CN107142235A - 一种表达猪生长激素基因的重组短短芽孢杆菌及构建方法与应用 - Google Patents
一种表达猪生长激素基因的重组短短芽孢杆菌及构建方法与应用 Download PDFInfo
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- CN107142235A CN107142235A CN201710467609.3A CN201710467609A CN107142235A CN 107142235 A CN107142235 A CN 107142235A CN 201710467609 A CN201710467609 A CN 201710467609A CN 107142235 A CN107142235 A CN 107142235A
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- brevibacillus brevis
- pig growth
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Abstract
本发明公开了一种表达猪生长激素基因的重组短短芽孢杆菌的构建方法是:(1)对NCBI上公布的猪生长激素序列根据短短芽孢杆菌密码子偏好性进行优化得到pGH基因和pNCM02质粒;(2)用限制性内切酶XbaI和ClaI对上述pGH基因和pNCM02质粒进行双酶切;(3)回收双酶切产物进行连接反应,连接产物转化大肠杆菌JM109感受态细胞,使用氨苄西林抗性筛选转化子,获得重组质粒pGH‑pNCM02;(4)提取重组质粒并电转化短短芽孢杆菌SP3,使用新霉素抗性筛选转化子,获得重组短短芽孢杆菌,表达猪生长激素。本发明有效解决了生长激素半衰期短的问题,达到了长效的效果。
Description
技术领域
本发明涉及一种表达猪生长激素基因的重组短短芽孢杆菌及构建方法与应用。
背景技术
短短芽孢杆菌是一种安全、无毒性的益生菌,同时具有蛋白分泌能力强和胞外蛋白酶活性低的特点,因此特别适合做为胞外分泌蛋白表达系统。
猪生长激素是由猪脑垂体前叶嗜酸性细胞分泌的蛋白质类单链激素,由191个氨基酸组成,分子量22kDa,等电点6,在猪基因组中该基因为单拷贝基因,无其他相似序列。猪生长激素可促进机体蛋白质合成及组织对循环系统中氨基酸的利用,增加肌肉组织蛋白含量,降低脂肪含量,促进骨骼发育,提高猪生长速度,同时生长激素对广泛的生理功能都有调节作用,可影响几乎所有的组织和细胞,是畜牧业生产中非常重要的激素之一。因此使用基因工程技术表达猪生长激素用于猪养殖业,可产生重大的经济效益。同时猪生长基因对仔猪、小猪、生长猪、母猪都有作用,长期注射猪生长激素可显著提高各阶段猪体重,且对哺乳母猪泌乳量的提高也有作用。猪生长激素是蛋白类激素,是不亲脂性物质,不会储存在脂肪组织内,半衰期短,因此不会存在药物残留,但这也给猪生物激素的使用带来新的问题,由于半衰期短,目前需要对猪生长激素每天注射、口服、皮下包埋,才能产生较好的效果,这显然给养猪生产造成了巨大的困难。
发明内容
本发明其目的就在于提供一种表达猪生长激素基因的重组短短芽孢杆菌及构建方法与应用,本发明有效解决了生长激素半衰期短的问题,达到了长效的效果。
为实现上述目的而采取的技术方案是,
一种表达猪生长激素基因的重组短短芽孢杆菌,所述短短芽孢杆菌为SP3。
一种表达猪生长激素基因的重组短短芽孢杆菌的构建方法,其特征在于,包括以下步骤:
(1)对NCBI上公布的猪生长激素序列根据短短芽孢杆菌密码子偏好性进行优化得到pGH基因和pNCM02质粒;
(2)用限制性内切酶XbaI和ClaI对上述pGH基因和pNCM02质粒进行双酶切;
(3)回收双酶切产物进行连接反应,连接产物转化大肠杆菌JM109感受态细胞,使用氨苄西林抗性筛选转化子,获得重组质粒pGH-pNCM02;
(4)提取重组质粒并电转化短短芽孢杆菌SP3,使用新霉素抗性筛选转化子,获得重组短短芽孢杆菌,表达猪生长激素。
一种表达猪生长激素基因的重组短短芽孢杆菌的应用,其特征在于,所述使用白油佐剂将短短芽孢杆菌表达并纯化的猪生长激素蛋白乳化后对仔猪皮下注射,有效解决了生长激素半衰期短的问题,达到了长效的效果。
有益效果
与现有技术相比本发明具有以下优点。
本发明使用白油佐剂将短短芽孢杆菌表达并纯化的猪生长激素蛋白乳化后对仔猪皮下注射,结果显示乳化对猪生长激素起到缓释作用,有效解决了生长激素半衰期短的问题,达到了长效的效果。
附图说明
以下结合附图对本发明作进一步详述。
图1为pGH-pNCM02重组质粒的PCR鉴定图 (引物pGH-F和pGH-R,产物为600bp);
图2为短短芽孢杆菌SP3表达pGH蛋白的western blot图(蛋白大小为22kDa);
图3为pNCM02质粒图谱;
图4为pNCM02质粒的多克隆位点图谱。
具体实施方式
一种表达猪生长激素基因的重组短短芽孢杆菌,所述短短芽孢杆菌为SP3。
所述的质粒为pNCM02;
一种表达猪生长激素基因的重组短短芽孢杆菌的构建方法,如图1-4所示,包括以下步骤:
(1)对NCBI上公布的猪生长激素序列根据短短芽孢杆菌密码子偏好性进行优化得到pGH基因和pNCM02质粒;
(2)用限制性内切酶XbaI和ClaI对上述pGH基因和pNCM02质粒进行双酶切;
(3)回收双酶切产物进行连接反应,连接产物转化大肠杆菌JM109感受态细胞,使用氨苄西林抗性筛选转化子,获得重组质粒pGH-pNCM02;
(4)提取重组质粒并电转化短短芽孢杆菌SP3,使用新霉素抗性筛选转化子,获得重组短短芽孢杆菌,表达猪生长激素。
一种表达猪生长激素基因的重组短短芽孢杆菌的应用,其特征在于,所述使用白油佐剂将短短芽孢杆菌表达并纯化的猪生长激素蛋白乳化后对仔猪皮下注射,有效解决了生长激素半衰期短的问题,达到了长效的效果。
实施例1
猪生长激素基因的获得
对NCBI上公布的猪生长激素序列(AAA31045)如序列表中序列1所示,根据短短芽孢杆菌密码子偏好性进行优化,上游添加6个组氨酸的核苷酸序列,下游添加终止密码子,并在两端分别添加XbaI和ClaI两酶切位点及保护性碱基,人工合成该序列,见序列表中序列2。
序列1
FPAMPLSSLFANAVLRAQHLHQLAADTYKEFERAYIPEGQRYSIQNAQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFLSRVFTNSLVFGTSDRVYEKLKDLEEGIQALMRELEDGSPRAGQILKQTYDKFDTNLRSDDALLKNYGLLSCFKKDLHKAETYLRVMKCRRFVESSCAF
序列2
GCTCTAGACATCATCATCATCATCATTTCCCGGCAATGCCGCTGAGCAGCCTGTTCGCGAACGCGGTGCTGCGCGCGCAACACCTGCACCAGCTGGCGGCAGATACGTATAAAGAATTCGAACGCGCGTACATCCCGGAAGGCCAACGCTATAGCATCCAGAACGCGCAAGCGGCGTTCTGCTTCAGCGAGACGATCCCGGCGCCGACGGGCAAAGATGAAGCGCAACAGCGCAGCGACGTGGAACTGCTGCGCTTCAGCCTGCTGCTGATCCAAAGCTGGCTGGGCCCGGTCCAGTTTCTGAGCCGCGTCTTCACGAACAGCCTGGTGTTTGGCACGAGCGACCGCGTGTATGAAAAACTGAAAGACCTGGAGGAAGGCATCCAGGCGCTGATGCGCGAACTGGAAGACGGCAGCCCGCGCGCAGGCCAAATCCTGAAACAGACGTATGACAAATTCGATACGAACCTGCGCAGCGACGACGCGCTGCTGAAAAACTACGGCCTGCTGAGCTGCTTTAAAAAAGATCTGCACAAAGCGGAGACGTACCTGCGCGTCATGAAATGCCGCCGCTTCGTGGAAAGCAGCTGCGCGTTTTAAATCGATCC
实施例2
构建短短芽孢杆菌重组质粒pGH-pNCM02
1、pGH基因和pNCM02质粒的双酶切反应
用限制性内切酶XbaI和ClaI对实施案例1中获得的基因和pNCM02质粒进行双酶切,双酶切反应体系如下:
体系 | 体积(μL) |
XbaI | 2 |
ClaI | 2 |
pGH基因/ pNCM02质粒 | 20 |
10×M buffer | 4 |
ddH2O | 12 |
总体积 | 40 |
于37℃温箱反应3h后,琼脂糖凝胶回收双酶切片段。
2、pGH基因和pNCM02质粒的连接反应
T4连接酶连接反应体系如下:
体系 | 体积(μL) |
pGH | 8 |
pNCM02 | 8 |
T4 | 2 |
10×buffer | 2 |
总体积 | 20 |
于4℃冰箱中过夜反应。
3、连接产物转化JM109大肠杆菌感受态细胞
3.1、取出-80℃冻存的大肠杆菌JM109感受态细胞(100μL装),握于手心中15sec快速融化后置于冰上;
3.2、吸取10μL连接产物轻轻加至大肠杆菌JM109感受态细胞底部,冰浴30min;
3.3、42℃水浴热击90sec,迅速放置冰上1-2min;
3.4、加入LB液体培养基900μL,置于37℃摇床培养120rpm缓慢复苏40min;
3.5、复苏的转化产物涂于含氨苄西林100ug/mL的LB固体平板上, 37℃培养18h。
4、转化子菌落PCR鉴定
使用pGH -F和pGH -R引物对转化子进行菌落PCR扩增鉴定,引物序列为:
pGH-F:TCTAGACATCATCATCATCATCATTT,
pGH-R:ATCGATTTAAAACGCGCAGCTGCTTTCCA
PCR体系为:pGH-F 5μL,pGH-R 5μL,dNTP 5μL,10×buffer 5μL,transT-Taq酶1μL,转化子菌液1μL,ddH2O 28μL,总体积50μL 。
PCR扩增程序为:预变性,95℃,5min;变性,95℃,30sec;退火,55℃,30sec;延伸,72℃,1min;总延伸,72℃,5min。
其中,变性,退火,延伸,30 cycle。
PCR扩增完成后,PCR原液进行琼脂糖凝胶电泳,于600bp处有清晰条带,PCR原液同时测序,测序结果与合成基因一致。
实施例3
构建重组短短芽孢杆菌
1、相关培养基及试剂的配制
T2液体培养基:葡萄糖1.0%,蛋白胨1.0%,牛肉膏0.5%,酵母提取物0.2%,pH 7.0;T2固体培养基为添加2%琼脂粉的T2液体培养基。
感受态制备培养基: T2培养基中添加 MnSO4 0.001%(w/v,下同),FeSO40.0001%,ZnSO4 0.0001%
感受态洗涤液1:蔗糖10%,HEPES 16mM,CaCl2 1mM,甘油15%,pH 7.0 。
感受态洗涤液2:PEG6000 15%,HEPES 1mM,pH 7.0 。
修复培养基:T2液体培养基中添加20mM MgCl2
2、短短芽孢杆菌SP3感受态的制备
2.1、在T2固体培养基平板上划线复苏短短芽孢杆菌SP3,37℃倒置培养18;
2.2、培养种子液:挑取短短芽孢杆菌SP3单菌落接种于5ml T2液体培养基中,30℃250rpm培养过夜。
2.3、20ml感受态制备培养基中加入200μL过夜培养种子液,30℃ 250rpm培养至OD600=2.5,4℃ 2500rpm离心10min,弃上清培养基。
2.4、加入20mL预冷感受态洗涤液1,缓慢重悬菌体, 4℃ 2500rpm离心10min,弃上清培养基。
2.5、加1mL预冷感受态洗涤液2,缓慢重悬菌体,分装成90μL每管,立即电转使用。
3、重组质粒pGH-pNCM02电转短短芽孢杆菌SP3
将质粒小量提取试剂盒提取的pGH-pNCM02重组质粒电转化短短芽孢杆菌SP3,电转化步骤如下:
3.1、90μL短短芽孢杆菌SP3感受态细胞中加入10μL pGH-pNCM02重组质粒,轻轻混匀;
3.2、将重组质粒和感受态混合物缓慢加入2mm电转杯底部,冰浴10min;
3.3、设置参数电压2000V、电容50uF、电阻200Ω,电转完成后迅速加入900μL预冷的修复培养基,轻轻混匀后转移至1.5mlEP管中,30℃摇床80rpm缓慢复苏1h;
3.4、复苏好的转化产物涂于含新霉素30ug/mL的T2固体培养基平板上,每个平板涂200μL,37℃倒置培养15-18h。
4、转化子菌落PCR鉴定
使用T2液体培养基对疑似阳性转化子单菌落进行扩大培养,新霉素添加量为30ug/mL,过夜摇混后常温2500rpm离心,菌体沉淀加适量溶菌酶缓慢重悬,55℃作用30min以破坏短短芽孢杆菌细胞壁,再用质粒小量提取试剂盒提质粒,以所提质粒为模板进行PCR扩增鉴定,测序鉴定,从而获得重组短短芽孢杆菌。
实施例4
重组短短芽孢杆菌SP3的表达
平板上挑取重组短短芽孢杆菌单菌落接种于含新霉素30ug/mL的3mL T2液体培养基中,30℃ 120rpm摇床培养48-64h,表达的猪生长激素被分泌至培养基上清。
实施例5
重组短短芽孢杆菌表达猪生长激素的纯化
1、饱和硫酸铵沉淀培养基上清中的猪生长激素
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,使猪生长激素蛋白充分沉淀。 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。
2、猪生长激素蛋白镍柱纯化
相关试剂如下:
母液1:29.22g氯化钠,2.42gTris-Base,ddH2O定溶至1L,调节pH=8.0母液2:6.06gTris-Base,ddH2O定溶至1L,调节pH=8.0
Binding buffer:0.1702g咪唑,溶于500 mL母液1。
Elution buffer:17.02g咪唑,溶于500 mL母液1。
Washing buffer: 2.3828g咪唑,溶于500 mL母液2。
先用Binding buffer平衡镍离子亲和层析柱,然后将待纯化的样品上样于镍柱,结合30min左右,收集留出液Flow Through,用Washing buffer洗3个柱体积,收集Washthrongh,最后用Elution buffer洗脱蛋白,收集Eluent.将Binding buffer,Washthrongh,Eluent。
将镍柱收集的蛋白用Milipore压力超滤浓缩装置通过30kDa超滤膜截留浓缩至10mL左右,将样品通过Gel filtration buffer平衡过的Superdex200分子筛进行分离。
实施例6
猪生长激素的western blot验证
1、相关试剂的配制
1.1、Tris-HCl缓冲液(1mol/L,pH8.8)、Tris-HCl缓冲液(0.5mol/L,pH6.8)购于北京诺博莱德科技有限公司
1.2、SDS-PAGE分离胶(12%)
成分 | 体积(ml) |
ddH2O | 6.6 |
30 %丙烯酰胺 | 8 |
1.5M Tris-HCl(PH=8.8) | 5 |
10 %SDS | 0.2 |
10 %过硫酸铵 | 0.2 |
TEMED | 0.008 |
总体积 | 20 |
1.3、SDS-PAGE浓缩胶
成分 | 体积(ml) |
ddH2O | 4.1 |
30 %丙烯酰胺 | 1.0 |
1.0M Tris-HCl(PH=6.8) | 0.75 |
10 %SDS | 0.06 |
10 %过硫酸铵 | 0.06 |
TEMED | 0.006 |
总体积 | 6 |
1.4、SDS-PAGE电泳缓冲液(Tris-Glycine):9.4g甘氨酸、1.51g Tris-base、0.5gSDS,ddH2O定容至500ml;
1.5、 Western Blot转膜缓冲液:14.4g甘氨酸、3.03gTris-base、200ml甲醇,ddH2O定容至1000ml;
1.6 、TBST洗涤液:称取2.42g Tris-Base、8.8g NaCl,量取0.5mL Tween-20,用ddH2O定容至1L,调节至pH=7.4;
1.7、封闭液:在洗涤液中加入1%的BSA(牛血清白蛋白第五组分),溶解后4℃保存;
2、纯化猪生长激素的western blot验证
将纯化的生长激素中添加SDS-PAGE loading buffer,沸水浴15min,进行westernblot,步骤如下:
2.1、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;
2.2、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;
2.3、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;
2.4、每孔加样20μL,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;
2.5、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。
2.6、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;
2.7、转膜完成后卸下转膜装置,用TBST洗涤液清洗膜3次,每次5min;
2.8、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;
2.9、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7
重组猪生长激素的猪体试验
1、猪生长激素蛋白的乳化
将已知浓度的纯化猪生长激素蛋白和白油按1:1比例进行混合,使用乳化器无菌乳化,最终蛋白浓度为5mg-20mg/mL。
2、重组猪生长激素的猪体试验
取16头21日龄健康断奶仔猪,平均体重7.21 kg,随机分为4组,每组4头,对照组1每头皮下注射生理盐水1 mL,连续注射20日;对照组2每头皮下注射白油1 mL,仅注射1次;试验组1每头皮下注射250ug重组猪生长激素1 mL,连续注射20日;试验组2每头皮下注射白油乳化的重组猪生长激素5mg,仅注射1次。第21日称重,通过体重来评价重组猪生长激素对猪生长的作用。
试验结果如下表所示:
组别 | 对照组1 | 试验组1 | 对照组2 | 试验组2 |
试验后平均体重(kg) | 16.13a | 21.54b | 16.36a | 19.89b |
注:同行肩标字母不同者表示差异显著(P<0.05)。
结果显示,注射1次乳化的猪生长激素和连接注射20天猪生长激素的仔猪体重并无显著性差异,且与对照组相比,可显著提高21 日龄断奶仔猪增重33.5%和21.6%。
Claims (4)
1.一种表达猪生长激素基因的重组短短芽孢杆菌,其特征在于,所述短短芽孢杆菌为SP3。
2.根据权利要求1所述的一种表达猪生长激素基因的重组短短芽孢杆菌,其特征在于,包含了权利要求1编码猪生长激素基因的质粒,所述的质粒为pNCM02。
3.根据权利要求1所述的一种表达猪生长激素基因的重组短短芽孢杆菌的构建方法,其特征在于,包括以下步骤:
(1)对NCBI上公布的猪生长激素序列根据短短芽孢杆菌密码子偏好性进行优化得到pGH基因和pNCM02质粒;
(2)用限制性内切酶XbaI和ClaI对上述pGH基因和pNCM02质粒进行双酶切;
(3)回收双酶切产物进行连接反应,连接产物转化大肠杆菌JM109感受态细胞,使用氨苄西林抗性筛选转化子,获得重组质粒pGH-pNCM02;
(4)提取重组质粒并电转化短短芽孢杆菌SP3,使用新霉素抗性筛选转化子,获得重组短短芽孢杆菌,表达猪生长激素。
4.根据权利要求1所述的一种表达猪生长激素基因的重组短短芽孢杆菌的应用,其特征在于,所述使用白油佐剂将短短芽孢杆菌表达并纯化的猪生长激素蛋白乳化后对仔猪皮下注射,有效解决了生长激素半衰期短的问题,达到了长效的效果。
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