CN114875055B - 一种嗜热链球菌重组菌的构建方法及应用 - Google Patents
一种嗜热链球菌重组菌的构建方法及应用 Download PDFInfo
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Abstract
本发明提供了一种嗜热链球菌重组菌的构建方法及应用,主要为敲除嗜热链球菌的乳糖透过酶基因,得到嗜热链球菌ΔlacS,将含有大肠杆菌半乳糖转运酶片段基因的载体pNZ8148‑E‑gala转化入嗜热链球菌ΔlacS,获得嗜热链球菌重组菌,该重组菌株相较于野生菌株,能够更有效的降低发酵酸奶中半乳糖的含量。
Description
技术领域
本发明属于分子生物学技术领域,具体涉及一种嗜热链球菌重组菌的构建方法及应用。
背景技术
酸奶因其营养价值高、风味独特以及良好的保健效果,成为当今世界广泛流行的发酵乳制品。国际乳品联合会、世界卫生组织和联合国粮农组织于1997年将酸奶定义如下:在添加或不添加乳粉或脱脂乳粉的杀菌乳或浓缩乳中,通过接种商业化的嗜热链球菌和保加利亚乳杆菌进行发酵而成的凝乳状产品,成品中必须含有一定量的活菌。
嗜热链球菌和保加利亚乳杆菌对乳糖的代谢过程基本相同。乳糖通过乳糖透过酶LacS进入细胞,随后在胞内β-半乳糖苷酶LacZ的作用下水解为葡萄糖和半乳糖。其中,葡萄糖进入糖酵解途径,半乳糖因不被利用而排至细胞外。对于半乳糖的代谢,保加利亚乳杆菌缺乏编码Leloir途径所需酶的完整基因,而绝大多数的嗜热链球菌,虽然具有编码Leloir途径所需酶的完整基因galKTEM,但是仍然无法代谢半乳糖。嗜热链球菌和保加利亚乳杆菌代谢半乳糖缺陷导致酸奶中残留大量的半乳糖。这不仅会降低发酵产物的品质,如高浓度的半乳糖会导致奶酪在加热过程中发生褐变,残留的半乳糖还会被异源发酵剂利用而生成二氧化碳,导致乳酪裂口、断裂等组织缺陷。乳品中残留的半乳糖还限制了半乳糖血症患者的食用。也有少数嗜热链球菌菌株在利用乳糖的同时能够代谢半乳糖。
中国专利文献CN111254106A(申请号:202010229538.5)公开了一种食品级嗜热链球菌表达系统及其在酸奶制备中的应用,该专利文献公开的食品级嗜热链球菌表达系统包含食品级宿主嗜热链球菌CN3070、食品级表达载体pST5240;所述食品级宿主嗜热链球菌JIM8232基因组上的乳糖转运酶STlacS基因而获得,丧失利用乳糖的能力,食品表达载体pS5240包括来自植物乳杆菌的乳糖转运酶LPlacS基因作为筛选标记,将食品表达载体pS5240转化入嗜热链球菌CH3070,可以在只含有乳糖为碳源的LM17培养基中生长,可以利用乳糖;与本发明提供嗜热链球菌重组菌有明显区别。
现有技术中,针对只利用半乳糖而不利于乳糖的嗜热链球菌没有报道。
发明内容
针对现有技术的不足,本发明提供了一种嗜热链球菌重组菌的构建方法及应用。
本发明的技术方案如下:
一种嗜热链球菌重组菌的构建方法,具体如下步骤:
(1)一株嗜热链球菌ΔlacS的构建,包括如下步骤:
①提取嗜热链球菌DSM 32596(Streptococcus thermophilus DSM 32596)的基因组DNA;
②以步骤①的基因组DNA为模板,利用核苷酸序列为SEQ ID NO.1和SEQ ID NO.2的引物对ST-LacS-up-F和ST-LacS-up-R进行PCR扩增乳糖透过酶基因的上游同源臂,利用核苷酸序列为SEQ ID NO.3和SEQ ID NO.4的引物对ST-LacS-down-F和ST-LacS-down-R进行PCR扩增乳糖透过酶基因的下游同源臂,然后利用重叠拼接PCR的方法,对上游同源臂和下游同源臂进行连接,制得乳糖透过酶基因敲除连接臂;
ST-LacS-up-F:
ggtaccgggccccccctcgagTTCCAACGGAAACTGGTGCT SEQ ID NO.1;
ST-LacS-up-R:
TTCGGAAACCTCCTATTATTTG SEQ ID NO.2;
ST-LacS-down-F:
CAAATAATAGGAGGTTTCCGAATCTATGAACATGACTGAAAAAAT SEQ ID NO.3;
ST-LacS-down-R:
agtggatcccccgggctgcagAAGACAATTCTCTTACCATTC SEQ ID NO.4。
③使用PstI和XhoI对质粒pGhost9进行双酶切,然后利用同源重组酶将步骤②中,制得的乳糖透过酶基因敲除连接臂连接到pGhost9上,将连接产物转化入感受态大肠杆菌XL-Blue1,挑取验证正确的转化子,提取重组质粒;
④将步骤③得到的重组质粒转化入嗜热链球菌DSM 32596中,通过转化子培养发生第一次同源交换,利用红霉素进行筛选,然后进行连续传代发生第二次同源交换,筛选红霉素标记丢失的菌株,利用核苷酸序列为SEQ ID NO.5和SEQ ID NO.6的引物对,对红霉素标记丢失的菌株进行检测,扩增出3085bp的目的产物,即为乳糖透过酶基因敲除菌株,命名为嗜热链球菌ΔlacS;
ST-LacS-test-F:TCGTGACTATGTGCATCC SEQ ID NO.5;
ST-LacS-test-R:GATATCAGCTGGTTTCGC SEQ ID NO.6。
(2)将载体pNZ8148-E-gala转化入嗜热链球菌ΔlacS中,所述载体pNZ8148-E-gala的核苷酸序列如SEQ ID NO.13所示。
根据本发明优选的,步骤②中,上游同源臂PCR扩增体系如下:Ex taq buffer 25μL,dNTP4μL,引物ST-LacS-up-F 2μL,引物ST-LacS-up-R 2μL,基因组DNA 1μL,Ex taq 1μL,双蒸水13μL;
PCR扩增条件为:95℃预变性3min;95℃解链30s,50℃退火30s,72℃延伸1min,重复30个循环;72℃维持10min;4℃保存。
根据本发明优选的,步骤②中,下游同源臂PCR扩增体系如下:Ex taq buffer 25μL,dNTP4μL,引物ST-LacS-down-F 2μL,引物ST-LacS-down-R 2μL,基因组DNA 1μL,Ex taq1μL,双蒸水13μL。
PCR扩增条件为:95℃预变性3min;95℃解链30s,50℃退火30s,72℃延伸1min,重复30个循环;72℃维持10min;4℃保存。
根据本发明优选的,步骤②中,上游同源臂和下游同源臂进行连接,PCR扩增体系为:Ex taq buffer 25μL,dNTP 4μL,引物ST-LacS-up-F 2μL,引物ST-LacS-down-R 2μL,上游同源臂1μL,下游同源臂1μL,Ex taq 1μL,双蒸水12μL。
PCR扩增条件为:95℃预变性3min;95℃解链30s,50℃退火30s,72℃延伸2min,重复30个循环;72℃维持10min;4℃保存。
根据本发明优选的,步骤③中,验证转化子的方法:将连接产物转化入感受态大肠杆菌XL-Blue1,涂布于含有250μg/mL红霉素的LB平板,30℃静置培养48小时后,挑取转化子接入新鲜的含有250μg/mL红霉素的LB液体管,30℃摇动培养24小时后,提取质粒,使用PstI和XhoI进行双酶切,酶切产物进行琼脂糖凝胶电泳,其中包含一条2000bp的条带,即为正确的转化子,提取验证正确的转化子的质粒。
根据本发明优选的,步骤④中,转化子的培养条件:转化子在含有2.5μg/mL红霉素的SM17平板,30℃培养。
根据本发明优选的,步骤④中,利用红霉素的筛选条件:重组质粒转化入嗜热链球菌DSM32596中,涂布于含有2.5μg/mL红霉素的SM17平板,30℃静置培养72小时后,挑取转化子于新鲜的含有2.5μg/mL红霉素的SM17液体培养基中,30℃静置培养至OD600为1.0,将培养温度调节成42℃培养,促使质粒与基因组发生第一次同源交换,42℃培养2小时后,涂布于含有2.5μg/mL红霉素的SM17平板,继续在42℃培养,长出的即为发生第一次同源交换菌株。
根据本发明优选的,步骤④中,连续传代的培养条件为第一次同源交换成功的菌株接种于不含有抗生素的SM17液体培养基中,在30℃下静置培养,12小时后转接至新鲜的不含有抗生素的SM17液体培养基中,重复此步骤20次,使菌株发生第二次同源交换。
根据本发明优选的,步骤④中,红霉素标记丢失的菌株筛选为连续传代后的菌液涂布于不含有抗生素的SM17平板,30℃培养12小时后,挑取长出的单菌落,分别点接于添加和不添加2.5μg/mL红霉素的SM17平板,继续30℃培养,将在无红霉素下生长,而有红霉素下不生长的菌株即为红霉素标记丢失的菌株。
进一步优选的,步骤④中SM17培养基每升组分如下:
动物蛋白胨2.5g,胰蛋白胨2.5g,大豆蛋白胨5g,牛肉提取物5g,酵母提取物2.5g,抗坏血酸0.5g,硫酸镁0.25g,beta-甘油磷酸钠五水合物19g,蔗糖20g,余量水。
根据本发明优选的,步骤④中,PCR检测体系为:Ex taq buffer 25μL,dNTP 4μL,引物ST-LacS-test-F 2μL,引物ST-LacS-test-R 2μL,基因组DNA 1μL,Ex taq 1μL,双蒸水13μL。
PCR检测反应条件为:95℃预变性3min;95℃解链30s,45℃退火30s,72℃延伸3min,重复30个循环;72℃维持10min;4℃保存。
根据本发明优选的,载体pNZ8148-E-gala的构建方法:以大肠杆菌BL21的基因组DNA为模板,使用核苷酸序列为SEQ ID NO.7和SEQ ID NO.8的引物对扩增大肠杆菌半乳糖转运酶片段,随后,使用核苷酸序列为SEQ ID NO.9和SEQ ID NO.10的引物对从质粒pMG36e中扩增出P32启动子片段,通过重叠延伸PCR将P32启动子和大肠杆菌半乳糖转运酶片段基因融合,使用核苷酸序列为SEQ ID NO.11和SEQ ID NO.12的引物线性化表达载体pNZ8148,再将融合片段使用同源重组酶插入到表达载体pNZ8148中,连接产物转化至大肠杆菌XL1-Blue,然后收集菌体提取质粒,得到重组质粒pNZ8148-E-gala,核苷酸序列如SEQ ID NO.13所示。
E-gala-F:TTCGGAGGAATTTTGAAATGCCTGACGCTAAAAAAC SEQ ID NO.7;
E-gala-R:CCTTCGTTTTCAGACTTTGCTTAATCGTGAGCGCCTAT SEQ ID NO.8;
P32-F:TCGAATTCGGTCCTCGGG SEQ ID NO.9;
P32-R:TTCAAAATTCCTCCGAATA SEQ ID NO.10;
pNZ8148-F:GCTCAAGCTTTCTTTGAACC SEQ ID NO.11;
pNZ8148-R:CCCGAGGACCGAATTCGAATTATGCTCGCGTTATCGAC SEQ ID NO.12。
一种食品级嗜热链球菌表达系统,包含上述嗜热链球菌ΔlacS和上述载体pNZ8148-E-gala,载体pNZ8148-E-gala转化入嗜热链球菌ΔlacS。
上述食品级嗜热链球菌表达系统在降低半乳糖中的应用。
上述食品级嗜热链球菌表达系统在制备酸奶中的应用。
根据本发明优选的,上述食品级嗜热链球菌表达系统在降低酸奶中半乳糖的应用。
有益效果
1、本申请发现嗜热链球菌DSM 32596去除乳糖透过酶基因后,菌株同时失去利用乳糖以及半乳糖的能力,进一步回补大肠杆菌来源的半乳糖转运酶,所获得嗜热链球菌重组菌恢复了半乳糖的利用能力。
2、本发明构建的重组菌株嗜热链球菌ΔlacS/pNZ8148-E-gala,相对于既能利用乳糖又能利用半乳糖的嗜热链球菌DSM 32596能更有效的降低酸奶中的半乳糖。
附图说明
图1为嗜热链球菌DSM 32596和嗜热链球菌ΔlacS的生长过程图片;
图中:(a)的培养基为LM17液体培养基;(b)的培养基为SM17液体培养基;(c)的培养基为M17-半乳糖液体培养基。
图2为回补载体pNZ8148-E-gala的示意图。
图3为回补载体pNZ8148-E-gala的PCR鉴定图;
图中:M为标准分子量Marker,1为扩增获得的大肠杆菌半乳糖转运酶基因。
图4为回补大肠杆菌半乳糖转运酶的重组菌株嗜热链球菌ΔlacS/pNZ8148-E-gala的生长过程图片。
具体实施方式
下面通过具体实施例对本发明做进一步详细阐述,但本发明所保护的范围不限于此。
实施例中未详见说明的内容,均按本领域现有技术。
主要材料的来源
本发明实验中使用和涉及到的细菌菌株、质粒、主要材料及试剂:
大肠杆菌XL-Blue1感受态细胞:购自北京全式金生物技术有限公司,普通市售已知菌株;
大肠杆菌BL21菌株:购自广东环凯微生物科技有限公司,普通市售已知菌株;
嗜热链球菌DSM 32596:购自广东环凯微生物科技有限公司,普通市售已知菌株;
质粒pGhost9:购自BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心,普通市售产品;
细菌基因组提取试剂盒:购自北京天根生物科技有限公司;
柱式质粒DNA小量抽提试剂盒:购自南京诺韦赞生物科技有限公司;
红霉素、琼脂糖、核酸染料等购自上海生工生物工程有限公司;
Ex taq及rtaq等聚合酶、限制性内切酶(PstI、XhoI,NdeI、BamHI)、T4DNA连接酶,DNAMarker,DNA凝胶回收试剂盒均购自北京宝日医生物技术有限公司提供;
实施例中所述的SM17液体培养基,每升组分如下:
动物蛋白胨2.5g,胰蛋白胨2.5g,大豆蛋白胨5g,牛肉提取物5g,酵母提取物2.5g,抗坏血酸0.5g,硫酸镁0.25g,beta-甘油磷酸钠五水合物19g,蔗糖20g,余量水。
实施例中所述的SM17平板,每升组分如下:
动物蛋白胨2.5g,胰蛋白胨2.5g,大豆蛋白胨5g,牛肉提取物5g,酵母提取物2.5g,抗坏血酸0.5g,硫酸镁0.25g,beta-甘油磷酸钠五水合物19g,蔗糖20g,琼脂粉20g,余量水。
LM17液体培养基,每升组分如下:
动物蛋白胨2.5g,胰蛋白胨2.5g,大豆蛋白胨5g,牛肉提取物5g,酵母提取物2.5g,抗坏血酸0.5g,硫酸镁0.25g,beta-甘油磷酸钠五水合物19g,乳糖20g,余量水。
M17-半乳糖液体培养基,每升组分如下:
动物蛋白胨2.5g,胰蛋白胨2.5g,大豆蛋白胨5g,牛肉提取物5g,酵母提取物2.5g,抗坏血酸0.5g,硫酸镁0.25g,beta-甘油磷酸钠五水合物19g,半乳糖20g,余量水。
实施例1
敲除lacS基因嗜热链球菌的生长特性
嗜热链球菌DSM 32596敲除lacS基因的步骤具体如下:
(1)将嗜热链球菌DSM 32596接种于LM17液体培养基中,42℃静止培养过夜后收集菌体,使用细菌基因组提取试剂盒提取基因组DNA。
(2)以步骤(1)的基因组DNA为模板,利用核苷酸序列为SEQ ID NO.1和SEQ IDNO.2的引物对ST-LacS-up-F和ST-LacS-up-R进行PCR扩增乳糖透过酶基因的上游同源臂,
ST-LacS-up-F:
ggtaccgggccccccctcgagTTCCAACGGAAACTGGTGCT SEQ ID NO.1;
ST-LacS-up-R:
TTCGGAAACCTCCTATTATTTG SEQ ID NO.2。
PCR扩增体系为:Ex taq buffer 25μL,dNTP 4μL,引物ST-LacS-up-F 2μL,引物ST-LacS-up-R 2μL,基因组DNA 1μL,Ex taq 1μL,双蒸水13μL;
PCR扩增条件为:95℃预变性3min;95℃解链30s,50℃退火30s,72℃延伸1min,重复30个循环;72℃维持10min;4℃保存。
利用核苷酸序列为SEQ ID NO.3和SEQ ID NO.4的引物对ST-LacS-down-F和ST-LacS-down-R进行PCR扩增乳糖透过酶基因的下游同源臂,
ST-LacS-down-F:
CAAATAATAGGAGGTTTCCGAATCTATGAACATGACTGAAAAAAT SEQ ID NO.3;
ST-LacS-down-R:
agtggatcccccgggctgcagAAGACAATTCTCTTACCATTC SEQ ID NO.4。
PCR扩增体系为:Ex taq buffer 25μL,dNTP 4μL,引物ST-LacS-down-F 2μL,引物ST-LacS-down-R 2μL,基因组DNA 1μL,Ex taq 1μL,双蒸水13μL。
PCR扩增条件为:95℃预变性3min;95℃解链30s,50℃退火30s,72℃延伸1min,重复30个循环;72℃维持10min;4℃保存。
然后,利用重叠拼接PCR的方法,对上游同源臂和下游同源臂进行连接,制得乳糖透过酶基因敲除连接臂。
PCR扩增体系为:Ex taq buffer 25μL,dNTP 4μL,引物ST-LacS-up-F 2μL,引物ST-LacS-down-R 2μL,上游同源臂1μL,下游同源臂1μL,Ex taq 1μL,双蒸水12μL。
PCR扩增条件为:95℃预变性3min;95℃解链30s,50℃退火30s,72℃延伸2min,重复30个循环;72℃维持10min;4℃保存。
(3)使用PstI和XhoI对质粒pGhost9进行双酶切,然后利用同源重组酶将步骤(2)中,制得的乳糖透过酶基因敲除连接臂连接到pGhost9上,将连接产物转化入感受态大肠杆菌XL-Blue1,涂布于含有250μg/mL红霉素的LB平板,30℃静置培养48小时后,挑取转化子接入新鲜的含有250μg/mL红霉素的LB液体管,30℃摇动培养24小时后,提取质粒,使用PstI和XhoI进行双酶切,酶切产物进行琼脂糖凝胶电泳,其中包含一条2000bp的条带,即为正确的转化子,提取验证正确的转化子的质粒。
(4)将步骤(3)得到的重组质粒转化入嗜热链球菌DSM 32596中,涂布于含有2.5μg/mL红霉素的SM17平板,30℃静置培养72小时后,挑取转化子于新鲜的含有2.5μg/mL红霉素的SM17液体培养基中,30℃静置培养至OD600为1.0,将培养温度调节成42℃培养,促使质粒与基因组发生第一次同源交换,42℃培养2小时后,涂布于含有2.5μg/mL红霉素的SM17平板,继续在42℃培养,长出的即为发生第一次同源交换菌株。
(5)将步骤(4)中第一次同源交换成功的菌株接种于不含有抗生素的SM17液体培养基中,在30℃下静置培养,12小时后转接至新鲜的不含有抗生素的SM17液体培养基中,重复此步骤20次,使菌株发生第二次同源交换。将菌液涂布于不含有抗生素的SM17平板,30℃培养12小时后,挑取长出的单菌落,分别点接于添加和不添加2.5μg/mL红霉素的SM17平板,继续30℃培养。将在无红霉素下生长,而有红霉素下不生长的菌株挑取至新鲜的培养基。30℃培养12小时后提取基因组DNA。利用核苷酸序列为SEQ ID NO.5和SEQ ID NO.6的引物对对第二次同源交换成功的菌株进行PCR检测,扩增出3085bp的目的产物,即为乳糖透过酶基因敲除菌株,命名为嗜热链球菌ΔlacS。
ST-LacS-test-F:TCGTGACTATGTGCATCC SEQ ID NO.5
ST-LacS-test-R:GATATCAGCTGGTTTCGC SEQ ID NO.6
PCR检测体系为:Ex taq buffer 25μL,dNTP 4μL,引物ST-LacS-test-F 2μL,引物ST-LacS-test-R 2μL,基因组DNA 1μL,Ex taq 1μL,双蒸水13μL。
PCR检测反应条件为:95℃预变性3min;95℃解链30s,45℃退火30s,72℃延伸3min,重复30个循环;72℃维持10min;4℃保存。
嗜热链球菌DSM 32596和嗜热链球菌ΔlacS分别接种至LM17、SM17和M17-半乳糖中培养24h,以分析其在不同碳源下的生长能力。如图1所示,当在含有乳糖的培养基中培养时,野生菌株的OD600值最终为1.84±0.02,pH从6.96±0.01降低至5.07±0.04;而敲除菌株的OD600值最终仅0.37±0.00,pH降至6.89±0.01。然而,这两种菌株在SM17培养基中获得了相似水平的生物量,OD600值分别为1.99±0.012和2.03±0.01,pH分别达到4.51±0.01和4.52±0.01。当菌株在半乳糖培养基中生长时,与在乳糖中的生长状况相似。其中,野生菌株的OD600最终为1.65±0.01,pH最终为4.79±0.01;敲除菌株几乎不在半乳糖中生长,OD600最终仅达到0.68±0.03,pH最终为6.48±0.02。这些结果表明,乳糖透过酶缺失的突变菌株嗜热链球菌ΔlacS失去了在乳糖和半乳糖中生长的能力,但是该菌株仍然可以利用蔗糖,这有利于转化体的筛选。
实施例2
表达大肠杆菌半乳糖转运酶载体的构建
表达大肠杆菌半乳糖转运酶载体的示意图如图2所示。以大肠杆菌BL21的基因组DNA为模板,使用引物E-gala-F和E-gala-R扩增大肠杆菌半乳糖转运酶片段。随后,使用引物P32-F和P32-R从质粒pMG36e中扩增出P32启动子片段。通过重叠延伸PCR将P32启动子和大肠杆菌半乳糖转运酶片段基因融合,使用核苷酸序列为SEQ ID NO.11和SEQ ID NO.12的引物线性化表达载体pNZ8148,再将融合片段使用同源重组酶插入到表达载体pNZ8148中,连接产物转化至大肠杆菌XL1-Blue,复苏后涂布于补充有10μg/mL氯霉素的LB固体培养基,37℃培养12h。挑取转化子在补充有10μg/mL氯霉素的LB固体培养基中划线扩培,然后收集菌体提取质粒,进行PCR验证。得到重组质粒pNZ8148-E-gala,其序列如SEQ ID NO.13所示。将重组质粒电转化至嗜热链球菌ΔlacS,获得重组菌株嗜热链球菌ΔlacS/pNZ8148-E-gala表达大肠杆菌半乳糖转运酶。通过补充含有5μg/mL氯霉素的SM17筛选得到的重组菌株,重组菌株的PCR验证结果如图3所示。
E-gala-F:TTCGGAGGAATTTTGAAATGCCTGACGCTAAAAAAC SEQ ID NO.7;
E-gala-R:CCTTCGTTTTCAGACTTTGCTTAATCGTGAGCGCCTAT SEQ ID NO.8;
P32-F:TCGAATTCGGTCCTCGGG SEQ ID NO.9;
P32-R:TTCAAAATTCCTCCGAATA SEQ ID NO.10;
pNZ8148-F:GCTCAAGCTTTCTTTGAACC SEQ ID NO.11;
pNZ8148-R:CCCGAGGACCGAATTCGAATTATGCTCGCGTTATCGAC SEQ ID NO.12。
实施例3
嗜热链球菌回补菌株的生长测定
将重组嗜热链球菌菌株ΔlacS/pNZ8148-E-gala在补充有5μg/mL氯霉素的SM17中培养过夜,然后分别接种至LM17和M17-半乳糖培养基中培养24h。每隔2h取样测定OD600以表征菌株的生长状况,同时使用pH计测定培养物的pH以表征菌株的pH变化。设置3组平行试验,误差线代表3次重复实验的标准偏差。菌株的生长状况如图4所示。带有大肠杆菌半乳糖转运酶的重组嗜热链球菌菌株ΔlacS/pNZ8148-E-gala在半乳糖培养基中恢复了生长能力,OD600和pH值分别为1.76±0.00和5.10±0.01;然而在乳糖培养基中几乎不生长,OD600和pH值分别为0.26±0.00和6.40±0.01。
这些结果表明,来自大肠杆菌的半乳糖转运酶片段可以对乳糖透过酶缺陷菌株的半乳糖转运缺陷进行回补,恢复菌株的半乳糖利用能力,但不能恢复菌株的乳糖利用能力。
实施例4
低半乳糖发酵牛乳的制备
将嗜热链球菌DSM 32596、重组嗜热链球菌菌株ΔlacS/pNZ8148-E-gala分别与保加利亚乳杆菌ATCC11842以1:1的比例接种(4%,v/v)至牛乳,42℃培养12h后测定酸奶中半乳糖含量。测定方法如下,称取1g发酵乳,加入45mM硫酸溶液5mL后用涡旋仪震荡混匀1min,随后在16℃培养箱中以240rpm的速度将溶液摇动30min,再用涡旋仪震荡混匀1min。将所得混合液在6,000×g,4℃的条件下离心20min,上清液使用0.22μm滤器过滤后通过HPLC检测样品中乳糖和半乳糖含量。HPLC配置为CBM-20A控制器,LC-20AT泵,SIL-20A自动进样器,CTO-10A柱温箱和RID-20A示差检测器。使用色谱柱HPX-87H,设置柱温箱温度为60℃,流动相为3mM硫酸,流速为0.5mL/min。
结果显示,嗜热链球菌DSM 32596与保加利亚乳杆菌发酵酸奶的半乳糖含量为7.6±0.4g/L;重组嗜热链球菌ΔlacS/pNZ8148-E-gala与保加利亚乳杆菌发酵酸奶的半乳糖含量更低为0.3±0.1g/L;即不利用乳糖而利用半乳糖的重组嗜热链球菌ΔlacS/pNZ8148-E-gala与保加利亚乳杆菌共培养可以更有效地降低酸奶中半乳糖的含量。
对比例1
表达大肠杆菌乳糖透过酶载体的构建
以大肠杆菌BL21的基因组DNA为模板,使用引物E-lacS-F和E-lacS-R扩增大肠杆菌乳糖透过酶片段。随后,使用引物P32-F和P32-R从质粒pMG36e中扩增出P32启动子片段。通过重叠延伸PCR将P32启动子和大肠杆菌乳糖透过酶片段基因融合,再将融合片段插入表达载体pNZ8148,连接产物转化至大肠杆菌XL1-Blue,复苏后涂布于补充有10μg/mL氯霉素的LB固体培养基,37℃培养12h。挑取转化子在补充有10μg/mL氯霉素的LB固体培养基中划线扩培,然后收集菌体提取质粒进行PCR验证。得到重组质粒pNZ8148-E-lacS,其序列如SEQID NO.16所示。将重组质粒电转化至嗜热链球菌ΔlacS,通过补充含有5μg/mL氯霉素的SM17来筛选重组菌株。结果显示,表达大肠杆菌乳糖透过酶的回补载体转化嗜热链球菌一直未成功,推测其原因可能是大肠杆菌来源的乳糖透过酶对嗜热链球菌具有毒性所致。
E-lacS-F:TTCGGAGGAATTTTGAAATGTACTATTTAAAAAACACAAAC SEQ ID NO.14;
E-lacS-R:CCTTCGTTTTCAGACTTTGCTTAAGCGACTTCATTCACCT SEQ ID NO.15;
P32-F:TCGAATTCGGTCCTCGGG SEQ ID NO.9;
P32-R:TTCAAAATTCCTCCGAATA SEQ ID NO.10;
pNZ8148-F:GCTCAAGCTTTCTTTGAACC SEQ ID NO.11;
pNZ8148-R:CCCGAGGACCGAATTCGAATTATGCTCGCGTTATCGAC SEQ ID NO.12。
本发明中,选择能够同时利用乳糖和半乳糖的嗜热链球菌作为出发菌株,将其乳糖透过酶基因敲除后,发明人发现基因敲除菌株除了不能利用乳糖外,同时也失去了利用半乳糖的能力;发明人尝试在嗜热链球菌敲除株中导入大肠杆菌的乳糖透过酶基因,结果一直未获得重组菌株,表明不同外源基因与嗜热链球菌的适配性存在差异。发明人将来源于大肠杆菌的专一性半乳糖转运酶基因导入到嗜热链球菌敲除株中,发现重组菌株恢复了利用半乳糖的能力,但是不能利用乳糖,该重组菌株相较于野生菌株,能够更有效的降低发酵酸奶中半乳糖的含量。
SEQUENCE LISTING
<110> 齐鲁工业大学
<120> 一种嗜热链球菌重组菌的构建方法及应用
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 41
<212> DNA
<213> 人工序列
<400> 1
ggtaccgggc cccccctcga gttccaacgg aaactggtgc t 41
<210> 2
<211> 22
<212> DNA
<213> 人工序列
<400> 2
ttcggaaacc tcctattatt tg 22
<210> 3
<211> 45
<212> DNA
<213> 人工序列
<400> 3
caaataatag gaggtttccg aatctatgaa catgactgaa aaaat 45
<210> 4
<211> 42
<212> DNA
<213> 人工序列
<400> 4
agtggatccc ccgggctgca gaagacaatt ctcttaccat tc 42
<210> 5
<211> 18
<212> DNA
<213> 人工序列
<400> 5
tcgtgactat gtgcatcc 18
<210> 6
<211> 18
<212> DNA
<213> 人工序列
<400> 6
gatatcagct ggtttcgc 18
<210> 7
<211> 36
<212> DNA
<213> 人工序列
<400> 7
ttcggaggaa ttttgaaatg cctgacgcta aaaaac 36
<210> 8
<211> 38
<212> DNA
<213> 人工序列
<400> 8
ccttcgtttt cagactttgc ttaatcgtga gcgcctat 38
<210> 9
<211> 18
<212> DNA
<213> 人工序列
<400> 9
tcgaattcgg tcctcggg 18
<210> 10
<211> 19
<212> DNA
<213> 人工序列
<400> 10
ttcaaaattc ctccgaata 19
<210> 11
<211> 20
<212> DNA
<213> 人工序列
<400> 11
gctcaagctt tctttgaacc 20
<210> 12
<211> 38
<212> DNA
<213> 人工序列
<400> 12
cccgaggacc gaattcgaat tatgctcgcg ttatcgac 38
<210> 13
<211> 4614
<212> DNA
<213> 人工序列
<400> 13
agatctagtc ttataactat actgacaata gaaacattaa caaatctaaa acagtcttaa 60
ttctatcttg agaaagtatt ggtaataata ttattgtcga taacgcgagc ataattcgaa 120
ttcggtcctc gggatatgat aagattaata gttttagcta ttaatctttt tttattttta 180
tttaagaatg gcttaataaa gcggttactt tggatttttg tgagcttgga ctagaaaaaa 240
acttcacaaa atgctatact aggtaggtaa aaaaatattc ggaggaattt tgaaatgcct 300
gacgctaaaa aacaggggcg gtcaaacaag gcaatgacgt ttttcgtctg cttccttgcc 360
gctctggcgg gattactctt tggcctggat atcggtgtaa ttgctggcgc actgccgttt 420
attgcagatg aattccagat tacttcgcac acgcaagaat gggtcgtaag ctccatgatg 480
ttcggtgcgg cagtcggtgc ggtgggcagc ggctggctct cctttaaact cgggcgcaaa 540
aagagcctga tgatcggcgc aattttgttt gttgccggtt cgctgttctc tgcggctgcg 600
ccaaacgttg aagtactgat tctttcccgc gttctactgg ggctggcggt gggtgtggcc 660
tcttataccg caccgctgta cctctctgaa attgcgccgg aaaaaattcg tggcagtatg 720
atctcgatgt atcagttgat gatcactatc gggatcctcg gtgcttatct ttctgatacc 780
gccttcagct acaccggtgc atggcgctgg atgctgggtg tgattatcat cccggcaatt 840
ttgctgctga ttggtgtctt cttcctgcca gacagcccac gttggtttgc cgccaaacgc 900
cgttttgttg atgccgaacg cgtgctgcta cgcctgcgtg acaccagcgc ggaagcgaaa 960
cgcgaactgg atgaaatccg tgaaagtttg caggttaaac agagtggctg ggcgctgttt 1020
aaagagaaca gcaacttccg ccgcgcggtg ttccttggcg tactgttgca ggtaatgcag 1080
caattcaccg ggatgaacgt catcatgtat tacgcgccga aaatcttcga actggcgggt 1140
tataccaaca ctaccgagca aatgtggggg accgtgattg tcggcctgac caacgtactt 1200
gccaccttta tcgcaatcgg ccttgttgac cgctggggac gtaaaccaac gctaacgctg 1260
ggcttcctgg tgatggctgc tggcatgggc gtactcggta caatgatgca tatcggtatt 1320
cactctccgt cggcgcagta tttcgccatc gccatgctgc tgatgtttat tgtcggtttt 1380
gccatgagtg ccggtccgct gatttgggta ctgtgctccg aaattcagcc gctgaaaggc 1440
cgcgattttg gcatcacctg ctccactgcc accaactgga ttgccaacat gatcgttggc 1500
gcaacgttcc tgaccatgct caacacgctg ggtaacgcca acaccttctg ggtgtatgcg 1560
gctctgaacg tactgtttat cctgctgaca ttgtggctgg taccggaaac caaacacgtt 1620
tcgctggaac atattgaacg taatctgatg aaaggtcgta aactgcgcga aataggcgct 1680
cacgattaag ctcaagcttt ctttgaacca aaattagaaa accaaggctt gaaacgttca 1740
attgaaatgg caattaaaca aattacagca cgtgttgctt tgattgatag ccaaaaagca 1800
gcagttgata aagcaattac tgatattgct gaaaaattgt aatttataaa taaaaatcac 1860
cttttagagg tggttttttt atttataaat tattcgtttg atttcgcttt cgatagaaca 1920
atcaaatcgt ttctgagacg ttttagcgtt tatttcgttt agttatcggc ataatcgtta 1980
aaacaggcgt tatcgtagcg taaaagccct tgagcgtagc gtggctttgc agcgaagatg 2040
ttgtctgtta gattatgaaa gccgatgact gaatgaaata ataagcgcag cgtccttcta 2100
tttcggttgg aggaggctca agggagtttg agggaatgaa attccctcat gggtttgatt 2160
ttaaaaattg cttgcaattt tgccgagcgg tagcgctgga aaatttttga aaaaaatttg 2220
gaatttggaa aaaaatgggg ggaaaggaag cgaattttgc ttccgtacta cgacccccca 2280
ttaagtgccg agtgccaatt tttgtgccaa aaacgctcta tcccaactgg ctcaagggtt 2340
tgaggggttt ttcaatcgcc aacgaatcgc caacgttttc gccaacgttt tttataaatc 2400
tatatttaag tagctttatt tttgttttta tgattacaaa gtgatacact aattttataa 2460
aattatttga ttggagtttt ttaaatggtg atttcagaat cgaaaaaaag agttatgatt 2520
tctctgacaa aagagcaaga taaaaaatta acagatatgg cgaaacaaaa agatttttca 2580
aaatctgcgg ttgcggcgtt agctatagaa gaatatgcaa gaaaggaatc agaacaaaaa 2640
aaataagcga aagctcgcgt ttttagaagg atacgagttt tcgctacttg tttttgataa 2700
ggtaattata tcatggctat taaaaatact aaagctagaa attttggatt tttattatat 2760
cctgactcaa ttcctaatga ttggaaagaa aaattagaga gtttgggcgt atctatggct 2820
gtcagtcctt tacacgatat ggacgaaaaa aaagataaag atacatggaa tagtagtgat 2880
gttatacgaa atggaaagca ctataaaaaa ccacactatc acgttatata tattgcacga 2940
aatcctgtaa caatagaaag cgttaggaac aagattaagc gaaaattggg gaatagttca 3000
gttgctcatg ttgagatact tgattatatc aaaggttcat atgaatattt gactcatgaa 3060
tcaaaggacg ctattgctaa gaataaacat atatacgaca aaaaagatat tttgaacatt 3120
aatgattttg atattgaccg ctatataaca cttgatgaaa gccaaaaaag agaattgaag 3180
aatttacttt tagatatagt ggatgactat aatttggtaa atacaaaaga tttaatggct 3240
tttattcgcc ttaggggagc ggagtttgga attttaaata cgaatgatgt aaaagatatt 3300
gtttcaacaa actctagcgc ctttagatta tggtttgagg gcaattatca gtgtggatat 3360
agagcaagtt atgcaaaggt tcttgatgct gaaacggggg aaataaaatg acaaacaaag 3420
aaaaagagtt atttgctgaa aatgaggaat taaaaaaaga aattaaggac ttaaaagagc 3480
gtattgaaag atacagagaa atggaagttg aattaagtac aacaatagat ttattgagag 3540
gagggattat tgaataaata aaagcccccc tgacgaaagt cgacggcaat agttaccctt 3600
attatcaaga taagaaagaa aaggattttt cgctacgctc aaatccttta aaaaaacaca 3660
aaagaccaca ttttttaatg tggtctttta ttcttcaact aaagcaccca ttagttcaac 3720
aaacgaaaat tggataaagt gggatatttt taaaatatat atttatgtta cagtaatatt 3780
gacttttaaa aaaggattga ttctaatgaa gaaagcagac aagtaagcct cctaaattca 3840
ctttagataa aaatttagga ggcatatcaa atgaacttta ataaaattga tttagacaat 3900
tggaagagaa aagagatatt taatcattat ttgaaccaac aaacgacttt tagtataacc 3960
acagaaattg atattagtgt tttataccga aacataaaac aagaaggata taaattttac 4020
cctgcattta ttttcttagt gacaagggtg ataaactcaa atacagcttt tagaactggt 4080
tacaatagcg acggagagtt aggttattgg gataagttag agccacttta tacaattttt 4140
gatggtgtat ctaaaacatt ctctggtatt tggactcctg taaagaatga cttcaaagag 4200
ttttatgatt tatacctttc tgatgtagag aaatataatg gttcggggaa attgtttccc 4260
aaaacaccta tacctgaaaa tgctttttct ctttctatta ttccttggac ttcatttact 4320
gggtttaact taaatatcaa taataatagt aattaccttc tacccattat tacagcagga 4380
aaattcatta ataaaggtaa ttcaatatat ttaccgctat ctttacaggt acatcattct 4440
gtttgtgatg gttatcatgc tggattgttt atgaactcta ttcaggaatt gtcagatagg 4500
cctaatgact ggcttttata atatgagata atgccgactg tactttttac agtcggtttt 4560
ctaatgtcac taacctgccc cgttagttga agaaggtttt tatattacag ctcc 4614
<210> 14
<211> 41
<212> DNA
<213> 人工序列
<400> 14
ttcggaggaa ttttgaaatg tactatttaa aaaacacaaa c 41
<210> 15
<211> 40
<212> DNA
<213> 人工序列
<400> 15
ccttcgtttt cagactttgc ttaagcgact tcattcacct 40
<210> 16
<211> 4473
<212> DNA
<213> 人工序列
<400> 16
agatctagtc ttataactat actgacaata gaaacattaa caaatctaaa acagtcttaa 60
ttctatcttg agaaagtatt ggtaataata ttattgtcga taacgcgagc ataattcgaa 120
ttcggtcctc gggatatgat aagattaata gttttagcta ttaatctttt tttattttta 180
tttaagaatg gcttaataaa gcggttactt tggatttttg tgagcttgga ctagaaaaaa 240
acttcacaaa atgctatact aggtaggtaa aaaaatattc ggaggaattt tgaaatgtac 300
tatttaaaaa acacaaactt ttggatgttc ggtttattct ttttctttta cttttttatc 360
atgggagcct acttcccgtt tttcccgatt tggctacatg acatcaacca tatcagcaaa 420
agtgatacgg gtattatttt tgccgctatt tctctgttct cgctattatt ccaaccgctg 480
tttggtctgc tttctgacaa actcgggctg cgcaaatacc tgctgtggat tattaccggc 540
atgttagtga tgtttgcgcc gttctttatt tttatcttcg ggccactgtt acaatacaac 600
attttagtag gatcgattgt tggtggtatt tatctaggct tttgttttaa cgccggtgcg 660
ccagcagtag aggcatttat tgagaaagtc agccgtcgca gtaatttcga atttggtcgc 720
gcgcggatgt ttggctgtgt tggctgggcg ctgtgtgcct cgattgtcgg catcatgttc 780
accatcaata atcagtttgt tttctggctg ggctctggct gtgcactcat cctcgccgtt 840
ttactctttt tcgccaaaac ggatgcgccc tcttctgcca cggttgccaa tgcggtaggt 900
gccaaccatt cggcatttag ccttaagctg gcactggaac tgttcagaca gccaaaactg 960
tggtttttgt cactgtatgt tattggcgtt tcctgcacct acgatgtttt tgaccaacag 1020
tttgctaatt tctttacttc gttctttgct accggtgaac agggtacgcg ggtatttggc 1080
tacgtaacga caatgggcga attacttaac gcctcgatta tgttctttgc gccactgatc 1140
attaatcgca tcggtgggaa aaacgccctg ctgctggctg gcactattat gtctgtacgt 1200
attattggct catcgttcgc cacctcagcg ctggaagtgg ttattctgaa aacgctgcat 1260
atgtttgaag taccgttcct gctggtgggc tgctttaaat atattaccag ccagtttgaa 1320
gtgcgttttt cagcgacgat ttatctggtc tgtttctgct tctttaagca actggcgatg 1380
atttttatgt ctgtactggc gggcaatatg tatgaaagca tcggtttcca gggcgcttat 1440
ctggtgctgg gtctggtggc gctgggcttc accttaattt ccgtgttcac gcttagcggc 1500
cccggcccgc tttccctgct gcgtcgtcag gtgaatgaag tcgcttaagc tcaagctttc 1560
tttgaaccaa aattagaaaa ccaaggcttg aaacgttcaa ttgaaatggc aattaaacaa 1620
attacagcac gtgttgcttt gattgatagc caaaaagcag cagttgataa agcaattact 1680
gatattgctg aaaaattgta atttataaat aaaaatcacc ttttagaggt ggttttttta 1740
tttataaatt attcgtttga tttcgctttc gatagaacaa tcaaatcgtt tctgagacgt 1800
tttagcgttt atttcgttta gttatcggca taatcgttaa aacaggcgtt atcgtagcgt 1860
aaaagccctt gagcgtagcg tggctttgca gcgaagatgt tgtctgttag attatgaaag 1920
ccgatgactg aatgaaataa taagcgcagc gtccttctat ttcggttgga ggaggctcaa 1980
gggagtttga gggaatgaaa ttccctcatg ggtttgattt taaaaattgc ttgcaatttt 2040
gccgagcggt agcgctggaa aatttttgaa aaaaatttgg aatttggaaa aaaatggggg 2100
gaaaggaagc gaattttgct tccgtactac gaccccccat taagtgccga gtgccaattt 2160
ttgtgccaaa aacgctctat cccaactggc tcaagggttt gaggggtttt tcaatcgcca 2220
acgaatcgcc aacgttttcg ccaacgtttt ttataaatct atatttaagt agctttattt 2280
ttgtttttat gattacaaag tgatacacta attttataaa attatttgat tggagttttt 2340
taaatggtga tttcagaatc gaaaaaaaga gttatgattt ctctgacaaa agagcaagat 2400
aaaaaattaa cagatatggc gaaacaaaaa gatttttcaa aatctgcggt tgcggcgtta 2460
gctatagaag aatatgcaag aaaggaatca gaacaaaaaa aataagcgaa agctcgcgtt 2520
tttagaagga tacgagtttt cgctacttgt ttttgataag gtaattatat catggctatt 2580
aaaaatacta aagctagaaa ttttggattt ttattatatc ctgactcaat tcctaatgat 2640
tggaaagaaa aattagagag tttgggcgta tctatggctg tcagtccttt acacgatatg 2700
gacgaaaaaa aagataaaga tacatggaat agtagtgatg ttatacgaaa tggaaagcac 2760
tataaaaaac cacactatca cgttatatat attgcacgaa atcctgtaac aatagaaagc 2820
gttaggaaca agattaagcg aaaattgggg aatagttcag ttgctcatgt tgagatactt 2880
gattatatca aaggttcata tgaatatttg actcatgaat caaaggacgc tattgctaag 2940
aataaacata tatacgacaa aaaagatatt ttgaacatta atgattttga tattgaccgc 3000
tatataacac ttgatgaaag ccaaaaaaga gaattgaaga atttactttt agatatagtg 3060
gatgactata atttggtaaa tacaaaagat ttaatggctt ttattcgcct taggggagcg 3120
gagtttggaa ttttaaatac gaatgatgta aaagatattg tttcaacaaa ctctagcgcc 3180
tttagattat ggtttgaggg caattatcag tgtggatata gagcaagtta tgcaaaggtt 3240
cttgatgctg aaacggggga aataaaatga caaacaaaga aaaagagtta tttgctgaaa 3300
atgaggaatt aaaaaaagaa attaaggact taaaagagcg tattgaaaga tacagagaaa 3360
tggaagttga attaagtaca acaatagatt tattgagagg agggattatt gaataaataa 3420
aagcccccct gacgaaagtc gacggcaata gttaccctta ttatcaagat aagaaagaaa 3480
aggatttttc gctacgctca aatcctttaa aaaaacacaa aagaccacat tttttaatgt 3540
ggtcttttat tcttcaacta aagcacccat tagttcaaca aacgaaaatt ggataaagtg 3600
ggatattttt aaaatatata tttatgttac agtaatattg acttttaaaa aaggattgat 3660
tctaatgaag aaagcagaca agtaagcctc ctaaattcac tttagataaa aatttaggag 3720
gcatatcaaa tgaactttaa taaaattgat ttagacaatt ggaagagaaa agagatattt 3780
aatcattatt tgaaccaaca aacgactttt agtataacca cagaaattga tattagtgtt 3840
ttataccgaa acataaaaca agaaggatat aaattttacc ctgcatttat tttcttagtg 3900
acaagggtga taaactcaaa tacagctttt agaactggtt acaatagcga cggagagtta 3960
ggttattggg ataagttaga gccactttat acaatttttg atggtgtatc taaaacattc 4020
tctggtattt ggactcctgt aaagaatgac ttcaaagagt tttatgattt atacctttct 4080
gatgtagaga aatataatgg ttcggggaaa ttgtttccca aaacacctat acctgaaaat 4140
gctttttctc tttctattat tccttggact tcatttactg ggtttaactt aaatatcaat 4200
aataatagta attaccttct acccattatt acagcaggaa aattcattaa taaaggtaat 4260
tcaatatatt taccgctatc tttacaggta catcattctg tttgtgatgg ttatcatgct 4320
ggattgttta tgaactctat tcaggaattg tcagataggc ctaatgactg gcttttataa 4380
tatgagataa tgccgactgt actttttaca gtcggttttc taatgtcact aacctgcccc 4440
gttagttgaa gaaggttttt atattacagc tcc 4473
Claims (14)
1.一种嗜热链球菌重组菌的构建方法,其特征在于,具体如下步骤:
(1)一株嗜热链球菌ΔlacS的构建,包括如下步骤:
① 提取嗜热链球菌DSM 32596(Streptococcus thermophilus DSM 32596)的基因组DNA;
② 以步骤①的基因组DNA为模板,利用核苷酸序列为SEQ ID NO.1和SEQ ID NO.2的引物对ST-LacS-up-F和ST-LacS-up-R进行PCR扩增乳糖透过酶基因的上游同源臂,利用核苷酸序列为SEQ ID NO.3和SEQ ID NO.4的引物对ST-LacS-down-F和ST-LacS-down-R进行PCR扩增乳糖透过酶基因的下游同源臂,然后利用重叠拼接PCR的方法,对上游同源臂和下游同源臂进行连接,制得乳糖透过酶基因敲除连接臂;
③ 使用PstI和XhoI对质粒pGhost9进行双酶切,然后利用同源重组酶将步骤②中,制得的乳糖透过酶基因敲除连接臂连接到pGhost9上,将连接产物转化入感受态大肠杆菌XL-Blue1,挑取验证正确的转化子,提取重组质粒;
④ 将步骤③得到的重组质粒转化入嗜热链球菌DSM 32596中,通过转化子培养发生第一次同源交换,利用红霉素进行筛选,然后进行连续传代发生第二次同源交换,筛选红霉素标记丢失的菌株,利用核苷酸序列为SEQ ID NO.5和SEQ ID NO.6的引物对,对红霉素标记丢失的菌株进行检测,扩增出3085 bp的目的产物,即为乳糖透过酶基因敲除菌株,命名为嗜热链球菌ΔlacS;
(2)将载体pNZ8148-E-gala转化入嗜热链球菌ΔlacS中,所述载体pNZ8148-E-gala的核苷酸序列如SEQ ID NO.13所示。
2. 如权利要求1所述的构建方法,其特征在于,步骤②中,上游同源臂PCR扩增体系如下:Ex taq buffer 25 µL,dNTP 4 µL,引物ST-LacS-up-F 2 µL,引物ST-LacS-up-R 2 µL,基因组DNA 1 µL,Ex taq 1 µL, 双蒸水13 µL;
PCR扩增条件为:95℃预变性3min;95℃解链30s,50℃退火30s,72℃延伸1min,重复30个循环;72℃维持10min;4℃保存;
步骤②中,下游同源臂PCR扩增体系如下:Ex taq buffer 25 µL,dNTP 4 µL,引物ST-LacS-down-F 2 µL,引物ST-LacS-down-R 2 µL,基因组DNA 1 µL,Ex taq 1 µL,双蒸水13µL;
PCR扩增条件为:95℃预变性3min;95℃解链30s,50℃退火30s,72℃延伸1min,重复30个循环;72℃维持10min;4℃保存;
步骤②中,上游同源臂和下游同源臂进行连接,PCR扩增体系为:Ex taq buffer 25 µL,dNTP 4 µL,引物ST-LacS-up-F 2 µL,引物ST-LacS-down-R 2 µL,上游同源臂 1 µL,下游同源臂 1 µL,Ex taq 1 µL,双蒸水12 µL;
PCR扩增条件为:95℃预变性3min;95℃解链30s,50℃退火30s,72℃延伸2min,重复30个循环;72℃维持10min;4℃保存。
3.如权利要求1所述的构建方法,其特征在于,步骤③中,验证转化子的方法:将连接产物转化入感受态大肠杆菌XL-Blue1,涂布于含有250 µg/mL红霉素的LB平板,30℃静置培养48小时后,挑取转化子接入新鲜的含有250 µg/mL红霉素的LB液体管,30℃摇动培养24小时后,提取质粒,使用PstI和XhoI进行双酶切,酶切产物进行琼脂糖凝胶电泳,其中包含一条2000 bp的条带,即为正确的转化子,提取验证正确的转化子的质粒。
4. 如权利要求1所述的构建方法,其特征在于,步骤④中,转化子的培养条件:转化子在含有2.5 µg/mL红霉素的SM17平板,30℃培养。
5. 如权利要求1所述的构建方法,其特征在于,步骤④中,利用红霉素的筛选条件:重组质粒转化入嗜热链球菌DSM 32596中,涂布于含有2.5 µg/mL红霉素的SM17平板,30℃静置培养72小时后,挑取转化子于新鲜的含有2.5 µg/mL红霉素的SM17液体培养基中,30℃静置培养至OD600为1.0,将培养温度调节成42℃培养,促使质粒与基因组发生第一次同源交换,42℃培养2小时后,涂布于含有2.5 µg/mL红霉素的SM17平板,继续在42℃培养,长出的即为发生第一次同源交换菌株。
6.如权利要求1所述的构建方法,其特征在于,步骤④中,连续传代的培养条件为第一次同源交换成功的菌株接种于不含有抗生素的SM17液体培养基中,在30℃下静置培养,12小时后转接至新鲜的不含有抗生素的SM17液体培养基中,重复此步骤20次,使菌株发生第二次同源交换。
7. 如权利要求1所述的构建方法,其特征在于,步骤④中,红霉素标记丢失的菌株筛选为连续传代后的菌液涂布于不含有抗生素的SM17平板,30℃培养12小时后,挑取长出的单菌落,分别点接于添加和不添加2.5 µg/mL红霉素的SM17平板,继续30℃培养,将在无红霉素下生长,而有红霉素下不生长的菌株即为红霉素标记丢失的菌株。
8.如权利要求5-7任一项所述的构建方法,其特征在于,步骤④中SM17培养基每升组分如下:
动物蛋白胨2.5 g,胰蛋白胨2.5 g,大豆蛋白胨5 g,牛肉提取物5 g,酵母提取物2.5g,抗坏血酸0.5 g,硫酸镁0.25 g,beta-甘油磷酸钠五水合物19 g,蔗糖20 g,余量水。
9. 如权利要求1所述的构建方法,其特征在于,步骤④中,PCR检测体系为:Ex taqbuffer 25 µL,dNTP 4 µL,引物ST-LacS-test-F 2 µL,引物ST-LacS-test-R 2 µL,基因组DNA 1 µL,Ex taq 1 µL, 双蒸水13 µL;
PCR检测反应条件为:95℃预变性3min;95℃解链30s,45℃退火30s,72℃延伸3min,重复30个循环;72℃维持10min;4℃保存。
10.如权利要求1所述的构建方法,其特征在于,步骤(2)中,载体pNZ8148-E-gala的构建方法:以大肠杆菌 BL21的基因组DNA为模板,使用核苷酸序列为SEQ ID NO.7和SEQ IDNO.8的引物对扩增大肠杆菌半乳糖转运酶片段,随后,使用核苷酸序列为SEQ ID NO.9和SEQ ID NO.10的引物对从质粒pMG36e中扩增出P32启动子片段,通过重叠延伸PCR将P32启动子和大肠杆菌半乳糖转运酶片段基因融合,使用核苷酸序列为SEQ ID NO.11和SEQ IDNO.12的引物线性化表达载体pNZ8148,再将融合片段使用同源重组酶插入到表达载体pNZ8148中,连接产物转化至大肠杆菌XL1-Blue,然后收集菌体提取质粒,得到重组质粒pNZ8148-E-gala,核苷酸序列如SEQ ID NO.13所示。
11.一种嗜热链球菌重组菌,其特征在于,包含权利要求1所述的嗜热链球菌ΔlacS和载体pNZ8148-E-gala,载体pNZ8148-E-gala转化入嗜热链球菌ΔlacS。
12.权利要求11所述嗜热链球菌重组菌在降低半乳糖中的应用。
13.权利要求11所述嗜热链球菌重组菌在制备酸奶中的应用。
14.如权利要求13所述的应用,其特征在于,所述嗜热链球菌重组菌在降低酸奶中半乳糖的应用。
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