct 240
gaaaccgata ctttccctct agtccacgaa cacatccagt ttgctcactg ctacaaagac 300
caagctgggt ggcaaaatgt ccttatgaga tttattaagg gacacggtac tctatatgat 360
ttggaatttt tggaaaatga ccaaggtaga agagttgctg cctttggatt ttacgctggg 420
ttcgcaggtg cagcccttgg tgtaagagac tgggcattca agcaaacgca ttctgacgat 480
gaagacttgc ctgcagtgtc gccttacccc aatgaaaagg cattggttaa agatgttacc 540
aaagattata aagaagcctt agccaccgga gccagaaagc caaccgtgtt aatcattggt 600
gcgctaggaa gatgtggttc cggtgccatc gatctgttgc acaaagttgg tattccagat 660
gctaacatat taaaatggga tatcaaagaa acttcccgtg gtggtccctt tgacgaaatt 720
ccacaagctg atatttttat caattgtata tatctatcga agccaattgc tcctttcact 780
aacatggaga aactgaataa tcctaacaga agactaagga ccgtggtgga cgtatcagca 840
gacactacca accctcacaa ccccatccca atatacactg tggctactgt gtttaacaaa 900
cctaccgttc tggtacctac cactgccggg cctaaattat ctgtcatctc tattgatcac 960
ttgccttctt tgctgccaag agaagcttca gaatttttct ctcatgatct cttaccatct 1020
ttagagctcc tacctcaaag aaaaactgct cctgtctggg ttagagccaa gaaattgttc 1080
gatagacatt gcgctcgtgt taaaagatct tcaagattgt ag 1122
<210> 4
<211> 1341
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 4
atgggaaaga acgttttgtt gctaggatct ggttttgttg cacaacctgt tatcgacaca 60
ttggctgcta atgatgacat caatgtcact gtcgcatgta gaacattagc caatgcgcaa 120
gcattggcca agccctctgg atccaaggct atttcattgg atgttaccga tgacagtgcc 180
ttagacaaag ttctggctga taacgatgtt gtcatctctt tgattccata caccttccat 240
ccaaatgtgg taaagagcgc catcagaaca aagaccgatg tcgtcacttc ctcttacatc 300
tcacctgcct taagagaatt ggaaccagaa atcgtaaagg caggtattac agttatgaac 360
gaaattgggt tggatccagg tatcgaccac ttgtatgcgg tcaagactat tgatgaagtt 420
cacagagctg gtggtaagct aaagtcattc ttgtcatact gtggtggttt accagctcct 480
gaagactctg ataatccatt aggatacaaa ttttcatggt cctccagagg tgtgctactg 540
gctttaagaa actctgctaa atactggaaa gacggaaaga ttgaaactgt ttcttccgaa 600
gacttaatgg ccactgctaa gccttacttc atctacccag gttatgcatt cgtttgctac 660
ccaaatagag actctaccct tttcaaggat ctttatcata ttccagaagc cgaaacggtc 720
attagaggta ctttgagata tcaaggtttc ccagaatttg ttaaggcttt agttgacatg 780
ggtatgttga aggatgatgc taacgaaatc ttcagcaagc caattgcctg gaacgaagca 840
ctaaaacaat atttaggtgc caagtctact tctaaagaag atttgattgc ttccattgac 900
tcaaaggcta cttggaaaga tgatgaagat agagaaagaa tcctttccgg gtttgcttgg 960
ttaggcttgt tctctgacgc aaagatcaca ccaagaggta atgctttaga cactctatgt 1020
gcacgtttag aagaactaat gcaatatgaa gacaatgaaa gagatatggt tgtactacaa 1080
cacaaattcg gtattgaatg ggctgatgga actaccgaaa caagaacatc cactttagtt 1140
gactatggta aggttggtgg ttacagttct atggccgcta ctgttggtta tccagttgcc 1200
attgcaacga aattcgtctt agatggtaca atcaagggac caggcttact agcgccatac 1260
tcaccagaga ttaatgatcc aatcatgaaa gaactaaagg acaagtacgg catctatcta 1320
aaggaaaaga cagtggctta a 1341
<210> 5
<211> 819
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 5
atggttaaaa cgactgaagt agtaagcgaa gtttcaaagg tggcaggtgt aagaccatgg 60
gcaggtatat tcgttgttga aattcaagag gatatactcg cggatgagtt tacgttcgag 120
gcattaatga gaactttgcc attggcgtct caagccagaa tcctcaataa aaaatcgttt 180
cacgatagat gttcaaatct atgcagccag ctgctgcagt tgtttggctg ctctatagta 240
acgggcttaa attttcaaga gctgaaattt gacaagggca gcttcggtaa gccattctta 300
gacaacaatc gttttcttcc atttagcatg accatcggtg aacaatatgt agctatgttc 360
ctcgtaaaat gtgtaagtac agatgaatac caggatgtcg gaattgatat cgcttctccg 420
tgcaattatg gcgggaggga agagttggag ctatttaaag aagtttttag tgaaagagaa 480
tttaacggtt tactgaaagc gtctgatcca tgcacaatat ttacttactt atggtccttg 540
aaggagtcgt atacaaaatt tactggaact ggccttaaca cagacttgtc actaatagat 600
tttggcgcta tcagcttttt tccggctgag ggagcttcta tgtgcataac tctggatgaa 660
gttccattga ttttccattc tcaatggttc aataacgaaa ttgtcactat ctgtatgcca 720
aagtccatca gtgataaaat caacacgaac agaccaaaat tatataatat cagcttatct 780
acgttgattg attatttcat cgaaaatgat ggtttataa 819
<210> 6
<211> 4179
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 6
atgactaacg aaaaggtctg gatagagaag ttggataatc caactctttc agtgttacca 60
catgactttt tacgcccaca acaagaacct tatacgaaac aagctacata ttcgttacag 120
ctacctcagc tcgatgtgcc tcatgatagt ttttctaaca aatacgctgt cgctttgagt 180
gtatgggctg cattgatata tagagtaacc ggtgacgatg atattgttct ttatattgcg 240
aataacaaaa tcttaagatt caatattcaa ccaacgtggt catttaatga gctgtattct 300
acaattaaca atgagttgaa caagctcaat tctattgagg ccaatttttc ctttgacgag 360
ctagctgaaa aaattcaaag ttgccaagat ctggaaagga cccctcagtt gttccgtttg 420
gcctttttgg aaaaccaaga tttcaaatta gacgagttca agcatcattt agtggacttt 480
gctttgaatt tggataccag taataatgcg catgttttga acttaattta taacagctta 540
ctgtattcga atgaaagagt aaccattgtt gcggaccaat ttactcaata tttgactgct 600
gcgctaagcg atccatccaa ttgcataact aaaatctctc tgatcaccgc atcatccaag 660
gatagtttac ctgatccaac taagaacttg ggctggtgcg atttcgtggg gtgtattcac 720
gacattttcc aggacaatgc tgaagccttc ccagagagaa cctgtgttgt ggagactcca 780
acactaaatt ccgacaagtc ccgttctttc acttatcgcg acatcaaccg cacttctaac 840
atagttgccc attatttgat taaaacaggt atcaaaagag gtgatgtagt gatgatctat 900
tcttctaggg gtgtggattt gatggtatgt gtgatgggtg tcttgaaagc cggcgcaacc 960
ttttcagtta tcgaccctgc atatccccca gccagacaaa ccatttactt aggtgttgct 1020
aaaccacgtg ggttgattgt tattagagct gctggacaat tggatcaact agtagaagat 1080
tacatcaatg atgaattgga gattgtttca agaatcaatt ccatcgctat tcaagaaaat 1140
ggtaccattg aaggtggcaa attggacaat ggcgaggatg ttttggctcc atatgatcac 1200
tacaaagaca ccagaacagg tgttgtagtt ggaccagatt ccaacccaac cctatctttc 1260
acatctggtt ccgaaggtat tcctaagggt gttcttggta gacatttttc cttggcttat 1320
tatttcaatt ggatgtccaa aaggttcaac ttaacagaaa atgataaatt cacaatgctg 1380
agcggtattg cacatgatcc aattcaaaga gatatgttta caccattatt tttaggtgcc 1440
caattgtatg tccctactca agatgatatt ggtacaccgg gccgtttagc ggaatggatg 1500
agtaagtatg gttgcacagt tacccattta acacctgcca tgggtcaatt acttactgcc 1560
caagctacta caccattccc taagttacat catgcgttct ttgtgggtga cattttaaca 1620
aaacgtgatt gtctgaggtt acaaaccttg gcagaaaatt gccgtattgt taatatgtac 1680
ggtaccactg aaacacagcg tgcagtttct tatttcgaag ttaaatcaaa aaatgacgat 1740
ccaaactttt tgaaaaaatt gaaagatgtc atgcctgctg gtaaaggtat gttgaacgtt 1800
cagctactag ttgttaacag gaacgatcgt actcaaatat gtggtattgg cgaaataggt 1860
gagatttatg ttcgtgcagg tggtttggcc gaaggttata gaggattacc agaattgaat 1920
aaagaaaaat ttgtgaacaa ctggtttgtt gaaaaagatc actggaatta tttggataag 1980
gataatggtg aaccttggag acaattctgg ttaggtccaa gagatagatt gtacagaacg 2040
ggtgatttag gtcgttatct accaaacggt gactgtgaat gttgcggtag ggctgatgat 2100
caagttaaaa ttcgtgggtt cagaatcgaa ttaggagaaa tagatacgca catttcccaa 2160
catccattgg taagagaaaa cattacttta gttcgcaaaa atgccgacaa tgagccaaca 2220
ttgatcacat ttatggtccc aagatttgac aagccagatg acttgtctaa gttccaaagt 2280
gatgttccaa aggaggttga aactgaccct atagttaagg gcttaatcgg ttaccatctt 2340
ttatccaagg acatcaggac tttcttaaag aaaagattgg ctagctatgc tatgccttcc 2400
ttgattgtgg ttatggataa actaccattg aatccaaatg gtaaagttga taagcctaaa 2460
cttcaattcc caactcccaa gcaattaaat ttggtagctg aaaatacagt ttctgaaact 2520
gacgactctc agtttaccaa tgttgagcgc gaggttagag acttatggtt aagtatatta 2580
cctaccaagc cagcatctgt atcaccagat gattcgtttt tcgatttagg tggtcattct 2640
atcttggcta ccaaaatgat ttttacctta aagaaaaagc tgcaagttga tttaccattg 2700
ggcacaattt tcaagtatcc aacgataaag gcctttgccg cggaaattga cagaattaaa 2760
tcatcgggtg gatcatctca aggtgaggtc gtcgaaaatg tcactgcaaa ttatgcggaa 2820
gacgccaaga aattggttga gacgctacca agttcgtacc cctctcgaga atattttgtt 2880
gaacctaata gtgccgaagg aaaaacaaca attaatgtgt ttgttaccgg tgtcacagga 2940
tttctgggct cctacatcct tgcagatttg ttaggacgtt ctccaaagaa ctacagtttc 3000
aaagtgtttg cccacgtcag ggccaaggat gaagaagctg catttgcaag attacaaaag 3060
gcaggtatca cctatggtac ttggaacgaa aaatttgcct caaatattaa agttgtatta 3120
ggcgatttat ctaaaagcca atttggtctt tcagatgaga agtggatgga tttggcaaac 3180
acagttgata taattatcca taatggtgcg ttagttcact gggtttatcc atatgccaaa 3240
ttgagggatc caaatgttat ttcaactatc aatgttatga gcttagccgc cgtcggcaag 3300
ccaaagttct ttgactttgt ttcctccact tctactcttg acactgaata ctactttaat 3360
ttgtcagata aacttgttag cgaagggaag ccaggcattt tagaatcaga cgatttaatg 3420
aactctgcaa gcgggctcac tggtggatat ggtcagtcca aatgggctgc tgagtacatc 3480
attagacgtg caggtgaaag gggcctacgt gggtgtattg tcagaccagg ttacgtaaca 3540
ggtgcctctg ccaatggttc ttcaaacaca gatgatttct tattgagatt tttgaaaggt 3600
tcagtccaat taggtaagat tccagatatc gaaaattccg tgaatatggt tccagtagat 3660
catgttgctc gtgttgttgt tgctacgtct ttgaatcctc ccaaagaaaa tgaattggcc 3720
gttgctcaag taacgggtca cccaagaata ttattcaaag actacttgta tactttacac 3780
gattatggtt acgatgtcga aatcgaaagc tattctaaat ggaagaaatc attggaggcg 3840
tctgttattg acaggaatga agaaaatgcg ttgtatcctt tgctacacat ggtcttagac 3900
aacttacctg aaagtaccaa agctccggaa ctagacgata ggaacgccgt ggcatcttta 3960
aagaaagaca ccgcatggac aggtgttgat tggtctaatg gaataggtgt tactccagaa 4020
gaggttggta tatatattgc atttttaaac aaggttggat ttttacctcc accaactcat 4080
aatgacaaac ttccactgcc aagtatagaa ctaactcaag cgcaaataag tctagttgct 4140
tcaggtgctg gtgctcgtgg aagctccgca gcagcttaa 4179
Claims (9)
1.用于治疗高赖氨酸血症的组合物,其特征在于,活性成分为代谢赖氨酸的工程菌I和II;
其中,工程菌I是以大肠杆菌为出发菌株,通过敲除ldcC1和ldcC2基因,并在大肠杆菌中表达来自酿酒酵母菌BY4741的LKR和SDR基因构建得到的;
优选地,所述大肠杆菌为Nissle 1917;
ldcC1基因为:
a1)SEQ ID NO:1所示的核苷酸序列;
b1)SEQ ID NO:1所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;
c1)在严格条件下与SEQ ID NO:1所示序列杂交且表达相同功能蛋白质的核苷酸序列,所述严格条件为在含0.1%sds的0.1×sspe或含0.1%sds的0.1×ssc溶液中,在65℃下杂交,并用该溶液洗膜;或
d1)与a1)、b1)或c1)的核苷酸序列具有90%以上同源性且表达相同功能蛋白质的核苷酸序列;
ldcC2基因为:
a2)SEQ ID NO:2所示的核苷酸序列;
b2)SEQ ID NO:2所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;
c2)在严格条件下与SEQ ID NO:2所示序列杂交且表达相同功能蛋白质的核苷酸序列,所述严格条件为在含0.1%SDS的0.1×SSPE或含0.1%SDS的0.1×SSC溶液中,在65℃下杂交,并用该溶液洗膜;或
d2)与a2)、b2)或c2)的核苷酸序列具有90%以上同源性且表达相同功能蛋白质的核苷酸序列;
LKR基因为:
a3)SEQ ID NO:3所示的核苷酸序列;
b3)SEQ ID NO:3所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;
c3)在严格条件下与SEQ ID NO:3所示序列杂交且表达相同功能蛋白质的核苷酸序列,所述严格条件为在含0.1%SDS的0.1×SSPE或含0.1%SDS的0.1×SSC溶液中,在65℃下杂交,并用该溶液洗膜;或
d3)与a3)、b3)或c3)的核苷酸序列具有90%以上同源性且表达相同功能蛋白质的核苷酸序列;
SDR基因为:
a4)SEQ ID NO:4所示的核苷酸序列;
b4)SEQ ID NO:4所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;
c4)在严格条件下与SEQ ID NO:4所示序列杂交且表达相同功能蛋白质的核苷酸序列,所述严格条件为在含0.1%SDS的0.1×SSPE或含0.1%SDS的0.1×SSC溶液中,在65℃下杂交,并用该溶液洗膜;或
d4)与a4)、b4)或c4)的核苷酸序列具有90%以上同源性且表达相同功能蛋白质的核苷酸序列;
工程菌II是以大肠杆菌为出发菌株,通过敲除ldcC1和ldcC2基因,并在大肠杆菌中表达来自酿酒酵母菌BY4741的lys5和lys2基因构建得到的;
优选地,所述大肠杆菌为Nissle 1917;
lys5基因为:
a5)SEQ ID NO:5所示的核苷酸序列;
b5)SEQ ID NO:5所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;
c5)在严格条件下与SEQ ID NO:5所示序列杂交且表达相同功能蛋白质的核苷酸序列,所述严格条件为在含0.1%SDS的0.1×SSPE或含0.1%SDS的0.1×SSC溶液中,在65℃下杂交,并用该溶液洗膜;或
d5)与a5)、b5)或c5)的核苷酸序列具有90%以上同源性且表达相同功能蛋白质的核苷酸序列;
lys2基因为:
a6)SEQ ID NO:6所示的核苷酸序列;
b6)SEQ ID NO:6所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;
c6)在严格条件下与SEQ ID NO:6所示序列杂交且表达相同功能蛋白质的核苷酸序列,所述严格条件为在含0.1%SDS的0.1×SSPE或含0.1%SDS的0.1×SSC溶液中,在65℃下杂交,并用该溶液洗膜;或
d6)与a6)、b6)或c6)的核苷酸序列具有90%以上同源性且表达相同功能蛋白质的核苷酸序列。
2.根据权利要求1所述的组合物,其特征在于,
工程菌I的构建方法包括:
分别构建靶向ldcC1和ldcC2基因的CRISPR-Cas9系统,共同导入大肠杆菌得到重组菌,然后将来自酿酒酵母菌BY4741的LKR和SDR基因通过质粒导入所述重组菌中,即得代谢赖氨酸的工程菌;其中,CRISPR-Cas9系统中靶向ldcC1基因的gRNA的核苷酸序列为:5′-CTGTTTAAATATGTTCGTGA-3′,靶向ldcC2基因的gRNA的核苷酸序列为:5′-CAATATGCGTATTCAGGATC-3′;
工程菌II的构建方法包括:
分别构建靶向ldcC1和ldcC2基因的CRISPR-Cas9系统,共同导入大肠杆菌得到重组菌,然后将来自酿酒酵母菌BY4741的lys5和lys2基因通过质粒导入所述重组菌中,即得代谢赖氨酸的工程菌;其中,CRISPR-Cas9系统中靶向ldcC1基因的gRNA的核苷酸序列为:5′-CTGTTTAAATATGTTCGTGA-3′,靶向ldcC2基因的gRNA的核苷酸序列为:5′-CAATATGCGTATTCAGGATC-3′。
3.根据权利要求1或2所述的组合物,其特征在于,工程菌I和II的活菌数比为1:1~1:4,优选1:1、1:2、1:3或1:4,更优选1:2。
4.复合菌剂的制备方法,其特征在于,将代谢赖氨酸的工程菌I和II按一定比例混合后发酵培养,发酵液经离心,菌体沉淀用去离子水重悬清洗2~3次,然后将菌体沉淀经海藻酸钠包被形成微球,即得;
其中,工程菌I和II同权利要求1或2中所述。
5.根据权利要求4所述的方法,其特征在于,代谢赖氨酸的工程菌I和II按OD600比为1:1~1:4混合,优选1:1、1:2、1:3或1:4,更优选1:2。
6.根据权利要求4或5所述的方法,其特征在于,两种菌体沉淀混合后,取0.1~2g混合物,向其中加入2%海藻酸钠溶液0.1~5mL,搅拌均匀得到海藻酸钠微生物胶体;将1mL胶体匀速滴入1~4%氯化钙溶液中,在30~37℃、转速100~700r/min条件下交联2~15min。
7.按照权利要求4-6任一项所述方法制备的复合菌剂。
8.权利要求1-3任一项所述组合物或权利要求7所述复合菌剂在制备用于改善或治疗基因缺陷型赖氨酸紊乱疾病的药物中的应用。
9.根据权利要求8所述的方法,其特征在于,所述疾病包括高赖氨酸血症。
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