CN105907784B - 用于表达t4连接酶的家蚕后部丝腺生物反应器双启动子通用型质粒及其应用与方法 - Google Patents
用于表达t4连接酶的家蚕后部丝腺生物反应器双启动子通用型质粒及其应用与方法 Download PDFInfo
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Abstract
本发明公开了一种用于表达T4连接酶的家蚕后部丝腺生物反应器双启动子通用型质粒及其应用与方法。质粒以piggyBac转座子为基础并带有Amp抗性基因,包含有作为外源基因的T4连接酶基因和作为标记基因的绿色荧光EGFP基因的功能表达框,质粒用分子生物学方法构建,且DDDDK与丝素轻链基因polyA之间含有ApaI和NheI专一性的两个酶切位点,采用ApaI和NheI双酶切通用型质粒,与T4连接酶基因连接后,与辅助质粒共同注射到家蚕受精卵内,利用转座子特性使绿色荧光蛋白基因和T4连接酶基因导入到家蚕基因组内,并稳定遗传和表达,获得转基因家蚕。本发明借助荧光标志基因筛选转基因家蚕,利用该家蚕丝腺细胞特异地合成分泌T4连接酶蛋白。
Description
技术领域
本发明涉及一种质粒及其应用与方法,尤其是涉及利用转基因技术的一种用于表达T4连接酶的家蚕后部丝腺生物反应器双启动子通用型质粒及其应用与方法。
背景技术
piggyBac转座子最初是从粉纹夜蛾(Trichoplusia ni)TN-368细胞株的基因组中分离得到的,是目前发现的转座活性最高的DNA转座子。piggyBac转座系统是一种非病毒载体,转座效率较高。与sleeping beauty相比,piggyBac载体容量较大,可携带18kb外源基因,可以实现多基因的共表达,且转座片段被切除后不会在原位点留下印迹(footprint),基因组可以实现切除后精确修复,在可逆转基因的应用中具有重要作用。
piggyBac转座子介导的转基因家蚕丝腺生物反应器的研究开展了十几年,世界各国的科学家致力于外源基因的表达,已经建成了以丝素蛋白轻链启动子(Fib-LPromoter)、丝素蛋白重链启动子(Fib-H Promoter)和丝胶蛋白1启动子(Ser1Promoter)表达外源蛋白的转基因家蚕丝腺生物反应器。但纵观十几年来国内外家蚕生物反应器的研究结果,转基因家蚕丝腺生物反应器的外源蛋白表达效率都很低,不管是丝胶启动子驱动表达的外源基因,还是丝素启动子驱动表达的外源基因,极大多数实验的表达量都没有在达到茧层量的1%左右,远远没有达到科学家们期望的象表达蚕丝蛋白那样的高效表达水平。
发明内容
为了解决背景技术中存在的问题,本发明的目的在于提出了一种用于表达T4连接酶的家蚕后部丝腺生物反应器双启动子通用型质粒及其应用与方法,利用转基因家蚕技术将T4连接酶基因导入家蚕基因组内,并在家蚕丝腺细胞中特异表达,开发出能合成分泌单一的T4连接酶的家蚕。
为了达到上述目的,本发明采用的技术方案的步骤如下:
一、一种用于表达T4连接酶的家蚕后部丝腺生物反应器双启动子通用型质粒:
所述的质粒为piggy-10004质粒,piggy-10004质粒的碱基序列如SEQ ID NO.1,是以piggyBac转座子为基础并带有Amp抗性基因,包括piggyBac转座子的两个转座臂PBL和PBR以及两个转座臂之间的两个分别包含有作为外源基因的T4连接酶基因和作为标记基因的绿色荧光EGFP基因的功能表达框。
一个功能表达框是A3启动子启动的绿色荧光蛋白基因表达框,即A3Promoter-EGFP-SV40,另一个功能表达框是包含家蚕丝素蛋白轻链基因启动子、18s rRNA启动子、丝素蛋白轻链基因信号肽、His 6序列、肠激酶酶切位点DDDDK、T4连接酶基因和家蚕丝素蛋白轻链基因3’末端的表达框,即Fibroin L chain Promoter-18s rRNA promoter-Fibroin Lchain signal peptide-His6-DDDDK-T4Ligase-Fibroin L chain PolyA。
所述的piggy-10004质粒是利用双启动子Fibroin L chain Promoter和18s rRNApromoter驱动T4连接酶基因的表达。
二、一种用于表达T4连接酶的家蚕后部丝腺生物反应器双启动子通用型质粒的制备方法,包括以下步骤:
1)以带有ApaI酶切位点序列的T4连接酶5’端序列和带有NheI酶切位点序列的T4连接酶3’端序列为引物,以包含A3基因启动子-绿色荧光蛋白基因-SV40-丝胶蛋白1基因启动子-T4连接酶基因-SV40([A3-EGFP-SV40]-[Ser1promoter-T4Ligase-SV40])的piggy-10522质粒为模板,piggy-10522质粒的碱基序列如SEQ ID NO.2,通过PCR扩增获得长度为1536bp的T4连接酶基因,T4连接酶基因的碱基序列如SEQ ID NO.3;
2)用ApaI和NheI双酶切家蚕后部丝腺生物反应器双启动子通用型质粒piggy-8480质粒,piggy-8480质粒的碱基序列如SEQ ID NO.4,得到长度为8478bp的包含[A3-EGFP-SV40]-[FL692promoter-18s rRNA promoter-FLSP-His6-DDDDK-FLPA和Amp抗性基因的基因序列;
3)连接步骤1)得到的T4连接酶基因和步骤2)得到的基因序列,得到含有[A3-EGFP-SV40]-[FL692promoter-18s rRNA-promoter-FLSP-His6-DDDDK-T4Ligase-FLPA]和Amp抗性基因的piggy-10004质粒,其碱基序列如SEQ ID NO.1。
三、本发明所述通用型质粒在T4连接酶表达的应用。
四、一种用于表达T4连接酶的家蚕后部丝腺生物反应器双启动子通用型质粒表达T4连接酶的方法,步骤如下:
采用分子生物学方法构建包含[A3-EGFP-SV40]-[Fibroin L chain Promoter-18s rRNA promoter-Fibroin L chain signal peptide-His 6-DDDDK-T4Ligase-FibroinL chain PolyA]两个功能表达框的piggy-10004质粒,piggy-10004质粒的碱基序列如SEQID NO.1,作为在家蚕后部丝腺中表达的T4连接酶基因载体,该质粒是以piggyBac转座子为基础包含有作为外源基因的T4连接酶基因和作为标记基因的绿色荧光EGFP基因表达框;
(1)采用显微注射的方法,将piggy-10004质粒及能够提供piggyBac转座酶的辅助质粒按浓度比1:1的比例导入家蚕产卵后6小时以内的受精卵内,利用piggy-10004质粒中的piggyBac转座子将T4连接酶基因插入到家蚕基因组内;
(2)蚕卵孵化后饲养至成虫,然后与非转基因家蚕交配制种续代,此代为G1代,在G1代蚕一龄第二天,通过荧光体视显微镜观察筛选体色表达绿色荧光EGFP标记基因的转基因家蚕,饲养至成虫再与非转基因家蚕交配制种续代成为G2代;
(3)G2代家蚕采用单蛾育,筛选出荧光体视显微镜下表达绿色荧光EGFP标记基因的家蚕,采用同蛾区蚕蛾相互交配制成G3代;
(4)G3代家蚕采用单蛾育,同蛾区表达绿色荧光EGFP标记基因的蚕蛾相互交配,制成G4代;
(5)从G4代开始并经连续3代进行选择和交配,育成绿色荧光基因和T4连接酶基因纯合、丝腺细胞能够合成分泌T4连接酶的转基因家蚕;
(6)通过家蚕丝腺细胞合成分泌T4连接酶,并随家蚕吐丝结茧行为进入蚕茧。
所述步骤(5)中连续3代进行选择和交配具体是采用绿色荧光表型纯一的蛾区饲养、单蛾育和同蛾区蚕蛾交配的方式。
所述的piggy-10004质粒是利用双启动子Fibroin L chain Promoter和18s rRNApromoter驱动T4连接酶基因的表达。
所述步骤(6)的T4连接酶基因在家蚕丝腺细胞特异表达,在家蚕丝素蛋白轻链信号肽的作用下分泌到丝腺腺腔,并随吐丝行为分泌到蚕茧。
本发明是先构建家蚕合成分泌T4连接酶基因的载体piggy-10004,再利用显微注射转基因家蚕技术将这种质粒(如图1所示)与能够提供piggyBac转座酶的辅助质粒(如图2所示)一起导入到家蚕受精卵内,依靠piggyBac转座子的转座特性,使绿色荧光蛋白基因和T4连接酶基因导入到家蚕基因组内,并得到稳定遗传和表达,从而创制成一种能够在家蚕丝腺细胞特异性合成分泌T4连接酶的转基因家蚕,自交使T4连接酶基因纯合,育成能分泌T4连接酶的转基因家蚕,然后利用该种家蚕合成分泌T4连接酶。
本发明具有的有益效果是:
本发明借助荧光标志基因筛选转基因家蚕,这种转基因家蚕能够在家蚕丝腺细胞特异地合成分泌T4连接酶,T4连接酶具有功能活性。本发明开发了一种新型的T4连接酶生产工艺,为大量生产T4连接酶奠定了基础。
附图说明
图1是本发明的piggy-10004质粒结构图。
图2是能够提供piggyBac转座酶的辅助质粒结构图。
具体实施方式
下面结合附图和实施例对本发明作进一步说明。
本发明的实施例如下:
A)制备本发明质粒:
以带有ApaI酶切位点序列的T4连接酶5’端序列和带有NheI酶切位点序列的T4连接酶3’端序列为引物,以含A3基因启动子-绿色荧光蛋白基因-SV40-丝胶基因启动子-T4连接酶基因-SV40([A3-EGFP-SV40]-[Ser promoter-T4Ligase-SV40])piggy-10522质粒(其碱基序列如SEQ ID NO.2)为模板,获得长度为1536bp的T4连接酶基因(其碱基序列如SEQID NO.3),且该序列5’端含有ApaI酶切位点,3’端含有NheI酶切位点。
再用ApaI和NheI双酶切家蚕后部丝腺生物反应器双启动子质粒piggy-8480质粒(其碱基序列如SEQ ID NO.4),得到含[A3-EGFP-SV40]-[FL692promoter-18s rRNApromoter-FLSP-His6-DDDDK-FLPA]和Amp抗性基因的8478bp的序列。
连接上述两个获得的目的片段,得到含有[A3-EGFP-SV40]-[FL692promoter-18srRNA-promoter-FLSP-His6-DDDDK-T4Ligase-FLPA]和Amp抗性基因的piggy-10004质粒(其碱基序列如SEQ ID NO.1)。
B)表达T4连接酶:
将上述构建的piggy-10004质粒(图1)及能够提供piggyBac转座酶的辅助质粒pHA3PIG质粒(图2)按1:1比率混合,2种质粒的总浓度为0.4μg/μl,质粒溶解在pH=7、0.5mM的磷酸缓冲液中,然后采用显微注射方法导入家蚕产卵后6小时以内的受精卵内,导入总体积为10μl。将显微注射的蚕卵在25℃、85%湿度条件下饲养至成虫,与非转基因家蚕杂交传代,是为G1代。在转基因实验的G1代一龄第二天,通过荧光显微镜(Olympus,SZX12,日本)观察获取表达EGFP标志基因的转基因阳性家蚕蛾区2个。
将阳性蚕饲养至成虫与非转基因家蚕杂交传代,是为G2。自第G2代以后的转基因家蚕均采用单蛾育,在一龄期通过荧光体视显微镜观察,挑选表达EGFP标志基因的转基因家蚕,饲养至成虫,同蛾区交配,进而培育得到G3代、G4代,使T4连接酶基因纯合。。
在G4代时,随机取2个蛾区的5龄第3天家蚕各1条,提取后部丝腺细胞基因组DNA为模板,采用反向PCR分析方法对转基因阳性家蚕外源基因插入位点分析结果表明,一个蛾区的外源基因插入在第7染色体scaffold 45处,插入在基因间隔区,另外一个蛾区的外源基因插入在第13染色体scaffold 1处,也是插入在基因间隔区,这个结果证明了带有外源基因的转座子已经被整合到家蚕基因组内。
表1转基因阳性家蚕外源基因插入位点分析结果
从G5代开始选择EGFP基因型纯合的蛾区饲养,采用同蛾区蚕蛾交配,育成EGFP基因纯合、后部丝腺细胞能够合成分泌T4连接酶的转基因家蚕新品种。
提取上述测定插入位点的2个家系的家蚕茧丝蛋白为材料,采用SDS-PAGE电泳和His 6抗体的Western blot技术分析证明了实验获得的转基因家蚕能够表达T4连接酶,而且能够随家蚕吐丝行为进入蚕茧。
比较这个带有双启动子的转基因家蚕T4连接酶表达量和只有1个丝素蛋白轻链启动子的转基因家蚕T4连接酶表达量,双启动子的表达量显著提高10%。
综上可看出,利用本发明方法用家蚕后部丝腺生物反应器双启动子通用型质粒快速、方便地构建转基因家蚕外源基因表达质粒,可以在家蚕丝腺细胞高效合成T4连接酶,T4连接酶可以像蚕丝一样由丝腺分泌进入腺腔,并进一步吐出蚕体。该性状已能够稳定表达并遗传。采用本方法能够大量生产T4连接酶,能够提高蚕桑经济效益,提高蚕农收入。
上述具体实施方式用来解释说明本发明,而不是对本发明进行限制,在本发明的精神和权利要求的保护范围内,对本发明作出的任何修改和改变,都落入本发明的保护范围。
Claims (7)
1.一种用于表达T4连接酶的家蚕后部丝腺生物反应器双启动子通用型质粒,其特征在于:所述的质粒为piggy-10004质粒,是以piggyBac转座子为基础并带有Amp抗性基因,包括piggyBac转座子的两个转座臂PBL和PBR以及两个转座臂之间的两个分别包含有作为外源基因的T4 连接酶基因和作为标记基因的绿色荧光EGFP基因的功能表达框;
一个功能表达框是A3启动子启动的绿色荧光蛋白基因表达框,即A3 Promoter -EGFP-SV40,另一个功能表达框是包含家蚕丝素蛋白轻链基因启动子、18s rRNA启动子、丝素蛋白轻链基因信号肽、His 6序列、肠激酶酶切位点DDDDK、T4连接酶基因和家蚕丝素蛋白轻链基因3’末端的表达框,即Fibroin L chain Promoter-18s rRNA promoter-Fibroin L chainsignal peptide-His 6-DDDDK-T4 Ligase- Fibroin L chain PolyA;
所述的piggy-10004质粒是利用双启动子Fibroin L chain Promoter和18s rRNApromoter驱动T4连接酶基因的表达。
2.权利要求1所述的一种用于表达T4连接酶的家蚕后部丝腺生物反应器双启动子通用型质粒的制备方法,其特征在于包括以下步骤:
1)以带有ApaI酶切位点序列的T4 连接酶5’端序列和带有NheI酶切位点序列的T4 连接酶3’端序列为引物,以包含A3基因启动子-绿色荧光蛋白基因-SV40-丝胶蛋白1基因启动子-T4 连接酶基因-SV40的piggy-10522质粒为模板,piggy-10522质粒的碱基序列如SEQID NO.2,获得长度为1536bp的T4 连接酶基因;
2)用ApaI和NheI双酶切piggy-8480质粒,piggy-8480质粒的碱基序列如SEQ ID NO.4,得到长度为8478bp的包含[A3-EGFP-SV40]-[FL692 promoter-18s rRNA promoter-FLSP-His6-DDDDK-FLPA 和Amp抗性基因的基因序列;
3)连接步骤1)得到的T4 连接酶基因和步骤2)得到的基因序列,得到含有[A3-EGFP-SV40]-[FL692 promoter-18s rRNA -promoter-FLSP-His6-DDDDK-T4 Ligase-FLPA] 和Amp抗性基因的piggy-10004质粒。
3.根据权利要求1所述通用型质粒的应用,其特征在于:用于T4连接酶表达的应用。
4.一种用于表达T4连接酶的家蚕后部丝腺生物反应器双启动子通用型质粒表达T4 连接酶的方法,其特征在于该方法的步骤如下:
(1)采用显微注射的方法,将权利要求1所述piggy-10004质粒及能够提供piggyBac转座酶的辅助质粒按浓度比1:1的比例导入家蚕产卵后6小时以内的受精卵内,利用piggy-10004质粒中的piggyBac转座子将T4连接酶基因插入到家蚕基因组内;
(2)蚕卵孵化后饲养至成虫,然后与非转基因家蚕交配制种续代,此代为G1代,在G1代蚕一龄第二天,通过荧光体视显微镜观察筛选体色表达绿色荧光EGFP标记基因的转基因家蚕,饲养至成虫再与非转基因家蚕交配制种续代成为G2代;
(3)G2代家蚕采用单蛾育,筛选出荧光体视显微镜下表达绿色荧光EGFP标记基因的家蚕,采用同蛾区蚕蛾相互交配制成G3代;
(4)G3代家蚕采用单蛾育,同蛾区表达绿色荧光EGFP标记基因的蚕蛾相互交配,制成G4代;
(5)从G4代开始并经连续3代进行选择和交配,育成绿色荧光基因和T4 连接酶基因纯合、丝腺细胞能够合成分泌T4连接酶的转基因家蚕;
(6)通过家蚕丝腺细胞合成分泌T4连接酶,并随家蚕吐丝结茧行为进入蚕茧。
5.根据权利要求4所述的一种用于表达T4连接酶的家蚕后部丝腺生物反应器双启动子通用型质粒表达T4 连接酶的方法,其特征在于:所述步骤(5)中连续3代进行选择和交配具体是采用绿色荧光表型纯一的蛾区饲养、单蛾育和同蛾区蚕蛾交配的方式。
6.根据权利要求4所述的一种用于表达T4连接酶的家蚕后部丝腺生物反应器双启动子通用型质粒表达T4 连接酶的方法,其特征在于:所述的piggy-10004质粒是利用双启动子Fibroin L chain Promoter和18s rRNA promoter驱动T4连接酶基因的表达。
7.根据权利要求4所述的一种用于表达T4连接酶的家蚕后部丝腺生物反应器双启动子通用型质粒表达T4 连接酶的方法,其特征在于:所述步骤(6)的T4连接酶基因在家蚕丝腺细胞特异表达,在家蚕丝素蛋白轻链信号肽的作用下分泌到丝腺腺腔,并随吐丝行为分泌到蚕茧。
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CN104278032A (zh) * | 2014-02-14 | 2015-01-14 | 上海美百瑞生物医药技术有限公司 | 一种新型双启动子结构单元 |
CN104593413A (zh) * | 2014-12-31 | 2015-05-06 | 浙江大学 | 利用家蚕后部丝腺合成分泌人血清白蛋白的方法 |
CN104846011A (zh) * | 2015-03-18 | 2015-08-19 | 浙江大学 | 利用家蚕后部丝腺合成分泌蜂王浆主蛋白1的方法 |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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CN104278032A (zh) * | 2014-02-14 | 2015-01-14 | 上海美百瑞生物医药技术有限公司 | 一种新型双启动子结构单元 |
CN104593413A (zh) * | 2014-12-31 | 2015-05-06 | 浙江大学 | 利用家蚕后部丝腺合成分泌人血清白蛋白的方法 |
CN104846011A (zh) * | 2015-03-18 | 2015-08-19 | 浙江大学 | 利用家蚕后部丝腺合成分泌蜂王浆主蛋白1的方法 |
Non-Patent Citations (1)
Title |
---|
CMV与SP双启动子增强外源基因在小鼠骨骼肌中的表达效率;章倩倩等;《中国生物化学与分子生物学报》;20070731;第23卷(第7期);摘要,第555页左栏第三段 * |
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