CN107916244A - 一种表达溶葡球菌素基因的重组乳酸乳球菌的构建方法及应用 - Google Patents
一种表达溶葡球菌素基因的重组乳酸乳球菌的构建方法及应用 Download PDFInfo
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Abstract
本发明公开了一种表达溶葡球菌素基因的重组乳酸乳球菌的构建方法及应用,该重组重组乳酸乳球菌的构建方法是:(1)按照宿主菌乳酸乳球菌的密码子偏好性进行优化而合成溶葡球菌素基因与乳酸乳球菌质粒;(2)将合成的溶葡球菌素基因与乳酸乳球菌质粒同时双酶切、回收、连接,转化至大肠杆菌CM1061感受态细胞中;(3)使用氯霉素抗性进行筛选后,经酶切鉴定、PCR鉴定得出正确的重组质粒后,提取重组质粒并电转至乳酸乳球菌中,得到重组乳酸乳球菌;(4)Western Blot鉴定重组乳酸乳球菌的表达结果。本发明既可发挥乳酸乳球菌的益生菌功效,又可发挥猪溶葡球菌素杀灭肠道内葡萄球菌,预防和治疗葡萄球菌病的功效。
Description
技术领域
本发明涉及一种表达溶葡球菌素基因的重组乳酸乳球菌的构建方法及应用。
背景技术
溶葡球菌素是一种对葡萄球菌溶菌作用的抗菌酶,具有水解葡萄球菌细胞壁肽聚糖 gly 五肽桥联的催化活性,近年来葡萄球菌尤其是金黄色葡萄球菌的耐药状况日益严重,溶葡球菌素的药用潜力更加受到人们重视,同时葡萄球菌病给畜禽养殖业的发展造成巨大危害,因此应用溶葡球菌素预防和治疗畜禽葡萄球菌病具有广阔的应用前景。
乳酸乳球菌做为一种益生菌,对机体的健康十分重要,它能产生多种代谢酶类和维生素,促进畜禽消化,抑制有害菌繁殖并改变肠道内环境,进而促进肠道健康,提高畜禽生产性能。
发明内容
本发明其目的就在于提供一种表达溶葡球菌素基因的重组乳酸乳球菌的构建方法及应用,本发明既可发挥乳酸乳球菌的益生菌功效,又可发挥猪溶葡球菌素杀灭肠道内葡萄球菌,预防和治疗葡萄球菌病的功效。
为实现上述目的而采取的技术方案是,
一种表达溶葡球菌素基因的重组乳酸乳球菌,重组乳酸乳球菌为Lysostaphin-pNZ8148-NZ9000。
一种表达溶葡球菌素基因的重组乳酸乳球菌的构建方法,包括以下步骤:
(1)按照宿主菌乳酸乳球菌的密码子偏好性进行优化而合成溶葡球菌素(Lysostaphin )基因与乳酸乳球菌( pNZ8148)质粒;
(2)用限制性内切酶分别对溶葡球菌素 (Lysostaphin )基因与乳酸乳球菌(pNZ8148)质粒进行双酶切,回收,再经 T4 DNA 连接酶进行连接后转化至大肠杆菌 MC1061感受态细胞中;
(3)使用氯霉素抗性进行筛选后,提取重组质粒并电转至乳酸乳球菌NZ9000中,PCR 鉴定正确,得到重组乳酸乳酸球菌 Lysostaphin-pNZ8148-NZ9000;
(4)Western Blot鉴定重组乳酸乳球菌的表达结果。
一种表达溶葡球菌素基因的重组乳酸乳球菌的应用,所述的重组乳酸乳球菌Lysostaphin-pNZ8148-NZ9000全培养基可直接饲喂动物,既可发挥乳酸乳球菌的益生菌功效,又可发挥猪溶葡球菌素杀灭肠道内葡萄球菌,预防和治疗葡萄球菌病的功效。
有益效果
与现有技术相比本发明具有以下优点。
本发明公开的优点是,可经诱导产生溶葡球菌素,该重组菌全培养基直接饲喂动物,既可发挥乳酸乳球菌的益生菌功效,又可发挥猪溶葡球菌素杀灭肠道内葡萄球菌,预防和治疗葡萄球菌病的功效。
附图说明
以下结合附图对本发明作进一步详述。
图1为筛选溶葡球菌素基因与 pNZ8148 质粒连接的阳性克隆子电泳图(引物Lysostaphin-F 和 Lysostaphin-R,产物约768bp);
图2为乳酸乳球菌 NZ9000 表达 Lysostaphin 蛋白的western blot图(蛋白大小在27KD 左右);
图3为 pNZ8148 质粒图谱。
具体实施方式
一种表达溶葡球菌素基因的重组乳酸乳球菌,重组乳酸乳球菌为Lysostaphin-pNZ8148-NZ9000。
所述的质粒为pNZ8148。
一种表达溶葡球菌素基因的重组乳酸乳球菌的构建方法,如图1-3所示,包括以下步骤:
(1)按照宿主菌乳酸乳球菌的密码子偏好性进行优化而合成溶葡球菌素(Lysostaphin )基因与乳酸乳球菌( pNZ8148)质粒;
(2)用限制性内切酶分别对溶葡球菌素 (Lysostaphin )基因与乳酸乳球菌(pNZ8148)质粒进行双酶切,回收,再经 T4 DNA 连接酶进行连接后转化至大肠杆菌 MC1061感受态细胞中;
(3)使用氯霉素抗性进行筛选后,提取重组质粒并电转至乳酸乳球菌NZ9000中,PCR 鉴定正确,得到重组乳酸乳酸球菌 Lysostaphin-pNZ8148-NZ9000;
(4)Western Blot鉴定重组乳酸乳球菌的表达结果。
一种表达溶葡球菌素基因的重组乳酸乳球菌的应用,所述的重组
乳酸乳球菌Lysostaphin-pNZ8148-NZ9000全培养基可直接饲喂动物,既可发挥乳酸乳球菌的益生菌功效,又可发挥猪溶葡球菌素杀灭肠道内葡萄球菌,预防和治疗葡萄球菌病的功效。
实施例 1
目的基因的合成
如序列中序列 1 所示, 溶葡球菌素成熟肽有 246 个氨基酸, 通过乳酸乳球菌密码子偏好性进行密码子优化, 在序列上游添加 6 个组氨酸的基因序列, 并在其上下游分别设计 NcoI 和 XbaI 两个限制性内切酶位点及保护性碱基,对优化好的序列进行人工合成,如序列表中序列 2 所示。
表 1 序列1
AATHEHSAQWLNNYKKGYGYGPYPLGINGGMHYGVDFFMNIGTPVKAISSGKIVEAGWSNYGGGNQIGLIENDGVHRQWYMHLSKYNVKVGDYVKAGQIIGWSGSTGYSTAPHLHFQRMVNSFSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIK
表 2 序列2
CATGCCATGGCATCATCATCATCATCATGCTGCTACTCATGAACATTCTGCTCAATGGTTAAATAATTATAAAAAAGGATATGGATATGGACCATATCCATTAGGTATTAATGGAGGTATGCATTATGGAGTTGATTTTTTTATGAATATTGGAACCCCAGTTAAAGCAATTTCTTCTGGTAAAATTGTGGAAGCAGGATGGTCAAATTATGGAGGTGGTAATCAAATTGGATTAATTGAAAATGATGGAGTGCATAGACAATGGTATATGCATTTATCAAAATATAATGTGAAAGTGGGGGATTATGTTAAAGCTGGACAAATTATTGGTTGGTCTGGATCTACAGGATATTCTACAGCTCCACATTTACATTTTCAACGTATGGTTAATAGCTTTTCAAATTCTACAGCACAAGATCCAATGCCATTTTTAAAATCAGCTGGTTATGGAAAAGCTGGTGGTACAGTTACTCCAACACCAAATACTGGATGGAAAACAAATAAATATGGGACTTTATATAAAAGCGAAAGCGCATCATTTACACCAAATACAGATATTATTACCCGTACAACTGGACCATTTAGATCAATGCCACAATCAGGAGTTTTAAAAGCTGGACAAACAATTCATTATGATGAAGTTATGAAACAAGATGGTCATGTTTGGGTTGGTTATACTGGTAATTCTGGACAAAGAATTTATCTTCCAGTTCGTACATGGAATAAATCAACTAATACACTTGGTGTTCTTTGGGGTACTATTAAATCTAGAGC
实施例 2
构建重组质粒 Lysostaphin-pNZ8148
使用 NcoI 和 XbaI 两种限制性内切酶分别对质粒 pNZ8148 和实施案例 1 中所合成的基因序列双酶切,琼脂糖凝胶电泳回收酶切产物,4℃连接过夜,将连接产物转化大肠杆菌 MC1061 感受态细胞,提取 Lysostaphin-pNZ8148,用 NcoI和 XbaI双酶切验证,重组质粒 Lysostaphin-pNZ8148 双酶切鉴定图如图 1 所示。以重组质粒为模板,扩增 PCR 鉴定重组质粒,PCR 引物如下所示,
Lysostaphin-F:CCATGGCATCATCATCATCATCATGCTGCTACTC
Lysostaphin-R:TCTAGATTTAATAGTACCCCAAAGAACACCAAGT
PCR 体系如下:
体系 体积(ul)
Lysostaphin-F 1
Lysostaphin-R 1
dNTP 1
10×buffer 2
transT -Taq 酶 0.5.
转化子菌液 2
ddH2O 12.5
总体积 20
扩增产物测序,测序结果与合成基因完全一致,从而获得了重组乳酸乳球菌株Lysostaphin-pNZ8148-MC1061 。
实施例 3
重组质粒 Lysostaphin-pNZ8148 对乳酸乳球菌 NZ9000 的电转化使用质粒小量提取试剂盒提取乳酸乳球菌重组质粒 Lysostaphin-pNZ8148,电转化乳酸乳球菌 NZ9000,涂布于 50ug/mL 氯霉素的 GM17 平板,于 72h 后挑取单菌落于 50ug/mL 氯霉素的 GM17 液体培养基中扩大培养,收集菌体后加入溶菌酶破坏乳酸乳球菌细胞壁,用质粒小量提取试剂盒提取质粒,再按实施案例2中的双
酶切反应和PCR反应鉴定,从而获得重组乳酸乳球菌Lysostaphin-pNZ8148-NZ9000。
实施例 4
重组乳酸乳酸球 Lysostaphin-pNZ8148-NZ9000 的表达
平板上挑取重组乳酸乳球菌 Lysostaphin-pNZ8148-NZ9000 单菌落接种于含氯霉素25ug/mL 的 5mL GM17 液体培养基中,30℃静置培养过夜;将过夜培养物分别吸取 2mL 各加入含氯霉素 25ug/mL 的 50mL GM17 液体培养基中,30℃培养到 OD600=0.4,向其中一瓶加 5uL 10ug/mL 的诱导剂 nisin (Sigma 公司,2.5%),继续静置诱导培养 24h,重组蛋白分泌到培养基上清中,培养完成的菌液最大转速离心 30min,弃菌体沉淀收集上清。
实施例 5
重组蛋白 Lysostaphin 的纯化及 Western Blot
培养完成的菌液最大转速离心 30min,弃菌体沉淀,将等体积的饱和硫酸铵溶液(4.1mol/L,25℃)边搅拌边慢慢加入,加完放磁力搅拌器上 4℃搅拌过夜,使蛋白充分沉淀。10000rpm 4℃离心 30min,弃上清保留沉淀,将沉淀溶于少量(10-20mL)PBS-0.2g/L 叠氮钠中,使沉淀溶解,再使用透析袋 4℃透析 48h,每隔 6 小时更换透析缓冲液,以彻底去除硫酸铵。再将透析完成的重组蛋白经过镍柱纯化,得到纯化蛋白。将纯化蛋白中添加SDS-PAGE loading buffer,沸水浴 15min,进行 western blot,步骤如下:
5.1、清洗 Bio-Rad 玻璃板,干燥后安装,按上配方配制分离胶,1mm 胶板中加入2ml分离胶液体,至梳齿下 1.5cm,覆盖少量 ddH2O,待凝固后倒掉覆盖的 ddH2O,用滤纸吸净残留的 ddH2O;
5.2、同样的方法按上配方配制浓缩胶,灌制于分离胶之上,立即插入 1mm 胶梳,室温下凝固;
5.3、待浓缩胶制备完成,拔下胶梳,立即将胶板固定于电泳装置上,电泳槽内加入电泳缓冲液,排尽凝胶底部玻璃板间的气泡;
5.4、每孔加样 20μ L,接通电源,设定电压 80V/cm,待染料进入浓缩胶后,调节电压至120V/cm,至溴酚兰染料距胶底部 2cm 处停止电泳;
5.5、拆下凝胶放于转膜缓冲液中待用,同时将滤纸, NC 膜,纤维垫放入转膜缓冲
液中。
5.6、按正极-纤维垫-滤纸-NC 膜-凝胶-滤纸-纤维垫的顺序安装膜转移装置,赶走气泡,注意两层滤纸的面积要小于凝胶和 NC 膜,100V 电泳 4h;
5.7、转膜完成后卸下转膜装置,用 TBST 洗涤液清洗膜 3 次,每次 5min;
5.8、将膜放在封闭液中,4℃封闭过夜,弃去封闭液,用洗涤液清洗 3 次,每次5min;
5.9、使用 TBST 1:5000 稀释 His-tag 单抗, 室温摇床孵育 2h, 洗涤液洗涤 3 次,每次 5min;孵育羊抗鼠 lgG-HRP 二抗 30min,TBST 洗涤 5 次,每次 5min;使用HRP-DAB底物显色试剂盒进行显色,结果见图2。
实施例 6
表达溶葡球菌素基因的重组乳酸乳酸球对小鼠肠道金黄色葡萄球菌数量的影响取 30只 4 周龄雌性 BALB/c 小鼠,分为试验组 1,试验组 2 和对照组,3 组小鼠分别灌服如下三种培养基,0.2 mL/日,连续灌服 2 周。
试验组 1: GM17 液体培养基培养表达溶葡球菌素基因的重组乳酸乳酸球经诱导剂nisin 诱导的全培养液(含重组乳酸乳球菌 1 亿/ mL,溶葡球菌素 0.1mg/ml)
试验组 2:GM17 液体培养基培养表达耐热α -淀粉酶基因的重组乳酸乳酸球菌的全培养液(含重组乳酸乳球菌 1 亿/ mL,不经诱导,不含溶葡球菌素);
对照组:GM17 液体培养基
小鼠自由采食、饮水,每日定时更换饲料、饮水。两周后处死小鼠,无菌条件下剪开腹腔取盲肠内容物 100mg,倍比稀释后分别涂布金黄色葡萄球菌选择性培养基(paird-parker)平板中,计算每克盲肠内容物中的细菌数量(CFU/g), 见下表。
试验组 1 试验组 2 对照组
1.7×105a 4.5×106b 5.9×106b
注:同行肩标字母不同者表示差异显著(P<0.05)。
结果显示,经诱导表达溶葡球菌素的重组乳酸乳球菌,与空白培养基对照组和不表达溶葡球菌素的重组乳酸乳球菌相比, 可以显著降低小鼠盲肠内容物中金黄色葡萄球菌的数量。
Claims (4)
1.一种表达溶葡球菌素基因的重组乳酸乳球菌,其特征在于,重组乳酸乳球菌为Lysostaphin-pNZ8148-NZ9000。
2.根据权利要求1所述的一种表达溶葡球菌素基因的重组乳酸乳球菌,其特征在于,包含了权利要求1所述质粒的乳酸乳球菌,所述的质粒为pNZ8148。
3.根据权利要求1所述的一种表达溶葡球菌素基因的重组乳酸乳球菌的构建方法,其特征在于,包括以下步骤:
(1)按照宿主菌乳酸乳球菌的密码子偏好性进行优化而合成溶葡球菌素(Lysostaphin )基因与乳酸乳球菌( pNZ8148)质粒;
(2)用限制性内切酶分别对溶葡球菌素 (Lysostaphin )基因与乳酸乳球菌(pNZ8148)质粒进行双酶切,回收,再经 T4 DNA 连接酶进行连接后转化至大肠杆菌 MC1061感受态细胞中;
(3)使用氯霉素抗性进行筛选后,提取重组质粒并电转至乳酸乳球菌NZ9000中,PCR 鉴定正确,得到重组乳酸乳酸球菌 Lysostaphin-pNZ8148-NZ9000;
(4)Western Blot鉴定重组乳酸乳球菌的表达结果。
4.根据权利要求1所述的一种表达溶葡球菌素基因的重组乳酸乳球菌的应用,其特征在于,所述的重组乳酸乳球菌Lysostaphin-pNZ8148-NZ9000全培养基可直接饲喂动物,既可发挥乳酸乳球菌的益生菌功效,又可发挥猪溶葡球菌素杀灭肠道内葡萄球菌,预防和治疗葡萄球菌病的功效。
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