CN113150104A - 特异性靶向鱼类卵细胞运输的蛋白及其制备方法和应用 - Google Patents
特异性靶向鱼类卵细胞运输的蛋白及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种特异性靶向鱼类卵细胞运输的蛋白,其氨基酸序列如SEQ ID NO:2所示。该蛋白可携带其他多肽(含荧光蛋白)物质靶向运输到卵细胞,进而实现卵细胞的靶向标记;其具有精准度高、毒性小、价格低廉、标记耗时短等优点,适用于鱼类卵巢卵子等相关研究。该蛋白的制备方法包括如下步骤:将特异性靶向鱼类卵细胞运输的蛋白的编码基因克隆至蛋白表达载体中,再将构建成功的蛋白表达载体质粒转入大肠杆菌中进行诱导表达,最终得到特异性靶向鱼类卵细胞运输的蛋白。该蛋白的应用方法包括如下步骤:将特异性靶向鱼类卵细胞运输的蛋白对雌性鱼类进行注射,使该蛋白进入卵子。本发明的制备方法和应用方法,操作简单,耗时短。
Description
技术领域
本发明属于生物技术领域,涉及一种特异性靶向鱼类卵细胞运输的蛋白及其制备方法和应用。
背景技术
目前,针对鱼类卵细胞的标记方法主要采用非特异性荧光染色、免疫荧光抗体标记以及制备Oct4-EGFP转基因鱼等方法,虽然具有一定的优点,但仍然存在不足之处,譬如:欠精准、有毒性、价格昂贵、耗时长等。因此,开发一种精准度高、毒性小、价格低廉,且标记耗时短的标记方法,对本领域具有十分重要的意义。
发明内容
本发明所要解决的技术问题是,克服以上背景技术中提到的不足和缺陷,通过建立特异性识别序列,提供一种可将其他多肽(含荧光蛋白)等物质靶向运输到卵细胞的特异性靶向鱼类卵细胞运输的蛋白,以及其制备方法和应用。
为解决上述技术问题,本发明提出的技术方案为:
一种特异性靶向鱼类卵细胞运输的蛋白,其氨基酸序列如SEQIDNO:2所示。
上述特异性靶向鱼类卵细胞运输的蛋白,优选的,所述特异性靶向鱼类卵细胞运输的蛋白的编码基因核苷酸序列如SEQIDNO:1所示。
优选的,还携带改良型荧光报告蛋白,所述改良型荧光报告蛋白的编码基因核苷酸序列如SEQIDNO:3所示,氨基酸序列如SEQIDNO:4所示;携带绿色荧光蛋白的特异性靶向鱼类卵细胞运输的蛋白,其氨基酸序列如SEQIDNO:8所示,其编码基因核苷酸序列如SEQIDNO:7所示。
基于一个总的发明构思,本发明还提供一种所述特异性靶向鱼类卵细胞运输的蛋白的制备方法,包括如下步骤:将所述特异性靶向鱼类卵细胞运输的蛋白的编码基因连接至蛋白表达载体中,再将构建成功的蛋白表达载体质粒转入大肠杆菌中进行诱导表达,最终得到所述特异性靶向鱼类卵细胞运输的蛋白。
上述的制备方法,优选的,所述特异性靶向鱼类卵细胞运输的蛋白携带改良型荧光报告蛋白时,其制备方法包括如下步骤:将所述特异性靶向鱼类卵细胞运输的蛋白的编码基因与改良型荧光报告蛋白的编码基因,通过Link序列连接到同一蛋白表达载体中,再将构建成功的蛋白表达载体质粒转入大肠杆菌中进行诱导表达,最终得到携带改良型荧光报告蛋白的特异性靶向鱼类卵细胞运输的蛋白。
优选的,所述诱导表达具体包括如下步骤:将构建成功的蛋白表达载体质粒转入大肠杆菌中,摇菌至菌液OD600值位于0.6~0.8,加终浓度为0.5~1mM的IPTG进行诱导表达,转速为140~180rpm,温度为16~25℃,6~12h后收取菌液,离心得到菌体沉淀,超声破碎后过滤,进行蛋白纯化,最终得到蛋白。
优选的,所述Link序列的核苷酸序列如SEQIDNO:5所示,氨基酸序列如SEQIDNO:6所示。
基于一个总的发明构思,本发明还提供所述特异性靶向鱼类卵细胞运输的蛋白的应用,其应用方法包括如下步骤:将所述特异性靶向鱼类卵细胞运输的蛋白对雌性鱼类进行注射,使该蛋白进入卵子。
上述的应用,优选的,所述注射的方式包括静脉注射、眼眶注射或眼泡注射,所述鱼类包括斑马鱼、鲤、鲫或金鱼。
优选的,所述特异性靶向鱼类卵细胞运输的蛋白还携带包括基因敲除在内的基因编辑体系。
与现有技术相比,本发明的有益效果为:
1、本发明的特异性靶向鱼类卵细胞运输的蛋白,可携带其他多肽(含荧光蛋白)等物质靶向运输到卵细胞,进而实现卵细胞的靶向标记;其具有精准度高、毒性小、价格低廉、标记耗时短等优点,适用于鱼类卵巢卵子等相关研究。
2、本发明的制备方法和应用方法,操作简单,耗时短。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是实施例3中利用特异性靶向蛋白序列+增强型绿色荧光蛋白序列表达融合蛋白进入金鱼卵细胞图;
图2是实施例4中利用特异性靶向蛋白序列+增强型绿色荧光蛋白序列表达融合蛋白进入鲤卵细胞图。
具体实施方式
为了便于理解本发明,下文将结合说明书附图和较佳的实施例对本发明做更全面、细致地描述,但本发明的保护范围并不限于以下具体实施例。
除非另有定义,下文中所使用的所有专业术语与本领域技术人员通常理解含义相同。本文中所使用的专业术语只是为了描述具体实施例的目的,并不是旨在限制本发明的保护范围。
除非另有特别说明,本发明中用到的各种原材料、试剂、仪器和设备等均可通过市场购买得到或者可通过现有方法制备得到。
实施例1:
一种本发明的特异性靶向鱼类卵细胞运输的蛋白,其氨基酸序列如SEQIDNO:2所示,其编码基因核苷酸序列如SEQIDNO:1所示。
该特异性靶向鱼类卵细胞运输的蛋白的制备方法,包括如下步骤:
(1)构建蛋白表达载体:将所述特异性靶向鱼类卵细胞运输的蛋白的编码基因连接至蛋白表达载体中,具体步骤为:分析多种鱼类卵黄蛋白原序列,分析得到其特异性识别区域,采用高保真RT-PCR从雌性斑马鱼肝脏的RNA材料中得到包含该序列的核酸序列,对序列进行密码子分析,同义突变以保证诱导能够顺利进行,将同义突变成功后的序列(如SEQIDNO:1所示)通过连接酶或者同源重组的方法连接至原核表达载体pET30a上;
(2)蛋白表达与纯化:将构建成功的蛋白表达载体质粒转入大肠杆菌,摇菌至菌液OD600值位于0.6-0.8区间内,加IPTG(终浓度0.5~1mM)诱导表达,转速为140rpm,温度为16~25℃,6~12h后收取菌液,离心得到菌体沉淀,超声破碎后使用0.22μm孔径过滤器过滤后,进行蛋白纯化,最终得到具有活性的高浓度的特异性靶向蛋白。
实施例2:
一种携带绿色荧光蛋白的特异性靶向鱼类卵细胞运输的蛋白,其氨基酸序列如SEQID NO:8所示,其编码基因核苷酸序列如SEQIDNO:7所示。
该特异性靶向鱼类卵细胞运输的蛋白的制备方法,包括如下步骤:
(1)构建融合蛋白表达载体:将特异性靶向鱼类卵细胞运输的蛋白的核苷酸序列(如SEQIDNO:1所示)与绿色荧光蛋白核苷酸序列(如SEQIDNO:3所示);将上述两序列通过Link序列(核苷酸序列如SEQIDNO:5所示)采用同源重组的方式连接到同一蛋白表达载体(pET30a或pET28a)上,确保两者能够融合表达并正确折叠(核苷酸序列如SEQID NO:7所示);
(2)融合蛋白表达与纯化:将构建成功的融合蛋白表达载体质粒转入大肠杆菌,摇菌至菌液OD600值位于0.6-0.8区间内,加IPTG(终浓度0.5~1mM)诱导表达,转速为140~180rpm,温度为16~25℃,6~12h后收取菌液,离心得到菌体沉淀,超声破碎后使用0.22μm孔径过滤器过滤后,进行蛋白纯化,最终得到具有活性的高浓度的特异性靶向蛋白-绿色荧光融合蛋白,其氨基酸序列如SEQIDNO:8所示。
实施例3:特异性靶向蛋白-绿色荧光融合蛋白靶向进入金鱼卵细胞
实施例2得到的特异性靶向蛋白-绿色荧光融合蛋白靶向进入金鱼卵细胞的应用,包括如下步骤:
(1)雌性眼泡金鱼选取:选择已达性成熟、健壮的雌性眼泡金鱼;
(2)多次眼部注射:MS222麻醉后,采用胰岛素注射针(规格:U40,32G,1mL)眼泡部位注射100~200μL纯化后的融合蛋白(蛋白浓度为1~5mg/mL);
(3)解剖观察:注射后的3~7天,经麻醉,取性腺,在体式荧光显微镜下观察(见图1)。
由图1可知,本发明所述的利用特异性靶向蛋白-绿色荧光融合蛋白对雌性眼泡金鱼进行注射,可携带绿色荧光蛋白进入卵子,适用于鱼类卵巢卵子等相关研究。
实施例4:特异性靶向蛋白-绿色荧光融合蛋白靶向进入鲤卵细胞
实施例2得到的特异性靶向蛋白-绿色荧光融合蛋白靶向进入鲤鱼卵细胞的应用,包括如下步骤:
(1)雌性鲤鱼选取;选择已达性成熟、健壮的雌性鲤鱼;
(2)多次静脉注射:采用一次性注射器(规格1mL)静脉注射500μL纯化后的融合蛋白(蛋白浓度为1~5mg/mL),注射2~3次,间隔时间2~3天;
(3)解剖观察:注射后的3~7天,经麻醉,取性腺,在体式荧光显微镜下观察(见图2)。
由图2可知,本发明所述的利用特异性靶向蛋白-绿色荧光融合蛋白对雌性鲤鱼进行注射,可携带绿色荧光蛋白进入卵子,适用于鱼类卵巢卵子等相关研究。
序列表
<110> 湖南师范大学
<120> 特异性靶向鱼类卵细胞运输的蛋白及其制备方法和应用
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Claims (10)
1.一种特异性靶向鱼类卵细胞运输的蛋白,其特征在于,其氨基酸序列如SEQ ID NO:2所示。
2.根据权利要求1所述特异性靶向鱼类卵细胞运输的蛋白,其特征在于,所述特异性靶向鱼类卵细胞运输的蛋白的编码基因核苷酸序列如SEQ ID NO:1所示。
3.根据权利要求1所述特异性靶向鱼类卵细胞运输的蛋白,其特征在于,还携带改良型荧光报告蛋白,携带荧光蛋白的特异性靶向鱼类卵细胞运输的蛋白的氨基酸序列如SEQ IDNO:8所示。
4.一种如权利要求1-3中任一项所述特异性靶向鱼类卵细胞运输的蛋白的制备方法,其特征在于,包括如下步骤:将所述特异性靶向鱼类卵细胞运输的蛋白的编码基因连接至蛋白表达载体中,再将构建成功的蛋白表达载体质粒转入大肠杆菌中进行诱导表达,最终得到所述特异性靶向鱼类卵细胞运输的蛋白。
5.根据权利要求4所述的制备方法,其特征在于,所述特异性靶向鱼类卵细胞运输的蛋白携带改良型荧光报告蛋白时,其制备方法包括如下步骤:将所述特异性靶向鱼类卵细胞运输的蛋白的编码基因与改良型荧光报告蛋白的编码基因,通过Link序列连接到同一蛋白表达载体中,再将构建成功的蛋白表达载体质粒转入大肠杆菌中进行诱导表达,最终得到携带改良型荧光报告蛋白的特异性靶向鱼类卵细胞运输的蛋白。
6.根据权利要求4或5所述的制备方法,其特征在于,所述诱导表达具体包括如下步骤:将构建成功的蛋白表达载体质粒转入大肠杆菌中,摇菌至菌液OD600值位于0.6~0.8,加终浓度为0.5~1mM的IPTG进行诱导表达,转速为140~180rpm,温度为16~25℃,6~12h后收取菌液,离心得到菌体沉淀,超声破碎后过滤,进行蛋白纯化,最终得到蛋白。
7.根据权利要求5所述的制备方法,其特征在于,所述Link序列的核苷酸序列如SEQ IDNO:5所示,氨基酸序列如SEQ ID NO:6所示。
8.一种如权利要求1-3中任一项所述或由权利要求4-7中任一项所述的制备方法制备的特异性靶向鱼类卵细胞运输的蛋白的应用,其特征在于,其应用方法包括如下步骤:将所述特异性靶向鱼类卵细胞运输的蛋白对雌性鱼类进行注射,使该蛋白进入卵子。
9.根据权利要求8所述的应用,其特征在于,所述注射的方式包括静脉注射、眼眶注射或眼泡注射,所述鱼类包括斑马鱼、鲤、鲫或金鱼。
10.根据权利要求8或9所述的应用,其特征在于,所述特异性靶向鱼类卵细胞运输的蛋白还携带包括基因敲除在内的基因编辑体系。
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CN103333812A (zh) * | 2013-06-09 | 2013-10-02 | 北京市水产科学研究所 | 表达鲟鱼卵母细胞成熟相关蛋白的重组巴斯德毕赤酵母 |
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CN103333812A (zh) * | 2013-06-09 | 2013-10-02 | 北京市水产科学研究所 | 表达鲟鱼卵母细胞成熟相关蛋白的重组巴斯德毕赤酵母 |
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