CN106434742A - 利用大豆表达犬瘟热蛋白的方法 - Google Patents
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Abstract
本发明公开了一种利用大豆表达犬瘟热蛋白的方法,属于分子生物学与基因工程技术领域,具体设计一种获得大豆种子表达系统进行犬瘟热病毒囊膜蛋白亚基疫苗的方法。本发明的目的是提供一种获得大豆种子表达系统进行犬瘟热病毒囊膜蛋白亚基疫苗的方法,通过基因修饰改造,将犬瘟热病毒主要囊膜蛋白H(主要抗原蛋白CDV)亚克隆到植物双元表达载体pTF101‑35s中,并通过农杆菌介导的大豆子叶节方法进行大豆转CDV基因研究,最终获得在大豆种子中表达CDV基因的转基因再生植株。
Description
技术领域
本发明属于分子生物学与基因工程技术领域,具体涉及一种利用大豆种子表达犬瘟热病毒的方法。
背景技术
随着植物生物技术的发展,特别是植物转基因技术的不断推进,利用植物系统表达具有经济价值疫苗或者药物的重组蛋白已经成为可能。在最近十年间,已经有越来越多的药物重组蛋白在植物体中表达利用,例如治疗型抗体、免疫刺激物、抗病毒药物、血液组分的疫苗等。相对于动物细胞、细菌和转基因动物,植物表达相对来说更加经济实用,而且仅需要种植不需要建立工厂和相关设备。这种蛋白加工方式仅仅是利用了土壤、空气和水,可以说是符合绿色、安全环保等特点,在社会节能减排的背景下更加具有优势。就生物安全的角度而言,植物表达也不存在散毒和污染环境等缺点,能够在距离市中心的地方进行蛋白生产加工。所以说利用植物系统表达具有经济价值疫苗或者药物的重组蛋白具有非常广阔的应用前景和机会。在众多植物中,大豆是一种种子含有丰富蛋白的植物。原产于我国,东北最为著名。由于大豆种子这种高蛋白特性,利用其作为表达疫苗重组蛋白的研究在疫苗重组产量上具备天然的优势和潜力。
发明内容
本发明的目的是提供一种一种利用大豆种子表达犬瘟热病毒的方法。
本发明通过以下方法实现上述目的:通过基因修饰改造,将犬瘟热病毒主要囊膜蛋白H(主要抗原蛋白CDV)亚克隆到植物双元表达载体pTF101-35s中,并通过农杆菌介导的大豆子叶节方法进行大豆转CDV基因研究,最终获得在大豆种子中表达CDV基因的转基因再生植株。
本发明主要包含以下步骤:
1.植物表达载体构建
根据NCBI上犬瘟热病毒囊膜蛋白H基因(GenBank登录号JN896331.1)序列信息,按照植物密码子偏爱性人工合成CDV基因。同时根据植物双元表达载体多克隆位点分子特征,分别在CDV-H基因上游引入内切酶SpeI酶切位点,下游引入内切酶SacI酶切位点。把CDV-H基因片段进行SpeI和SacI酶切后连接pTF101-35s载体上形成pTF101-35s-CDV。利用PrimerPremier 5.0软件设计具体引物序列如下:pTF101-35S-CDVF:5'-GCAGAACAGCACTAGTGCTGAAGAACCAAGACAAGC-3’;pTF101-35S-CDVR:5'-GATCGGGGAAATTCGAGCTCCATGTTGCGCTCGATGTGCA-3'。同时针对CDV基因的编码区,设计PCR分子检测扩增特异性引物如下:CDV-F:5'-GCTGAAGAACCAAGACAAGC-3';CDV-R:5'-CATGTTGCGCTCGATGTGCA-3'。
2.农杆菌介导的大豆遗传转化
以农杆菌株EHA101为工程菌株,将pTF101-35s-CDV通过大豆子叶节方法转入受体材料Williams82中。农杆菌介导大豆子叶节转化法基本流程如下:(1)农杆菌28℃培养16h后收集菌体,转入YEP液体培养基中,直至OD600值0.5~0.7备用。(2)选取大豆受体品种P3成熟种子,氯气灭菌16h。灭菌后种子放入GM萌发培养基(培养基配方参照OLHOFT等[11])弱光萌发16h。(3)切子叶节,从胚轴处将大豆种子一分为二,切时刀尖蘸工程菌液。将切开后的子叶节放入工程菌液中,轻柔晃动30min,转入共培养基中,避光培养(23℃,3~5d)。(4)共培养后,将伸长的胚轴切去约2/3,保留约5mm的胚轴,插入加筛选剂的SIM伸长培养基中,诱导丛生芽生长,培养条件25℃,光照16h·d-1,光照强度2000lx。(5)在SIM培养基中培养7d后,转入SEM筛选培养基中,间隔15d继代1次,筛选3~4轮,得到分生苗。(6)将已经伸长的分生苗从外置体上切下,转入生根培养基中生根。(7)生根健全的转化苗,经炼苗(3~5d)后移入盆中栽培。
3.转基因植株的草丁膦抗性检测
转化载体中以草丁膦作为筛选标记,因此在后代可采用草丁膦涂抹叶片的方法快速检测转基因苗。具体方法:用棉签蘸取适量草丁膦(浓度为150mg·L-1)溶液,轻柔擦拭半片叶子,同叶未涂抹的叶子作为对照,在正常光照情况下3~5d后观察叶片生长情况。
4.T0-T3转基因植株的PCR检测
利用CDV基因引物对T0-T3代转化植株基因组DNA进行PCR扩增检测。使用CTAB法提取转化植株及对照P3的DNA。PCR反应体系(20μl):DNA模板50ng,10×buffer 2.0μl,2.5mMdNTP 1μl,10μM引物各0.5μl,5U·μl-1Taq酶0.5μl,加ddH2O补至20μl。PCR反应程序:95℃,变性5min,94℃,变性30s,56℃退火30s,72℃延伸1min,30个循环,72℃延伸5min。PCR扩增产物通过1%琼脂糖凝胶电泳分离,电泳结果利用凝胶成像系统进行拍照和分析。
5.T3转基因植株的Southern分析
为了检测CDV基因整合到大豆基因组中的拷贝数,选取PCR检测呈阳性的不同3个T3家系的3个单株,进行基因组DNA的Southern blot分析。Southern blot分析使用的是Roche的地高辛试剂盒KitⅡ型,实验具体流程参照说明书。以克隆到载体上的CDV基因的全长(1824bp)作为Southern杂交探针,使用SacI和HindIII两种内切酶进行基因组DNA酶切。
本方法的有益效果在于获得了能在大豆中表达犬瘟热蛋白的转基因大豆材料及其后代。
附图说明
图1为CDV基因质粒载体图谱及电泳检测结果;A为pTF101-35S-CDV载体图谱;B为相应载体酶切电泳检测结果图,其中M:Trans2K TM Plus II DNA Marker;1:经SpeI和SacI双酶切的pTF101-35S-CDV载体。
图2为农杆菌介导的大豆子叶节遗传转化法
图3为T0代转CDV基因大豆阳性植株的BASTA抗性分析(A)和PAT/bar基因试纸条检测结果(B)
图4为利用PCR方法检测T0代转基因后代目的基因PCR扩增结果;上排为目的基因检测结果,下排为相应单株DNA用Actin引物检测的结果。M:Trans2KTM plus DNA Marker;“+”质粒阳性对照;“-”阴性对照;1-15依次为CDV转基因后代材料
图5为转基因T3植株的Southern blot杂交,HindIII酶切结果。其中M:Trans15KDNA Marker;“+”质粒阳性对照;“-”阴性对照;1-9:经过PCR检测的转基因植株
图6为转基因T3植株的Southern blot杂交,SacI酶切结果。其中M:Trans15K DNAMarker;“+”质粒阳性对照;“-”阴性对照;1-9:经过PCR检测的转基因植株
图7为外源基因犬瘟热病毒囊膜蛋白CDV基因在大豆中表达Western检测结果
具体实施方式
下述实施例中的受体材料为大豆品种Williams,由吉林省农业科学院生物所大豆遗传转化课题组保存。
大肠杆菌菌株为E.coli DH5α,农杆菌菌株为EHA101,植物双元表达载体为pTF101-35s等菌株由吉林省农业科学院生物技术研究所大豆遗传转化课题组保存。
Taq DNA聚合酶、限制性内切酶、T4-DNA连接酶等购自大连宝生物有限公司;DNA片段回收试剂盒购自康为试剂生物公司,其他试剂均为国产分析纯产品。
实施例1植物表达载体构建
根据NCBI上犬瘟热病毒囊膜蛋白CDV基因(GenBank登录号JN896331.1)序列,按照植物密码子偏爱性人工合成CDV基因。根据植物双元表达载体多克隆位点分子特征,分别在CDV基因上游引入内切酶SpeI酶切位点,下游引入内切酶SacI酶切位点,同时选择载体上两个酶切位点两侧20-25bp序列作为接头序列设计引物,把带有载体接头序列的CDV基因片段进行SpeI和SacI酶切后连接pTF101-35s载体上形成pTF101-35s-CDV。扩增目的基因含有载体接头序列利用Primer Premier 5.0软件设计,具体引物序列如下:pTF101-35S-CDVF:5'-GCAGAACAGCACTAGTGCTGAAGAACCAAGACAAGC-3’;pTF101-35S-CDVR:5'-GATCGGGGAAATTCGAGCTCCATGTTGCGCTCGATGTGCA-3'。同时针对CDV基因的编码区,设计PCR分子检测扩增特异性引物如下:CDV-F:5'-GCTGAAGAACCAAGACAAGC-3';CDV-R:5'-CATGTTGCGCTCGATGTGCA-3'。
实施例2农杆菌介导的大豆遗传转化
以农杆菌株EHA101为工程菌株,将pTF101-35s-CDV通过大豆子叶节方法转入受体材料Williams82中。农杆菌介导大豆子叶节转化法基本流程如下:
(1)农杆菌28℃培养16h后收集菌体,转入YEP液体培养基中,直至OD600值0.5~0.7备用。
(2)选取大豆受体品种P3成熟种子,氯气灭菌16h。灭菌后种子放入GM萌发培养基(培养基配方参照OLHOFT等[11])弱光萌发16h。
(3)切子叶节,从胚轴处将大豆种子一分为二,切时刀尖蘸工程菌液。将切开后的子叶节放入工程菌液中,轻柔晃动30min,转入共培养基中,避光培养(23℃,3~5d)。
(4)共培养后,将伸长的胚轴切去约2/3,保留约5mm的胚轴,插入加筛选剂的SIM伸长培养基中,诱导丛生芽生长,培养条件25℃,光照16h·d-1,光照强度2000lx。
(5)在SIM培养基中培养7d后,转入SEM筛选培养基中,间隔15d继代1次,筛选3~4轮,得到分生苗。
(6)将已经伸长的分生苗从外置体上切下,转入生根培养基中生根。
(7)生根健全的转化苗,经炼苗(3~5d)后移入盆中栽培。
实施例3转基因植株的草丁膦抗性检测
转化载体中以bar作为筛选标记,因此在后代可采用商品化的PAT/bar转基因检测试纸条和草丁膦涂抹叶片的方法快速检测转基因苗。PAT/bar转基因检测试纸条检测方法可以参考QuickStix Kit for Roundup Ready Leaf&Seed(EnviroLogix,AS013,USA)进行。草丁膦涂抹叶片具体操作方法如下:用棉签蘸取适量草丁膦(浓度为150mg·L-1)溶液,轻柔擦拭半片叶子,同叶未涂抹的叶子作为对照,在正常光照情况下3~5d后观察叶片生长情况。
实施例4 T0-T3代转基因植株的目的基因的PCR检测
利用CDV基因引物对不同世代转化植株基因组DNA进行PCR扩增检测。使用CTAB法提取转化植株及对照P3的DNA。PCR反应体系(20μl):DNA模板50ng,10×buffer 2.0μl,2.5mM dNTP 1μl,10μM引物各0.5μl,5U·μl-1Taq酶0.5μl,加ddH2O补至20μl。PCR反应程序:95℃,变性5min,94℃,变性30s,56℃退火30s,72℃延伸1min,30个循环,72℃延伸5min。PCR扩增产物通过1%琼脂糖凝胶电泳分离,电泳结果利用凝胶成像系统进行拍照和分析。
实施例5转基因植株的外源目的基因的Southern分析
为了检测CDV基因整合到大豆基因组中的拷贝数,选取再生苗PCR检测呈阳性的12个单株,进行基因组DNA的Southern blot分析。Southern blot分析使用的是Roche的地高辛试剂盒KitⅡ型,实验具体流程参照说明书。以克隆到载体上的CDV基因的全长(861bp)作为Southern杂交探针,使用EcoRI和HindIII两种内切酶进行基因组DNA酶切。
实施例6外源目的基因Western Blot检测
提取经过Southern检测的单拷贝材料中,利用植物总蛋白提取试剂盒(Sigma,PE0230,USA)提取T3代大豆未成熟种子中的蛋白。将变性后的蛋白样品,采用分离胶为12%、浓缩胶为5%进行SDS-PAGE凝胶电泳;采用湿转的方式将胶上的分离蛋白转到硝酸纤维素膜上,电泳(85V,1h);将转完的膜用3%的脱脂牛奶封闭1h,按1:5000比例加一抗,1:10000比例加二抗,最后用HRP底物进行显色。
实施例7目的基因克隆验证
以人工合成的CDV基因为模板,以pTF101-35S-CDVFR为引物,通过PCR扩增得到含有载体接头序列和CDV基因全长PCR片段2023bp,然后通过同源重组的方法克隆到pTF101-35s载体中(图1A),构建好的质粒经过英俊生物技术公司测序和酶切检测(图1B),验证正确的植物表达载体转入农杆菌EHA101中,用于下一步大豆遗传转化。
实施例8转目的基因阳性材料的获得
在本研究中,选择带有目的基因CDV的农杆菌菌株EHA101,利用根癌农杆菌介导的大豆子叶节遗传转化法对大豆受体品种Williams进行遗传转化,共切取2126个大豆外植体,发生诱导的外植体有1001块,最终得到T0代转基因再生植株85株(图2),其中经bar试纸条检测呈阳性为61株(图3A),转化率为6.1%(阳性苗/诱导的外植体。这些初步检测呈阳性的再生植株移栽后,利用草丁膦涂抹叶片的方法快速检测,其中不抗除草剂的叶片明显变黄干枯,阳性植株叶片正常。检测49株转基因植株中,得到36株抗草丁膦的转化苗(见图3B)。
实施例9转基因植株中外源基因的分子检测
通过PCR对36株T0代转基因苗检测目的基因CDV,结果表明30株T0代转基因植株含有外源目的基因(图4)。将这些T0代阳性转基因材料繁殖培育,最终获得26个能够正常结实的T0代大豆转基因材料,见表。选取其中种子结实数大于等于20粒的TO代转基因材料,在下两年度田间繁殖。我们对在T3代材料进行了southern分析,每个家系随机检测3株材料。结果发现CDV8027所有单株均表现全长cDNA单拷贝插入到基因组中(图5),CDV8026呈现2拷贝。在获得的单拷贝的转基因目的基因已经真正的整合到植物基因组中。杂交带大小有所不同,说明外源基因整合位点不同。
实施例10 Western检测结果
我们对单拷贝转基因家系CDV8027中的材料进行了外源蛋白表达情况检测,结果发现来源于犬瘟热病毒囊膜蛋白CDV基因能够在转基因株系CDV8023中能够正常表达(图6)。
Claims (2)
1.一种利用大豆表达犬瘟热蛋白的方法,包括以下步骤:
A.植物表达载体构建根据NCBI上犬瘟热病毒囊膜蛋白H基因(GenBank登录号JN896331.1)序列信息,按照植物密码子偏爱性人工合成CDV基因;根据植物双元表达载体多克隆位点分子特征,分别在CDV-H基因上游引入内切酶SpeI酶切位点,下游引入内切酶SacI酶切位点;把CDV-H基因片段进行SpeI和SacI酶切后连接pTF101-35s载体上形成pTF101-35s-CDV;
B.农杆菌介导的大豆遗传转化以农杆菌株EHA101为工程菌株,将pTF101-35s-CDV通过大豆子叶节方法转入受体材料Williams82中。
2.根据权利要求1所述的用大豆表达犬瘟热蛋白的方法,其特征在于,所述农杆菌介导的大豆遗传转化通过大豆子叶节转化法实现,基本流程如下:
(1)农杆菌28℃培养16h后收集菌体,转入YEP液体培养基中,直至OD600值0.5~0.7备用;
(2)选取大豆受体品种P3成熟种子,氯气灭菌16h,灭菌后种子放入萌发培养基培养,弱光萌发16h;
(3)切子叶节,从胚轴处将大豆种子一分为二,切时刀尖蘸工程菌液,将切开后的子叶节放入工程菌液中,轻柔晃动30min,转入共培养基中,避光培养,23℃,培养3~5天;
(4)共培养后,将伸长的胚轴切去约2/3,保留约5mm的胚轴,插入加筛选剂的SIM伸长培养基中,诱导丛生芽生长,培养条件25℃,光照16h/d,光照强度2000lx;
(5)在SIM培养基中培养7天后,转入筛选培养基中,间隔15天继代1次,筛选3~4轮,得到分生苗;
(6)将已经伸长的分生苗从外置体上切下,转入生根培养基中生根;
(7)生根健全的转化苗,经炼苗3~5d天后移入盆中栽培。
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