CN111019958A - 过表达utp-葡萄糖-1-磷酸-尿苷转移酶基因及其重组工程菌的构建方法和应用 - Google Patents
过表达utp-葡萄糖-1-磷酸-尿苷转移酶基因及其重组工程菌的构建方法和应用 Download PDFInfo
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Abstract
本发明公开了过表达UTP‑葡萄糖‑1‑磷酸‑尿苷转移酶基因及其重组工程菌的构建方法和应用,特点是该基因来自于嗜酸乳杆菌ATCC 4356编码UTP‑葡萄糖‑1‑磷酸‑尿苷转移酶的基因,其核苷酸序列如序列表中SEQ ID No:1所示;构建方法包括以下步骤:通过克隆嗜酸乳杆菌ATCC4356的UTP‑葡萄糖‑1‑磷酸‑尿苷转移酶基因片段连接到pMG36e表达载体上,利用电转化的方法导入到肉葡萄球菌或者乳酸乳球菌中,通过红霉素抗性筛选并鉴定获得带有目的基因的重组工程菌,即过表达UTP‑葡萄糖‑1‑磷酸‑尿苷转移酶基因的工程菌,优点是显著提高菌体生长、酶活性、冻干存活率。
Description
技术领域
本发明属于生物工程及微生物发酵技术领域,尤其是涉及过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因及其重组工程菌的构建方法和应用。
背景技术
肉葡萄球菌呈椭圆形或球形,无鞭毛,不能运动,被分为凝血酶阳性或阴性葡萄球菌。其中金黄色葡萄球菌是凝固酶阳性葡萄球菌的原型物种,它是具有医学和兽医重要性的病原体;凝血酶阴性葡萄球菌有肉葡萄球菌。与其他葡萄球菌相比,肉葡萄球菌不产生溶血素、毒素等,在葡萄球菌属中被确认为食品级菌株。肉葡萄球菌可以产生不同的酶,例如硝酸盐还原酶,过氧化氢酶,超氧化物歧化酶,脂肪酶和蛋白酶,这些影响了发酵肉制品的肉色和风味形成,具有护色增香的功能。所以肉葡萄球菌在肉制品风味形成的加工中起着关键性的作用,因而该菌株常被作为发酵香肠的初始发酵剂的商业菌种广泛应用于肉制品发酵行业。
乳酸乳球菌是一种乳酸菌,是乳球菌属中最为典型和最重要的菌株之一,一般呈形卵圆形或球形,为兼性厌氧菌。其生长最佳生长温度为30℃,它们可以在10℃和45℃之间生长。乳酸乳球菌安全性可靠已在酸奶、泡菜、乳酪、酸奶油、大豆酸奶等各种食品加工中得到了广泛应用。乳酸乳球菌已得到其全部基因组。在分子方面,乳酸乳球菌由于具有易操作、培养时间短、自身只分泌蛋白等特点被选择作为克隆载体宿主菌的研究日益增多。
UTP-葡萄糖-1-磷酸-尿苷转移酶(UTP-glucose-1-phosphateuridylytransferase,)是糖代谢中一种重要的酶,在一定条件下它可以催化葡萄糖一磷酸和尿苷三磷酸的可逆反应生成尿苷二磷酸葡萄糖和焦磷酸,其产物尿苷二磷酸葡萄糖(UDPG)是可以用作合成细胞外聚糖或脂多糖的前体,是生物体代谢途径中的关键代谢产物在物质合成过程中发挥着至关重要的作用,参与蔗糖、纤维素、胼胝质等的合成。而UDPG的主要来源是通过UTP-葡萄糖-1-磷酸-尿苷转移酶的催化下生成的,因此该酶催化的这一反应将关系到下游的二糖或多糖的代谢。目前,通过过表达技术在植物中提高植物的生长报道已经很多,而将嗜酸乳杆菌编码UTP-葡萄糖-1-磷酸-尿苷转移酶的基因在肉葡萄球菌或者乳酸乳球菌过表达,而影响肉葡萄球菌或乳酸乳球菌的生长、酶活性及冻干存活率尚未见报道。因此,通过分子生物学技术改造菌株来提高肉葡萄球菌或乳酸乳球菌的生长、酶活性及冻干存活率具有重要的经济价值和社会意义。
发明内容
本发明所要解决的技术问题是提供一种能显著提高菌体生长速率、酶活性和冻干存活率的过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因及其重组工程菌的构建方法和应用。
本发明解决上述技术问题所采用的技术方案为:
1、一种过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因(LBA1719),所述基因来自于嗜酸乳杆菌(Lactobacillus acidophilus )ATCC 4356编码UTP-葡萄糖-1-磷酸-尿苷转移酶的基因,其核苷酸序列如序列表中SEQ ID No:1所示。
2、过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因(LBA1719)的重组工程菌的构建方法,具体步骤如下:
(1)重组表达载体pMG-LBA1719的构建
以嗜酸乳杆菌(Lactobacillus acidophilus )ATCC 4356基因组DNA为模板,设计PCR引物,扩增UTP-葡萄糖-1-磷酸-尿苷转移酶基因LBA1719,其核苷酸序列如序列表中SEQ IDNo:1所示;将表达载体pMG36e用限制性内切酶HindⅢ单酶切,并去磷酸化,然后进行琼脂糖凝胶电泳,切胶回收后,将PCR扩增产物插入到表达载体pMG36e组成型启动子P32下游多克隆位点,在50℃反应15min,构建重组表达载体pMG-LBA1719;
(2)重组工程菌的构建
将pMG-LBA1719过表达质粒经质粒小量提取试剂盒提取浓缩后电转化导入至肉葡萄球菌或乳酸乳球菌中,37℃复苏2h后,涂布红霉素抗性平板,再在37℃培养36h,筛选转化子;转化子经PCR进行验证,从而获得过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因的重组工程菌。
PCR引物的序列如下所示:LBA1719上游扩增引物:TCGACCTGCAGGCATGCAATGCACCATCATCACCATCATATGAAAGTAAGAAAAGCCATC;LBA1719下游扩增引物:GTTTTCAGACTTTGCAAGCTTTATTCCTTTTCAAGCTTCTT。
PCR扩增程序如下:(1)94℃ 4min;(2)98℃ 10sec, 60℃ 5sec,72℃ 1min;重复30个循环;(3)72℃ 5min;PCR反应体系如下表所示:5×PrimeSTAR Buffer 10 μL,dNTPMixture 4 μL,20mM 正向引物1 μL,20mM 反向引物1 μL,模板DNA 2 μL,PrimeSTAR HSDNA 聚合酶 0.5 μL,用蒸馏水补足至 50μL。
步骤(1)中所述的PCR扩增产物和所述的表达载体pMG36e摩尔比为2∶1。
所述的肉葡萄球菌电转化具体步骤如下:
(1)肉葡萄球菌感受态的制备
将一环肉葡萄球菌接种至20mL LB培养基中,37℃过夜培养得到种子液;将5mL种子液接入100mL含0.5wt%葡萄糖和0.5wt%甘氨酸的LB培养基中,培养至OD=0.8;于3000rpm,离心10min,收集菌体,无菌水洗3次之后,用20mL预冷的A液洗涤细胞2次,于3000rpm,离心10min,收集菌体,即得到感受态嗜酸乳杆菌细胞;然后用预冷的0.5mL A液重悬,80μL分装备用,再直接进行下一步实验,或-80℃保存感受态细胞;其中所述的A液为含有10wt%甘油和10wt%蔗糖的超纯水溶液;
(2)肉葡萄球菌的电转
将感受态肉葡萄球菌细胞和1~5μg过表达质粒pMG-LBA1719转移到冰冷的0.1cm间隙的电极杯中,以1.5kv,25μF,200 Ω,5.1ms脉冲转化;然后迅速往电极杯中加入1mL预冷MRS培养基,于37℃复苏2h,即感受态肉葡萄球菌导入过表达质粒pMG-LBA1719。
所述的乳酸乳球菌电转化具体步骤如下:
(1)乳酸乳球菌感受态的制备
在含0.5wt%葡萄糖的M17固体培养基上将乳酸乳球菌NZ9000 菌株置于30℃恒温培养箱中,培养36 h,挑单菌落至50 mL含0.5wt%葡萄糖的M17液体培养基中,30℃过夜活化,得到种子液;再将种子液以体积比2%的接种量将种子液接种于100 mL M17液体培养基中扩大培养,培养至OD600=0.2-0.3,于4℃,5000 rpm/10 min离心,收集菌体,用预冷A液洗沉淀3次后,再用1 mL 预冷A液重悬菌体,置于冰上马上用于乳酸乳球菌电转,其中所述的A液为含有10wt%甘油和10wt%蔗糖的超纯水溶液;
(2)乳酸乳球菌的电转
将乳酸乳球菌NZ9000感受态细胞和1~5μg过表达质粒pMG-LBA1719混匀,转移至预冷的电极杯中(0.1 cm间隙);电转条件设置为:1.2 kv,200Ω,25 μF,5.1 ms;然后迅速往电极杯中加入900 μL含有0.5wt%葡萄糖和2wt%甘氨酸的预冷M17液体培养基,于30℃培养箱中静置复苏2 h;将复苏后的菌液均匀涂布在含有浓度为1 μg/mL红霉素的M17琼脂平板上,于30℃恒温培养箱培养,直至M17琼脂平板上长出乳白色菌落,即感受态乳酸乳球菌成功导入过表达质粒pMG-LBA1719。
上述过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因的重组工程菌在制备冻干保护剂方面的应用。
与现有技术相比,本发明的优点在于:本发明首次公开了一株过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因及其重组肉葡萄球菌S. aureus-2的构建方法,通过克隆嗜酸乳杆菌ATCC4356(Lactobacillus acidophilus ATCC4356)的UTP-葡萄糖-1-磷酸-尿苷转移酶基因(LBA1719)片段连接到pMG36e表达载体上,利用电转化的方法导入到肉葡萄球菌或者乳酸乳球菌中,通过红霉素抗性筛选并鉴定获得带有目的基因的重组工程菌,即过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因(LBA1719)的重组工程菌。该重组肉葡萄球菌S. aureus-2比S. aureus-0株的酶活提高了35.49%;该重组乳酸乳球菌比空白对照组菌株的酶活提高了37.84%,并对其做冷冻干燥处理,其冻干存活率比空载乳酸乳球菌菌株提高了13.53%。其首次构建了一株能显著提高菌体生长、酶活性、冻干存活率的过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因的重组肉葡萄球菌和重组乳酸乳球菌,为制备高活性发酵剂和微生态制剂奠定研究基础和技术支持。
附图说明
图1为过表达UTP-葡萄糖-1-磷酸-尿苷转移酶(LBA1719)基因PCR产物进行琼脂糖凝胶电泳检测结果;Lane1、2、3、4、5、6均为目的基因LBA1719 PCR扩增后产物;
图2为表达载体pMG36e经HindⅢ单酶切后琼脂糖凝胶电泳检测结果;Lane1、2为pMG36e经HindⅢ单酶切并去磷酸化的验证结果图;
图3为pMG-LBA1719重组质粒图谱;
图4为重组肉葡萄球菌S. aureus-2与空载肉葡萄球菌工程菌S. aureus-0的生长曲线结果图;
图5为重组肉葡萄球菌S. aureus-2与空载肉葡萄球菌S. aureus-0的酶活测定结果图;
图6为重组乳酸乳球菌L.lactis-2与空载乳酸乳球菌工程菌L.lactis-0的生长曲线结果图;
图7为重组乳酸乳球菌L.lactis-2与空载乳酸乳球菌工程菌L.lactis-0的酶活测定结果图;
图8为用无菌水做冻干保护剂获得的重组乳酸乳球菌L.lactis-2与空载乳酸乳球菌工程菌L.lactis-0的的冻干存活率测定结果图。
具体实施方式
以下结合附图实施例对本发明作进一步详细描述。
具体实施例一
一株过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因(LBA1719),该基因来自于嗜酸乳杆菌(Lactobacillus acidophilus )ATCC 4356编码UTP-葡萄糖-1-磷酸-尿苷转移酶的基因,其核苷酸序列如序列表中SEQ ID No:1所示。
具体实施例二
重组表达载体pMG-LBA1719的构建
1、以嗜酸乳杆菌(Lactobacillus acidophilus )ATCC 4356基因组DNA为模板,设计PCR引物扩增UTP-葡萄糖-1-磷酸-尿苷转移酶(LBA1719)基因片段,其核苷酸序列如序列表中SEQ ID No:1所示;PCR引物的序列如下所示:LBA1719上游扩增引物:TCGACCTGCAGGCATGCAATGCACCATCATCACCATCATATGAAAGTAAGAAAAGCCATC;LBA1719下游扩增引物:GTTTTCAGACTTTGCAAGCTTTATTCCTTTTCAAGCTTCTT。
2、以嗜酸乳杆菌(Lactobacillus acidophilus)ATCC 4356(该嗜酸乳杆菌已保藏于中国普通微生物菌种保藏管理中心,保藏中心登记入册编号为1.1878)基因组DNA为模板,进行如下PCR扩增反应:(1)94℃ 4min;(2)98℃ 10sec, 60℃ 5sec,72℃ 1min;重复30个循环;(3)72℃ 5min。 PCR反应体系如下所示:5×PrimeSTAR Buffer 10μL,dNTPMixture 4μL,20mM 上游扩增引物 1μL,20mM 下游扩增引物 1μL,DNA模板2μL ,PrimeSTARHS DNA 聚合酶 0.5μL ,ddH2O 31.5μL,总体积为50μL。图1为目的基因PCR产物进行琼脂糖凝胶电泳检测结果,其中Lane1、2、3、4、5、6为基因LBA1719的 PCR扩增后产物,由图1的电泳条带位置可以看出产物分子量与该基因长度基本一致。
3、将表达载体pMG36e用限制性内切酶HindⅢ单酶切,并去磷酸化,然后进行琼脂糖凝胶电泳,切胶回收后,将PCR扩增产物插入到表达载体pMG36e组成型启动子P32下游多克隆位点,PCR产物和表达载体以2∶1的摩尔比用无缝克隆试剂盒在50℃反应15min,构建重组表达载体pMG-LBA1719。
图2为表达载体pMG36e经HindⅢ单酶切后琼脂糖凝胶电泳检测结果,由图2可知载体酶切完全,并且条带位置正确。
图3为pMG-LBA1719重组质粒图谱,是将PCR产物插入表达载体pMG36e中,成功构建了重组表达质粒pMG-LBA1719。
具体实施例三
1、肉葡萄球菌基因工程菌S. aureus-2、S. aureus-0的构建
将具体实施例二制备得到的pMG-LBA1719过表达质粒经质粒小量提取试剂盒(EM101-01,北京全式金生物技术有限公司)提取浓缩后电击转化导入至蓉欧葡萄球菌,37℃复苏2h后,涂布红霉素抗性平板,再在37℃培养36h,筛选转化子,转化子经PCR验证,从而获得过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因LBA1719的重组肉葡萄球菌S. aureus-2。同时将未处理的表达载体pMG36e经质粒小量提取试剂盒提取及浓缩后,电击转化导入至肉葡萄球菌,37℃复苏2h后,涂布红霉素抗性平板,再在37℃培养36h,筛选转化子,转化子经PCR验证,从而获得空载肉葡萄球菌工程菌S. aureus-0。
上述质粒提取浓缩详细步骤如下:向提取的pMG36e质粒中(质粒浓度为200μg/μL)中加入DNA溶液1/10倍体积乙酸钠(3mol/L,pH=5.2)充分混匀,使其终浓度为0.3 mol/L;加入DNA溶液2倍体积用冰预冷的无水乙醇,充分混匀置于-20℃条件下30min;离心(12,000×g,10min),小心移除上清液,吸走管壁上的液滴;加入1/2离心管容量的70wt%乙醇溶液,离心(12,000×g,2min);将EP管开盖,于室温下使残留液体挥发至干;加入适量50μL ddH2O溶解DNA;上述电转化具体步骤如下:
(1)肉葡萄球菌感受态的制备:
将一环肉葡萄球菌接种至20mL LB培养基中,37℃过夜培养得到种子液;将5mL种子液接入100mL含0.5wt%葡萄糖和0.5wt%甘氨酸的LB培养基中,培养至OD≈0.8;于3000rpm,离心10min,收集菌体,无菌水洗3次之后,用20mL预冷的A液(含有10wt%甘油和10wt%蔗糖的超纯水溶液)洗涤细胞2次,于3000rpm,离心10min,收集菌体,即得到感受态肉葡萄球菌细胞;然后用预冷的0.5mL A液重悬,80μL分装备用,再直接进行下一步实验,或-80℃保存感受态细胞;
(2)肉葡萄球菌的电转
将步骤(1)制备得到的感受态肉葡萄球菌细胞和1~5μg过表达质粒pMG-LBA1719转移到冰冷的0.1cm间隙的电极杯中,以1.5kv,25μF,200 Ω,5.1ms脉冲转化;然后迅速往电极杯中加入1mL预冷MRS培养基,于37℃复苏2h;将菌液涂布在抗性平板上,于37℃培养直到平板上出现菌落。
2、结果分析
(1)菌体生长
如图4所示,在肉葡萄球菌中过表达UTP-葡萄糖-1-磷酸-尿苷转移酶时,培养到 8h时,菌体进入对数期,18h处于对数后期,稳定前期,过表达UTP-葡萄糖-1-磷酸-尿苷转移酶的肉葡萄球菌高于对照组,显著提高重组肉葡萄球菌的生长速度,S. aureus-2比S. aureus-0提高了35.49%;
(2)肉葡萄球菌工程菌的酶活测定
将上述制备的重组肉葡萄球菌S. aureus-2接种于LB肉汤培养基中,于37℃静止培养活化18h,制备种子培养液,将种子培养液以2%(v/v)的接种量接种于LB肉汤培养基中扩大培养18h,于5000rpm离心15min后弃上清,菌体用生理盐水洗3次后,超声破碎提取蛋白,于10,000×g离心20min后,取其上清用UTP-葡萄糖-1-磷酸-尿苷转移酶试剂盒检测酶活(如图5所示),同理测定空载肉葡萄球菌工程菌S. aureus-0的酶活(如图5所示)。由图5可知,LBA1719基因过表达重组基因工程菌的酶活为445.72 IU/L,比S. aureus-0菌株酶活328.97IU/L提高了35.49%。
具体实施例四
1、乳酸乳球菌基因工程菌L. lactis-2、L. lactis-0的构建
将具体实施例二制备得到的pMG-LBA1719过表达质粒经质粒小量提取试剂盒(EM101-01,北京全式金生物技术有限公司)提取浓缩后电击转化导入至乳酸乳球菌NZ9000,30℃复苏2h后,涂布红霉素抗性平板,再在30℃培养36h,筛选转化子,转化子经PCR验证,从而获得过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因LBA1719的重组乳酸乳球菌L. lactis-2。同时将未处理的表达载体pMG36e经质粒小量提取试剂盒提取及浓缩后,电击转化导入至乳酸乳球菌NZ9000,30℃复苏2h后,涂布红霉素抗性平板,再在30℃培养36h,筛选转化子,转化子经PCR验证,从而获得空载乳酸乳球菌L. lactis-0。
上述质粒提取浓缩详细步骤如下:分别从-80℃冰箱取出工程菌重组乳酸乳球菌,分别接种到M17液体培养基(1 μg/mL红霉素),于30℃,活化8 h,得种子液,然后再以2%(v/v)的接种量将种子液接到已灭菌的100mL M17液体培养基(1 μg/mL红霉素)中,培养12 h,得到的菌液分别进行质粒提取,步骤如下:
(1)首先向试剂盒所带的吸附柱中加200 µL buffer CBS, 12,000rpm / 1min,弃去废液;
(2)将上述培养的液体分别取3 mL,离心取沉淀,每个菌株做三个平行,并做好标记;
(3)向收集沉淀中加入250 μL SolutionⅠ,并用枪头轻轻吹打直至无菌块;
(4)加250 μL SolutionⅡ,马上温和上下翻转6次,是溶液变成透亮的蛋清状溶液;
(5)加350 μL Solution Ⅲ,翻转8 次,静置4 min,离心,取上清于(1)处理后的吸附柱中,6000 rpm /1 min,弃废液;
(6)分两次加W1 Solution 、Wash Solution 500 μL,每次均要离心,弃废液;
(7)去除残液,离心;
(8)加ddH2O 80 μL,静置离心,得到重组表达质粒;
(9)取所提质粒进行凝胶电泳和送往华大基因公司测序,剩余质粒用于电转乳酸乳球菌或放于-20℃冰箱备用。注:本实验所提到的离心条件为:4℃,12,000 rpm离心1 min。
上述电转化具体步骤如下:
(1)乳酸乳球菌感受态的制备:
在含0.5wt%葡萄糖的M17固体培养基上将乳酸乳球菌NZ9000 菌株置于30℃恒温培养箱中,培养36 h,挑单菌落至50 mL含0.5wt%葡萄糖的M17液体培养基中,30℃过夜活化,得到种子液;再将种子液以体积比2%的接种量将种子液接种于100 mL M17液体培养基中扩大培养,培养至OD600=0.2-0.3,于4℃,5000 rpm/10 min离心,收集菌体,用预冷A液洗沉淀3次后,再用1 mL 预冷A液重悬菌体,置于冰上马上用于乳酸乳球菌电转,其中所述的A液为含有10wt%甘油和10wt%蔗糖的超纯水溶液;
(2)乳酸乳球菌的电转
将乳酸乳球菌NZ9000感受态细胞和1~5μg过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因的表达质粒pMG-LBA1719混匀,转移至预冷的电极杯中(0.1 cm间隙);电转条件设置为:1.2 kv,200Ω,25 μF,5.1 ms;然后迅速往电极杯中加入900 μL含有0.5wt%葡萄糖+2wt%甘氨酸的预冷M17液体培养基,于30℃培养箱中静置复苏2 h;将复苏后的菌液均匀涂布在含浓度为1 μg/mL红霉素的M17琼脂平板上,于30℃恒温培养箱培养,直至M17琼脂平板上长出乳白色菌落,即感受态乳酸乳球菌成功导入过表达质粒pMG-LBA1719。
2、结果分析
(1)乳酸乳球菌工程菌的生长曲线测定
将上述制备的重组乳酸乳球菌L. lactis -2接种于M17肉汤培养基中,于30℃静止培养活化12h,制备种子培养液,再按体积比为1%(v/v)的接种量将种子液接种到M17液体培养基(0.5%葡萄糖)中,培养36h。每间隔2 h取一次样,测定各样品在600 nm处的吸光值,并绘制重组乳酸乳球菌的生长曲线。注:为减少误差,每次测定取样前必需将菌液混匀,各做三个平行,结果说明在乳酸乳球菌中过表达UGPase促进菌体生长,如图6所示;
(2)乳酸乳球菌工程菌的酶活测定
将上述制备的重组乳酸乳球菌L. lactis -2接种于M17肉汤培养基中,于30℃静止培养活化12h,制备种子培养液,将种子培养液以2%(v/v)的接种量接种于M17肉汤培养基中扩大培养12h,于5000rpm离心15min后弃上清,菌体用生理盐水洗3次后,超声破碎提取蛋白,于10,000×g离心20min后,取其上清用试剂盒检测UTP-葡萄糖-1-磷酸-尿苷转移酶的酶活,同理测定空载乳酸乳球菌工程菌L. lactis-0的酶活(如图7所示)。由图7可知,LBA1719基因过表达重组基因工程菌的酶活为30.14U/L,相比于L.lactis-0酶活性(25.51U/L)提高了18.14%;
(3)乳酸乳球菌工程菌冻干存活率的测定方法
用无菌水做冻干保护剂,比较重组乳酸乳球菌L. lactis -2与重组空载菌L. lactis-0的冻干存活率:将乳酸乳球菌NZ9000工程菌 L.lactis-0,L.lactis-2分别法活化、扩大,培养12 h,分别得到2种重组乳酸乳球菌的发酵液;将每瓶工程菌发酵液调成相同的OD600值后,分别各取20 mL,做好标记,4℃,6000×g离心10 min收集菌体沉淀;无菌生理盐水洗涤沉淀3次,再用20mL灭菌生理盐水悬浮菌体,吹打均匀后各取100 μL进行稀释涂平板,剩下的菌液迅速放置-80℃冰箱进行预冻;取预冻好的菌液进行冷冻干燥,冷冻干燥条件为:-49℃,9 Pa,24 h。冷冻干燥完成后取样品,各管分别加19.9 mL生理盐水,重悬混匀菌体,然后各取100 μL进行稀释后涂板计数。剩下的放入-80℃冰箱过夜预冻;将预冻好的样品冷冻干燥(-49℃,9Pa)24h,取出样品,每管加900μL无菌水重悬菌体,取100μL进行计数。冻干存活率=冻干后活菌数/冻干前活菌数×100。结果如图8所示,用无菌水做冻干保护剂时,L.lactis-2冻干存活率为70.67%,比L.lactis-0(冻干存活率为57.14%)提高了13.53%。
上述说明并非对本发明的限制,本发明也并不限于上述举例。本技术领域的普通技术人员在本发明的实质范围内,做出的变化、改型、添加或替换,也应属于本发明的保护范围。
序 列 表
<110> 宁波大学
<120> 过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因及其重组工程菌的构建方法和应用
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 898
<212> DNA
<213> UTP-葡萄糖-1-磷酸-尿苷转移酶基因LBA1719
<400> 1
TTATTCCTTTTCAAGCTTCTTGCCTAAGTCGATAATATATTTCTTCAAAGCATCGCGAGTTTCAGGGTGTCTTAAACCATATTGAATTGAGGTTTCAAGGTAGCCTTCCTTGTTACCTACATCAAAACGTTGACCTCTAAAGACATGTGCAAATACTCGTTGAGTCTGGTTCAAAGTATCGATGGCATCAGTTAATTGAATTTCACCACCGCGACCTGGCTTTTGATTCTCAAGAATTTCAAAAATTTCCGGTGTGAGTAAGTAACGTCCAATAATTGCAAAATCACTTGGTGCCTTGTCAACGGCAGGCTTTTCAACAAATTTCTTTACATTGTAAAGATCTTTATCCACCTTGCCTTCAGGATCAATCACACCGTACTTGGAAACTTCCTTATGCGGAACCTGCATAACAGCAATTGTTGATGCATGAATCTTATCGTAATCATTCATCAATTGCTTAGTTAATGTAATTTTGTCCTTCATTAAGTCATCACCAAGCATAACAACGAATGGTTCTTCTCCCACAAAGCTACGTGCACGATAAATTGCATCACCTAAGCCAGCTGGATGTGGTTGTCGAGTGTAGTATAAATTAATTCCCAAGTTAGTAATATCTTGAGTTAATTTCAATAACTTAACCTTACCAGTGGCTTCCAAGTCTTGTTCAAGTTCTGGGTTAGAATCAAAGTGATTTTCAATTGCTCGCTTATTCTTACCAGTAACAATTAAAATATCTTCAATTCCTGCAGCTTTAGCTTCTTCAACAATAAATTGAATTGTTGGCTTATCGACAATTGGTAACATTTCCTTAGGCATAGCTTTAGTAGCTGGTAGAAAACGGGTCCCCAAACCTGCAGCTGGAATGATGGCTTTTCTTACTTTCATAGTTAGACCTCCA 898
<210> 2
<211> 60
<212> DNA
<213> LBA1719上游扩增引物
<400> 2
TCGACCTGCAGGCATGCAATGCACCATCATCACCATCATATGAAAGTAAGAAAAGCCATC 60
<210> 3
<211> 41
<212> DNA
<213> LBA1719下游扩增引物
<400> 3
GTTTTCAGACTTTGCAAGCTTTATTCCTTTTCAAGCTTCTT 41
Claims (8)
1.一种过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因,其特征在于:所述基因来自于嗜酸乳杆菌( Lactobacillus acidophilus)ATCC 4356编码UTP-葡萄糖-1-磷酸-尿苷转移酶的基因,其核苷酸序列如序列表中SEQ ID No:1所示。
2.一种过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因的重组工程菌的构建方法,其特征在于具体步骤如下:
(1)重组表达载体pMG-LBA1719的构建
以嗜酸乳杆菌(Lactobacillus acidophilus )ATCC 4356基因组DNA为模板,设计PCR引物,扩增UTP-葡萄糖-1-磷酸-尿苷转移酶基因LBA1719,其核苷酸序列如序列表中SEQ IDNo:1所示;将表达载体pMG36e用限制性内切酶HindⅢ单酶切,并去磷酸化,然后进行琼脂糖凝胶电泳,切胶回收后,将PCR扩增产物插入到表达载体pMG36e组成型启动子P32下游多克隆位点,在50℃反应15min,构建重组表达载体pMG-LBA1719;
(2)重组工程菌的构建
将pMG-LBA1719过表达质粒经质粒小量提取试剂盒提取浓缩后电转化导入至肉葡萄球菌或乳酸乳球菌中,37℃复苏2h后,涂布红霉素抗性平板,再在37℃培养36h,筛选转化子;转化子经PCR进行验证,从而获得过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因的工程菌。
3.根据权利要求2所述的过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因的重组工程菌的构建方法,其特征在于PCR引物的序列如下所示:LBA1719上游扩增引物:TCGACCTGCAGGCATGCAATGCACCATCATCACCATCATATGAAAGTAAGAAAAGCCATC;LBA1719下游扩增引物:GTTTTCAGACTTTGCAAGCTTTATTCCTTTTCAAGCTTCTT。
4.根据权利要求3所述的过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因的重组工程菌的构建方法,其特征在于PCR扩增程序如下:(1)94℃ 4min;(2)98℃ 10sec, 60℃ 5sec,72℃1min;重复30个循环;(3)72℃ 5min;PCR反应体系如下表所示:5×PrimeSTAR Buffer 10 μL,dNTP Mixture 4 μL,20mM 正向引物1 μL,20mM 反向引物1 μL,模板DNA 2 μL,PrimeSTAR HS DNA 聚合酶 0.5 μL,用蒸馏水补足至 50μL。
5.根据权利要求4所述的过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因的重组工程菌的构建方法,其特征在于:步骤(1)中所述的PCR扩增产物和所述的表达载体pMG36e摩尔比为2∶1。
6.根据权利要求5所述的过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因的重组工程菌的构建方法,其特征在于所述的肉葡萄球菌电转化具体步骤如下:
(1)肉葡萄球菌感受态的制备
将一环肉葡萄球菌接种至20mL LB培养基中,37℃过夜培养得到种子液;将5mL种子液接入100mL含0.5wt%葡萄糖和0.5wt%甘氨酸的LB培养基中,培养至OD=0.8;于3000rpm,离心10min,收集菌体,无菌水洗3次之后,用20mL预冷的A液洗涤细胞2次,于3000rpm,离心10min,收集菌体,即得到感受态嗜酸乳杆菌细胞;然后用预冷的0.5mL A液重悬,80μL分装备用,再直接进行下一步实验,或-80℃保存感受态细胞,其中所述的A液为含有10wt%甘油和10wt%蔗糖的超纯水溶液;
(2)肉葡萄球菌的电转
将感受态肉葡萄球菌细胞和1~5μg过表达质粒pMG-LBA1719转移到冰冷的0.1cm间隙的电极杯中,以1.5kv,25μF,200 Ω,5.1ms脉冲转化;然后迅速往电极杯中加入1mL预冷MRS培养基,于37℃复苏2h,即感受态肉葡萄球菌导入过表达质粒pMG-LBA1719。
7.根据权利要求5所述的过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因的重组工程菌的构建方法,其特征在于所述的乳酸乳球菌电转化具体步骤如下:
(1)乳酸乳球菌感受态的制备
在含0.5wt%葡萄糖的M17固体培养基上将乳酸乳球菌NZ9000 菌株置于30℃恒温培养箱中,培养36 h,挑单菌落至50 mL含0.5wt%葡萄糖的M17液体培养基中,30℃过夜活化,得到种子液;再将种子液以体积比2%的接种量将种子液接种于100 mL M17液体培养基中扩大培养,培养至OD600=0.2-0.3,于4℃,5000 rpm/10 min离心,收集菌体,用预冷A液洗沉淀3次后,再用1 mL 预冷A液重悬菌体,置于冰上马上用于乳酸乳球菌电转,其中所述的A液为含有10wt%甘油和10wt%蔗糖的超纯水溶液;
(2)乳酸乳球菌的电转
将乳酸乳球菌NZ9000感受态细胞和1~5μg过表达质粒pMG-LBA171混匀,转移至预冷的电极杯中;电转条件设置为:1.2 kv,200Ω,25 μF,5.1 ms;然后迅速往电极杯中加入900 μL含有0.5wt%葡萄糖和2wt%甘氨酸的预冷M17液体培养基,于30℃培养箱中静置复苏2 h;将复苏后的菌液均匀涂布在含有浓度为1 μg/mL红霉素的M17琼脂平板上,于30℃恒温培养箱培养,直至M17琼脂平板上长出乳白色菌落,即感受态乳酸乳球菌成功导入过表达质粒pMG-LBA1719。
8.权利要求1-7中任一项所述的过表达UTP-葡萄糖-1-磷酸-尿苷转移酶基因的重组工程菌在制备冻干保护剂方面的应用。
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