CN110041436A - 长效重组fsh融合蛋白在母猪批次化生产中的应用 - Google Patents
长效重组fsh融合蛋白在母猪批次化生产中的应用 Download PDFInfo
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Abstract
本发明公开了长效重组FSH融合蛋白在母猪批次化生产中的应用。所述长效重组FSH融合蛋白包括人促卵泡素Fc融合蛋白(hFSH‑Fc)、猪促卵泡素Fc融合蛋白(pFSH‑Fc)、人促卵泡素CTP融合蛋白(hFSH‑CTP)和猪促卵泡素CTP融合蛋白(pFSH‑CTP)。所述重组FSH融合蛋白可显著延长蛋白的体内半衰期,将其用于经产母猪和后备母猪中,可诱导卵泡同期发育,将所述长效重组FSH融合蛋白用于母猪批次化生产中能够显著促进母猪同期发情,还能显著提高后备和经产母猪的发情率、总受胎率、总分娩率、窝均产仔数,显著提高母猪繁殖性能。
Description
技术领域
本发明涉及分子生物学和兽药领域。更具体地,本发明涉及长效重组FSH融合蛋白在母猪批次化生产中的应用。
背景技术
随着中国经济发展,人民生活水平提高,对肉的需求也越来越大,养殖业由此迅猛发展,我国是养猪大国,据中国统计年鉴数据显示,至2017年12月,国内有超过3427万头存栏母猪。
由于母猪的繁殖周期不一致等因素,养殖厂通常需要大量的饲养畜舍和设施以及人力物力来保证繁殖需求,导致养殖成本居高不下,进而限制了畜牧业的大规模集约化养殖。至少50年前研究人员就试图调控猪的发情周期,以期能做到猪场繁殖的设施及人员利用最大化。同期发情主要是借助外源激素剌激卵巢,使其按照预定的要求发生变化,使处理母畜的卵巢生理机能都处于相同阶段,从而达到同期发情。对于母猪来说,同期发情可使母猪分娩时间集中,便于母猪集中断奶,全进全出、进入下一个繁殖周期,有效地提高母猪的繁殖率,减少病原的传播,利于规模化猪场实行严格的防疫制度和保证生物安全体系。
目前我国猪场大多仍采用传统的连续性生产管理模式,导致繁殖效率低、疾病防控难的问题,而国际上,养猪行业通常采用批次化生产的形式提高繁殖效率、实现疫病防控和健康养殖。
母猪批次化生产是围绕养猪生产由现有的生产模式到不同周批次的生产模式转变,并对不同生产模式下的繁殖生产同期化和批次化的关键工艺技术方案进行系统研究与应用。母猪批次化生产是养猪业发展的必然趋势,它是利用繁殖调控技术,精确控制母猪同期发情、同期排卵、同期配种和同期分娩等环节,按计划组织批次生产,是一种母猪繁殖的高效技术管理体系。常见的批次化模式有1周批、2周批、3周批和4周批等。其中,3周批和4周批模式对母猪、栏舍以及人工的综合利用效率更高,养殖工人的工作时间安排也更为合理和人性化。批次化繁殖管理能使猪场做到真正意义上的全进全出,更好的控制疾病传播,提高母畜的利用率、缩短母畜的非生产天数(NPD),大大提高畜牧业经济效益,尤其是对当前养猪生产造成重大危害的猪繁殖与呼吸障碍综合征(PRRS)、伪狂犬、猪流行性腹泻(PED)等劣性传染病,具有很好的防控作用。
目前,批次化生产在欧美等国应用广泛,其中德式(德国、东欧等国)母猪批次化生产技术更加适合中国现有母猪养殖模式,国内的母猪批次化生产多借鉴德式生产系统并结合实际加以改良。在母猪批次化生产的技术系统中,包括了定时输精技术、同期分娩技术及整体的栏舍规划设计等多个部分。其中,批次化生产的关键核心技术是定时输精,它是以母猪的同期发情、同期排卵为基础的一项繁殖新技术,可精确的控制母猪同期发情、同期排卵和同期配种。母猪的发情、排卵主要是由体内促卵泡素(FSH)和促黄体素(LH)等的生殖激素精确调控,因此,必须通过促进卵泡发育和排卵的激素处理调控母猪同期发情、同期排卵,才能够实现定时输精和批次化生产。
批次化生产的流程描述:批次化生产是猪场由传统模式到批次化生产模式的转变,首先应改善猪场现有状况,包括考虑现有栏舍是否满足批次化的需求、确定生产批次等,以达到实施批次化生产管理的基本条件;以经产母猪为基础母猪群,按照每批次生产的数量进行后备母猪补充,导入批次化生产,主要通过定时输精技术和分娩同期化技术,达到同期发情、同期排卵、同期配种和同期分娩的目的。传统猪场转为批次化生产猪场具体包括以下几个步骤:
(1)栏舍设计:统计传统猪舍产床和定位栏的数量。
(2)确定批次:根据栏舍的情况将母猪进行分批,确定生产的批次。
(3)猪群调整:根据每批生产的需要,对经产母猪同期断奶和后备母猪同期发情进行调整。
(4)定时输精:定时输精是批次化生产关键步骤,通过外源促性腺激素(gonadotropins)(现常用的产品为孕马血清促性腺激素,Pregnant Mare SerumGonadotropin,简称PMSG)的处理,可促进母猪卵泡发育的同步化,从而控制母猪定时同期发情。再通过促排卵激素(如促性腺激素释放激素GnRH)的调节,可使母猪排卵同步化,从而实现定时输精。效果稳定的生殖激素产品是保障定时输精同步化效果的关键。
(5)同期分娩:母猪经过妊娠期后,利用氯前列醇钠PGc使每一批的母猪能够在同一时间段内分娩完成。
其中步骤(1)-(3)为猪场前期管理的调整,步骤(4)-(5)为批次化关键技术步骤,具体参见图1。
目前我国母猪批次化繁殖的研发与应用尚处于起步阶段,主要问题在于缺乏质量稳定的诱导发情和卵泡发育的关键激素产品。目前常用的关键激素产品PMSG为生化提取来源,从怀孕母马的血液中提取,受原料来源限制导致成本高,同时由于原料成分复杂和纯化难度大,存在纯度和效价低、质量不稳定等问题,在实际应用过程中不能满足母猪繁殖生产的需求,成为制约母猪批次化繁殖技术的推广应用的瓶颈问题。
具体来讲,PMSG是一种糖蛋白激素,由孕马的子宫内膜杯状结构分泌,妊娠40-170天存在,兼有FSH(促卵泡激素)和LH(黄体生成素)双重活性。PMSG半衰期较长,达到40-120小时左右,单次注射就能满足母猪诱导发情的需求,目前是母猪繁殖管理的常用产品。但是,其原料怀孕母马血清极其稀缺且成本昂贵,只有在母马怀孕的第60-170天之间才能采集到血清原料,集中采血后必须建立冷库冰冻保存,成本居高不下,产品价格昂贵;且怀孕母马的数量日益减少,成本日益增加,使养殖户的养殖成本及经济负担大大增加。另外,因为PMSG分子含有较高的己糖和唾液酸,在动物体内半衰期太长,用于动物超数排卵时,容易引发母畜的多种不良效应,如卵巢囊肿,妊娠初期胚胎的提前退化。此外,从怀孕母马采集血清制备PMSG,经常因采血过多导致母马流产、胎马死亡,出于动物保护和伦理,欧洲大型养殖业已不再使用孕马血清。同时,由于血清原料成分复杂、纯化难度极大,提取后的产品存在纯度和效价低、质量不稳定等问题,实际使用过程中也出现药效不稳定的现象,用于其它动物时会产生抗体,存在一定的免疫原性,不能长时间使用;另一方面,PMSG天然含有促卵泡素(FSH)和促黄体素(LH)两种活性成分,且比例固定,无法调节两种活性的比例,限制了其应用范围。
PMSG在雌性动物体内作用于卵巢的靶细胞受体,包括FSH受体和LH受体。FSH对于雌性动物作用于卵巢,促进卵泡的发育成熟排卵及黄体生成,LH促进雌性动物排卵。基于PMSG、FSH和LH的作用机制,可以看出通过直接使用FSH和LH的效果比使用PMSG的效果更加显著。
促卵泡素(FSH)在促进母畜发情和繁殖方面具有PMSG相似功效,其药理作用为通过促进母畜卵巢卵泡的生长,促进卵泡颗粒细胞的增生和雌激素合成与分泌,不断升高的雌激素作用于大脑皮层,在卵泡发育成熟排卵的同时引起母畜发情。现有上市的FSH为垂体FSH,从猪垂体中提取获得,其半衰期大约是6小时左右,母猪卵泡发育周期约为3-4天,且在该过程中需要促卵泡素的持续刺激,使用垂体FSH需要进行多次注射,操作复杂,成本较高,不能满足定时输精和批次化生产的需求,且对动物有较大的应激。因此,目前垂体FSH只是在牛羊的育种超数排卵方面进行应用,而没有用于促进母猪卵泡发育和诱导发情。目前市场上极少使用垂体FSH产品。
在人用药物中,随着技术的进步,通过哺乳动物细胞表达和生产的重组蛋白药物已经得到广泛应用,与生化提取产品比较,其具有原料来源稳定、降低了病毒污染风险、更加安全有效且成本显著降低等显著优势。
其中,中国仓鼠卵巢细胞(Chinese Hamster Ovary Cell,CHO)是用于真核生物外源基因表达最为成功的宿主细胞,已有越来越多的药用蛋白在其中获得了高效表达,很多人用重组蛋白药物已上市。与其它表达系统相比,该系统具有许多优点,如拥有完备的翻译后加工过程,包括糖基化、羟基化,使表达的外源真核基因产物能够保持其天然结构及活性,且使表达产物分泌到胞外,有利于外源蛋白的分离纯化。
不同哺乳动物种属之间的FSH蛋白的氨基酸序列同源性很高,例如,人FSHα链和β链与猪FSHα链和β链的氨基酸序列同源性分别为83%和96%,人FSH与牛FSH的氨基酸序列同源性高达88%,提示不同来源的hFSH在其他哺乳动物繁殖领域的潜在应用。目前已有利用CHO细胞生产的人用药物重组hFSH(Human follicle-stimulating hormone,简称hFSH)上市,但仍然存在如下问题:首先,现有方法生产的重组hFSH表达量太低,制备过程复杂,生产成本太高;其次,其半衰期短,需要频繁注射给药。但重组蛋白药物在国内兽药领域基本为空白,目前兽药市场上只有一种非疫苗重组蛋白药物,是使用大肠杆菌进行生产。因此,利用分子生物学和细胞培养方法,开发具有生物活性和更长半衰期的FSH药物是本领域的一个挑战。
更为重要的是,目前还未见长效重组FSH融合蛋白在母猪批次化生产中应用的相关报导。
发明内容
发明要解决的问题
本发明旨在提供一系列长效重组人促卵泡素(Human follicle-stimulatinghormone,简称hFSH)融合蛋白和长效重组猪促卵泡素(Porcine follicle-stimulatinghormone,简称pFSH)融合蛋白在母猪批次化生产中的应用。
用于解决问题的方案
本发明人鉴于上述现有技术中存在的问题,进行了深入地研究,发现通过Fc融合、CTP等长效化手段对FSH进行修饰,借助哺乳动物细胞进行表达生产,并将制备得到的重组FSH融合蛋白应用于母猪的批次化生产中,从而完成了本发明。即,本发明如下所述:
本发明提供了重组FSH融合蛋白在母猪批次化生产中的应用,其特征在于,所述融合蛋白任选自:
(i)重组hFSH-Fc融合蛋白的氨基酸序列从N端到C端依次包含信号肽、hFSHβ亚基、CTP、hFSHα亚基、肽接头和Fc,其中所述Fc为IgG2-hFc变体或IgG1-hFc变体;或,
(ii)重组hFSH-Fc融合蛋白的氨基酸序列从N端到C端依次包含信号肽、pFSHβ亚基、CTP、pFSHα亚基、肽接头和Fc,其中所述IgG Fc变体为pFc、IgG2-hFc变体或IgG1-hFc变体中的任一种;或,
(iii)重组hFSH-CTP融合蛋白由hFSHα亚基和hFSHβ-CTP亚基组成,其中hFSHβ-CTP亚基的氨基酸序列从N端到C端包含hFSHβ亚基和CTP;或,
(iv)重组pFSH-CTP融合蛋白由pFSHα亚基和pFSHβ-CTP亚基组成,其中pFSHβ-CTP亚基的氨基酸序列从N端到C端依次包含pFSHβ亚基和CTP。
其中(i)和(ii)中所述的肽接头含有2-20个氨基酸,所述肽接头存在于hFSHα亚基和IgG Fc变体之间,且肽接头含有两个或更多选自甘氨酸、丝氨酸、丙氨酸和苏氨酸的氨基酸;优选的,所述肽接头的氨基酸序列为GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer。
在一些优选的实施方案中,上文所述(i)和(ii)中所述的CTP的氨基酸序列为来自HCGβ链羧酸基末端的33个氨基酸残基序列,如SEQ ID NO:20所示;其中(iii)和(iv)中所述的CTP的氨基酸序列为来自hCGβ链羧基末端的33个氨基酸残基,如SEQ ID NO:20所示。其中,SEQ ID NO:20所示的氨基酸序列由SEQ ID NO:19所示的核苷酸序列编码。
在进一步优选的实施方案中,其中(i)中所述融合蛋白的氨基酸序列如SEQ IDNO:2或SEQ ID NO:4所示;其中(ii)中所述融合蛋白的氨基酸序列如SEQ ID NO:6、SEQ IDNO:8或SEQ ID NO:10所示;其中(iii)中所述融合蛋白中包含的hFSHβ-CTP亚基的氨基酸序列如SEQ ID NO:12所示,所述融合蛋白中包含的hFSHα亚基的氨基酸序列如SEQ ID NO:14所示;其中(iv)中所述融合蛋白中包含的pFSHβ-CTP亚基的氨基酸序列如SEQ ID NO:16所示,所述融合蛋白中包含的pFSHα亚基的氨基酸序列如SEQ ID NO:18所示。
在具体的实施方案中,所述母猪为经产母猪或后备母猪。
本发明还提供了上文所述的重组FSH融合蛋白的制备方法,其特征在于:
(1)当所述重组FSH融合蛋白为(i)或(ii)时,其步骤包括:
a)构建编码重组hFSH-Fc或pFSH-Fc融合蛋白的基因表达载体
采用人工合成方法获得编码人hFSH-Fc或pFSH-Fc融合蛋白的基因,插入到哺乳动物细胞表达载体,获得含有hFSH-Fc或pFSH-Fc融合蛋白基因的表达质粒;
b)重组hFSH-Fc或pFSH-Fc融合蛋白在哺乳动物宿主细胞中的稳定表达
将含有hFSH-Fc或pFSH-Fc融合蛋白的表达载体转染到哺乳动物宿主细胞,筛选稳定表达hFSH-Fc或pFSH-Fc融合的细胞株;
c)高密度细胞培养重组hFSH-Fc或pFSH-Fc融合蛋白
d)重组hFSH-Fc或pFSH-Fc融合蛋白的纯化:包括使用Protein A亲和层析和疏水层析柱进行纯化,其中所述的疏水柱包括Butyl Sepharose 4 Fast Flow、OctylSepharose 4 Fast Flow、Phenyl Sepharose 6 Fast Flow、Butyl-S Sepharose 6 FastFlow、Butyl Sepharose 4B、Octyl Sepharose CL-4B、Phenyl Sepharose CL-4B,优选为Phenyl Sepharose 6Fast Flow;
(2)当所述重组FSH融合蛋白为(iii)或(iv)时,其步骤包括:
a)构建编码重组hFSH-CTP或pFSH-CTP融合蛋白的基因表达载体
采用人工合成方法获得编码人hFSH-CTP或pFSH-CTP融合蛋白的基因,插入到哺乳动物细胞表达载体,获得含有hFSH-CTP或pFSH-CTP融合蛋白基因的表达质粒;
b)重组hFSH-CTP或pFSH-CTP融合蛋白在哺乳动物宿主细胞中的稳定表达
将含有hFSH-CTP或pFSH-CTP融合蛋白的表达载体转染到哺乳动物宿主细胞,筛选稳定表达hFSH-CTP或pFSH-CTP融合的细胞株;
c)高密度细胞培养重组hFSH-CTP或pFSH-CTP融合蛋白;
d)重组hFSH-CTP或pFSH-CTP融合蛋白的纯化:包括使用分段硫酸铵沉淀、沉淀复溶及超滤换液、阴离子柱层析和疏水柱层析进行纯化。
在优选的实施方案中,上文所述制备方法中所述的基因表达载体为pCDNA3、pCMV/ZEO、pIRES、pDR、pBK、pSPORT或pCMV-DHFR,优选为pCDNA3,更优选为经过基因改造后的pCDNA3;其中所述的细胞转染方法包括电穿孔转染方法、磷酸钙转染、脂质体转染和原生质融合,优选为电穿孔转染方法;其中所述的哺乳动物宿主细胞包括CHO、HEK293、BHK、NS0和Sp2/0细胞,优选为CHO细胞,更优选为DHFR酶缺陷型CHO悬浮细胞(CHO DHFR-)。
发明的效果
本发明提供的长效重组促卵泡素(FSH)融合蛋白由哺乳细胞动物表达生产,并通过Fc融合、CTP等长效化手段延长其半衰期,具有促进卵泡发育,调控母猪同期发情的生理作用,从而实现母猪的批次化生产。与现有批次化生产中最常用的从怀孕马血清中生化提取的PMSG等产品相比,效价和纯度高,且具有产量高、成本可控、无病毒感染等优点,本发明提供的FSH融合蛋白仅有FSH单一活性成分,能与HCG灵活配比使用,应用范围更广,其可取代PMSG用于母猪批次化生产,对提高母猪繁殖效率和养殖水平具有重要意义。
具体来说,本发明提供的长效重组FSH融合蛋白具有以下优异效果:
1.本发明提供的重组FSH融合蛋白可显著延长蛋白的体内半衰期。其中,重组FSH-Fc融合蛋白是猪垂体FSH半衰期的约15倍;重组FSH-CTP融合蛋白的半衰期是猪垂体FSH的5倍以上。
2.与目前批次化生产中最常用的具有同等效果的PMSG进行比较,本发明提供的长效重组FSH融合蛋白用于母猪批次化生产能够显著提高后备母猪和经产母猪的发情率、总受胎率、、窝均产仔数以及显著缩短非生产天数。将所述融合蛋白用于母猪批次化生产,预计可视母猪的PSY(每头母猪年断奶仔猪头数)提高至少1.0头以上,从而大大提高我国养猪业新增经济效益。
附图说明
图1示出了母猪批次化生产流程图。
图2a示出了hFSH-Fc和pFSH-Fc单链的结构示意图,图2b示出了hFSH-Fc和pFSH-Fc二聚化的结构示意图。
图3a和图3b分别示出了所构建的编码hFSH-vIgG2-hFc和hFSH-vIgG1-hFc融合蛋白的真核表达质粒图谱。
图4a、图4b和图4c示出了所构建的编码pFSH-pFc、pFSH-vIgG2-hFc和pFSH-vIgG1-hFc融合蛋白的真核表达质粒图谱。
图5示出了重组融合蛋白hFSH-vIgG2-hFc、hFSH-vIgG1-hFc和猪垂体FSH的SDS-PAGE电泳图。其中,重组融合蛋白hFSH-vIgG2-hFc、hFSH-vIgG1-hFc和猪垂体FSH分别位于电泳图的第1、第2和第3泳道,第4泳道为Marker。
图6示出了重组融合蛋白hFSH-vIgG2-hFc和hFSH-vIgG1-hFc的SEC-HPLC图谱。其中,1为hFSH-vIgG1-hFc的SEC-HPLC图谱,2为hFSH-vIgG2-hFc的SEC-HPLC图谱。
图7示出了猪垂体FSH和重组融合蛋白pFSH-pFc、pFSH-vIgG2-hFc和pFSH-vIgG1-hFc的SDS-PAGE电泳图。其中,猪垂体FSH和重组融合蛋白pFSH-pFc、pFSH-vIgG2-hFc、pFSH-vIgG1-hFc分别位于电泳图的第1泳道和第3-5泳道,第2泳道为Marker。
图8示出了重组融合蛋白pFSH-pFc、pFSH-vIgG2-hFc和pFSH-vIgG1-hFc的SEC-HPLC图谱。其中,1为pFSH-vIgG2-hFc的SEC-HPLC图谱,2为pFSH-pFc的SEC-HPLC图谱,3为pFSH-vIgG1-hFc的SEC-HPLC图谱。
图9示出了hFSH-CTP和pFSH-CTP单链的结构示意图。
图10示出了所构建编码hFSH-CTP融合蛋白的真核表达质粒图谱。
图11示出了所构建编码pFSH-CTP融合蛋白的真核表达质粒图谱。
图12示出了猪垂体FSH、重组融合蛋白hFSH-CTP和pFSH-CTP的SDS-PAGE电泳图。其中,猪垂体FSH、重组融合蛋白hFSH-CTP和pFSH-CTP分别位于电泳图的第1泳道和第3-4泳道,第2泳道为Marker。
图13示出了hFSH-hFc的SEC-HPLC图谱。其中,1为重组融合蛋白hFSH-CTP的SEC-HPLC图谱,2为重组融合蛋白pFSH-CTP的SEC-HPLC图谱。
图14示出了各重组FSH-Fc融合蛋白和猪垂体FSH的药时曲线。
图15示出了各重组FSH-CTP融合蛋白和猪垂体FSH的药时曲线。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,按照常规条件如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring Harbor LaboratoryPress,1989)中所述的条件,或按照制造厂商所建议的条件。
实施例1重组FSH-Fc融合蛋白的制备
1.1构建编码重组hFSH-Fc融合蛋白的基因表达载体
基因序列设计是基于CHO细胞偏爱密码子进行优化,采用人工合成的方法合成经过优化的含有编码信号肽及hFSH蛋白β链成熟肽段、CTP和hFSH蛋白α链成熟肽段的融合基因,将所合成的DNA片段插入转移载体如pUC57中的EcoRV限制性酶切位点间,获得了hFSH质粒(即phFSH),使用DNA测序方法验证插入序列的正确性。
表达载体根据pcDNA3.0改造而成。先将pcDNA3.0原本的NeoR/KanR(新霉素/卡那霉素)去掉,换成抗凋亡基因BIRC2,BIRC2基因通过与活化的Caspase-3/7结合,依赖E3泛素连接酶的活性,介导Caspase-3/7降解,从而抑制细胞凋亡。再将其中的氨苄青霉素及其表达结构去掉,插入新的卡那霉素抗性基因,由T7启动子诱导表达。再在多克隆位点的EcoRI酶切位点后插入IRES和DHFR基因。DHFR基因可以使工程细胞在二氢叶酸还原酶抑制剂MTX存在下存活和生长,并可通过不断提高MTX的浓度,使得外源DHFR基因附近的目的基因得到扩增,从而提高目的基因的拷贝数。人工合成的IRES元件即内部核糖体进入位点,用于启动DHFR在CHO细胞中的表达。将此改造后的质粒命名为pUH3.0。
分别人工合成含有BamHI(5’端)和EcoRI(3’端)酶切位点的编码柔性肽接头(Linker,简称“L”)和Fc变体(包括vIgG2-hFc和vIgG1-hFc)片段的融合基因L-vIgG2-hFc和L-vIgG1-hFc。将获得的融合基因片段分别插入到转移载体PUC19中的BamHI和EcoRI限制性酶切位点间,分别得到含Fc变体的质粒pL-vIgG2-hFc和pL-vIgG1-hFc。通过DNA测序验证L-vIgG2-hFc和L-vIgG1-hFc的基因序列正确性。
为制备hFSH-L-Fc融合基因,用限制性内切酶SpeI和BamHI双酶切phFSH,凝胶电泳后胶回收编码信号肽及hFSH蛋白β链成熟肽段、CTP和hFSHα链成熟肽段的融合基因片段,经纯化的上述基因片段分别插入到pL-vIgG2-hFc和pL-vIgG1-hFc质粒中肽接头的5'-端,T4连接酶连接分别构成phFSH-L-vIgG2-hFc和phFSH-L-vIgG1-hFc质粒。所构建的融合基因由hFSHβ、CTP、hFSHα、肽接头和Fc变体基因组成,其单链结构如图2a所示,二聚化结构如图2b所示。
限制性内切酶SpeI/EcoRI双酶切phFSH-L-vIgG2-hFc和phFSH-L-vIgG1-hFc质粒,DNA凝胶纯化得到hFSH-L-vIgG2-hFc和hFSH-L-vIgG1-hFc片段。将纯化好的hFSH-L-Fc片段插入到上文所述改造后的哺乳动物细胞表达质粒pUH3.0的相应酶切位点间,最终获得含融合基因的表达质粒pUH3.0-hFSH-vIgG2-hFc、pUH3.0-hFSH-vIgG1-hFc,将它们统称为pUH3.0-hFSH-Fc质粒,所述质粒的结构分别如图3a和图3b所示。其中,所述pUH3.0-hFSH-vIgG2-hFc质粒中包含的hFSH-L-vIgG2-hFc的核苷酸序列和氨基酸序列分别如SEQ ID NO:1-2所示;所述pUH3.0-hFSH-vIgG1-hFc质粒中包含的hFSH-L-vIgG1-hFc的核苷酸序列和氨基酸序列分别如SEQ ID NO:3-4所示。该质粒含哺乳动物细胞高效表达外源基因蛋白所需的启动子CMV;该质粒含有卡那霉素抗性基因,可以在细菌中进行抗性筛选。此外,当宿主细胞是DHFR基因表达缺陷型时,改造后的pUH3.0表达载体所含有的小鼠的二氢叶酸还原酶(DHFR)基因,使其在氨甲蝶呤(MTX)存在时,能共扩增pFSH-Fc融合基因和DHFR基因。
用CTP肽段连接hFSH的α链和β链,可便于两条链正确折叠。通过肽接头(较佳地为柔性接头)进行hFSH和Fc片段的偶联可提高蛋白的生物活性,对本发明而言,优选的是长度为约20个或更少(但不能少于2个)氨基酸的肽接头,当然由1个氨基酸构成的肽接头也在本发明的保护范围内,宜使用含有或由2个或更多选自以下氨基酸构成的肽接头:甘氨酸、丝氨酸、丙氨酸和苏氨酸。本发明实施例的肽接头含有Gly-Ser肽构件,其氨基酸序列为GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer。
1.2构建编码重组pFSH-Fc融合蛋白的基因表达载体
将1.1节中构建所述编码重组hFSH-Fc融合蛋白的基因表达载体中所涉及的“hFSH蛋白β成熟肽段”和“hFSH蛋白α成熟肽段”的编码基因分别替换为“pFSH蛋白β成熟肽段”和“pFSH蛋白α成熟肽段”的编码基因,按照和1.1节中同样的构建方法,构建获得含融合基因的表达质粒pUH3.0-pFSH-pFc、pUH3.0-pFSH-vIgG2-hFc和pUH3.0-pFSH-vIgG1-hFc,将它们统称为pUH3.0-pFSH-Fc质粒,质粒的结构分别如图4a、图4b和图4c所示。其中,所述pUH3.0-pFSH-pFc质粒中包含的pFSH-L-pFc的核苷酸序列和氨基酸序列分别如SEQ ID NO:5-6所示;所述pUH3.0-pFSH-vIgG2-hFc质粒中包含的pFSH-L-vIgG2-hFc的核苷酸序列和氨基酸序列分别如SEQ ID NO:7-8所示;所述pUH3.0-pFSH-vIgG1-hFc质粒中包含的pFSH-L-vIgG1-hFc的核苷酸序列和氨基酸序列分别如SEQ ID NO:9-10所示。
1.3重组FSH-Fc融合蛋白在哺乳动物细胞中的稳定表达
将上述1.1节中构建的表达质粒pUH3.0-hFSH-Fc和1.2节中构建的表达质粒pUH3.0-pFSH-Fc分别转染入DHFR酶缺陷型CHO宿主细胞(CHO–DHFR-),图2b显示了重组二聚化hFSH-Fc融合蛋白和pFSH-Fc融合蛋白的示意图。采用电穿孔方法进行转染,使用960μFd电容的Gene Pulser Electroporator(Bio-Rad Laboratories,Hercules,CA),将其电场设置为250V,在比色杯内的2×107~5×107个细胞中加入10μg用PvuI线性化的质粒DNA。在转染两天后,将培养基改成含1μM MTX卡那霉素或100μM MSX(如用GS筛选标记)的生长培养基,由于存在抗凋亡元件,克隆效率相对于普通载体大幅度提高,在2-4周即可获得经抗性初筛的转染子。采用ELISA的方法,用抗hFSH抗体检测hFSH-Fc和pFSH-Fc的表达。利用DHFR扩增选择性标记基因提高重组二聚化蛋白的表达水平,为此在含有递增浓度MTX的生长培养基中,用DHFR基因共扩增转染的重组二聚化蛋白基因。用极限稀释法亚克隆能在高达10μM MTX或1mM MSX(如用GS筛选标记)培养基中生长的转染子。由于存在抗凋亡元件,克隆效率相对于普通载体大幅度提高,在2-4周即可获得亚克隆。对亚克隆细胞系的分泌率作进一步分析。筛选分泌水平超过约10(较佳地约20)μg/106(即百万)个细胞/24小时的细胞株,获得稳定高表达重组hFSH-Fc融合蛋白和重组pFSH-Fc融合蛋白的细胞株。
1.4重组hFSH-Fc融合蛋白和重组pFSH-Fc融合蛋白的生产和纯化
采用上述1.3节中得到的高产量细胞株,首先在培养皿中进行无血清驯化培养,然后转移到摇瓶中进行悬浮驯化培养,驯化过程中同时进行培养基的筛选,加入不同的成分观察细胞的生长状态、生长趋势,以及表达产物的活性和唾液酸等生化指标,优选的细胞培养条件为:基础培养基加入100μM Cu2+,feeding培养基加入2mM ManNAc(N-乙酰基-D-氨基甘露糖),该方法可使重组hFSH-Fc融合蛋白和重组pFSH-Fc融合蛋白的糖基化程度增加,唾液酸含量提高约20%。驯化成功后,进行细胞扩增,扩增到足够量,进行7L生物反应器监测培养,在细胞密度超过1×107个/mL时开始降温至33℃培养,批次生长周期为20天。取1mLCHO细胞培养上清,经0.45μm针头过滤器过滤后上样,采用POROS A 20μm ProteinA色谱柱上样后用流动相A(PBS溶液,pH7.0)冲平,用流动相B(100mM甘氨酸溶液,pH3.5)进行洗脱,流速为2ml/min,柱温为室温,检测波长为280nm。通过上述ProteinA-HPLC方法测定重组hFSH-Fc融合蛋白和重组pFSH-Fc融合蛋白的表达量,结果显示:重组hFSH-vIgG2-hFc和重组hFSH-vIgG1-hFc细胞株表达的累积产量分别为2.08g/L、0.86g/L;重组pFSH-vIgG2-hFc、重组pFSH-vIgG1-hFc、重组pFSH-pFc细胞株表达的累积产量分别为1.84g/L、0.89g/L、0.75g/L。
重组hFSH-Fc融合蛋白和重组pFSH-Fc融合蛋白的纯化包括以下步骤:
1)Protein A亲和层析:离心,收集上清,根据本发明蛋白偶联Fc片段的特性,利用亲和层析方法,将上清液加样到磷酸盐缓冲液盐水(PBS)平衡的Protein A柱;待重组融合蛋白结合于ProteinA后,用PBS洗涤该柱,直至OD280值低于0.01,再用20mM pH为4.0的醋酸钠缓冲液洗脱结合的重组hFSH-Fc融合蛋白和重组pFSH-Fc融合蛋白,最后用pH10.0的1MTris-HCl缓冲液中和活性收集液。纯化的hFSH-Fc蛋白和pFSH-Fc纯度可达到98%以上。
2)疏水柱层析:用超滤方法将上述Protein A活性收集液更换为20mM Tris-HCl-1.5M NaCl(pH8.0)缓冲液,将该样品加样到用20mM Tris-HCl-1.5M NaCl(pH8.0)平衡过的phenyl-6Fast Flow柱,先用相同的平衡液淋洗,再用20mM Tris-HCl-1.35M NaCl(pH8.0)淋洗,最后用20mM Tris-HCl-0.5M NaCl(pH8.0)缓冲液洗脱。
ELISA结果显示了重组hFSH-Fc融合蛋白和重组pFSH-Fc融合蛋白在CHO细胞中的成功表达。如图5和图7所示,在还原条件下SDS-PAGE胶电泳图谱,猪垂体FSH(商业产品)和本发明的重组hFSH-Fc融合蛋白(包括hFSH-vIgG2-hFc和hFSH-vIgG1-hFc)、重组pFSH-Fc融合蛋白(包括pFSH-pFc、pFSH-vIgG2-hFc和pFSH-vIgG1-hFc)分别在42kDa和128kDa显示相对应的目标蛋白杂交条带,用抗hFSH抗体检测并结合ProteinA-HPLC分析方法,验证了本发明得到的重组hFSH融合蛋白和重组pFSH融合蛋白中含有FSH蛋白和Fc片段。利用上述纯化工艺获得的纯化hFSH-Fc融合蛋白和pFSH-Fc融合蛋白通过SEC-HPLC检测其纯度,如图6和图8中的结果显示,纯化的hFSH-Fc融合蛋白和pFSH-Fc融合蛋白纯度可达到98%以上。
实施例2重组FSH-CTP融合蛋白的制备
2.1构建编码重组hFSH-CTP融合蛋白的基因表达载体
基因序列设计是基于CHO细胞偏爱密码子进行优化,采用人工合成的方法合成经过优化的含有编码hFSH蛋白β链成熟肽段、CTP、bGH终止信号、CMV启动子和hFSHα链成熟肽段的DNA片段,其两端带有SpeI和EcoRI酶切位点。将所合成的DNA片段插入转移载体如pUC57中的EcoRV限制性酶切位点间,获得了hFSH-CTP质粒(即phFSH-CTP),使用DNA测序方法验证插入序列的正确性。
表达载体根据pcDNA3.0改造而成。先将pcDNA3.0原本的NeoR/KanR(新霉素/卡那霉素)去掉,换成抗凋亡基因BIRC2,BIRC2基因通过与活化的Caspase-3/7结合,依赖E3泛素连接酶的活性,介导Caspase-3/7降解,从而抑制细胞凋亡。再将其中的氨苄青霉素及其表达结构去掉,插入新的卡那霉素抗性基因,由T7启动子诱导表达。再在多克隆位点的EcoRI酶切位点后插入IRES和DHFR基因。DHFR基因可以使工程细胞在二氢叶酸还原酶抑制剂MTX存在下存活和生长,并可通过不断提高MTX的浓度,使得外源DHFR基因附近的目的基因得到扩增,从而提高目的基因的拷贝数。人工合成的IRES元件即内部核糖体进入位点,用于启动DHFR在CHO细胞中的表达。将此改造后的质粒命名为pUH3.0。
用限制性内切酶SpeI和EcoRI双酶切phFSH质粒,凝胶电泳后胶回收切下的DNA片段。再用限制性内切酶SpeI/EcoRI双酶切改造后的哺乳动物细胞表达质粒pUH3.0,将上述酶切纯化后的DNA片段用T4连接酶连接到线性化后的载体中,最终获得含融合基因的表达质粒pUH3.0-hFSH-CTP,使用DNA测序方法验证插入序列的正确性。所构建的融合基因由hFSHβ、CTP、bGH终止信号、CMV启动子和hFSHα的DNA片段组成,其单链结构如图9所示,所述质粒的结构如图10所示。
该质粒含哺乳动物细胞高效表达外源基因蛋白所需的启动子CMV;该质粒含有卡那霉素抗性基因,可以在细菌中进行抗性筛选。此外,当宿主细胞是DHFR基因表达缺陷型时,改造后的pUH3.0表达载体所含有的小鼠的二氢叶酸还原酶(DHFR)基因,使其在氨甲蝶呤(MTX)存在时,能共扩增pFSH-CTP融合基因和DHFR基因。
FSHα和β两个亚基分别由两个CMV启动子来启动表达,β亚基由bGH终止信号来终止转录,而α亚基后面连接IRES元件来转录DHFR基因,并由SV40终止信号来终止转录。
FSHβ亚基羧基末端连接了一个CTP肽段。CTP是人绒毛膜促性腺激素(hCG)蛋白β亚基羧基端的一个包含33个氨基酸的肽段结构,位于hCGβ蛋白亚基的113~145位。CTP上有4个潜在的O-糖基化修饰位点,分别发生在S121、S127、S132和S138位,O-糖基化修饰能够增加提高了蛋白分子中唾液酸含量的比例,显著延长蛋白在体内的半衰期。其中,所述hFSH蛋白β链成熟肽段和CTP片段的核苷酸序列和氨基酸序列分别如SEQ ID NO:11-12所示;所述hFSHα亚基的核苷酸和氨基酸序列分别如SEQ ID NO:13-14所示。
2.2构建编码重组pFSH-CTP融合蛋白的基因表达载体
将2.1节中构建所述编码重组hFSH-CTP融合蛋白的基因表达载体中所涉及的“hFSH蛋白β成熟肽段”和“hFSH蛋白α成熟肽段”的编码基因分别替换为“pFSH蛋白β成熟肽段”和“pFSH蛋白α成熟肽段”的编码基因,按照和2.1节中同样的构建方法构建获得含融合基因的表达质粒pUH3.0-pFSH-CTP,质粒的结构如图11所示。其中,所述pFSH蛋白β链成熟肽段和CTP片段的核苷酸序列和氨基酸序列分别如SEQ ID NO:15-16所示;所述pFSHα亚基的核苷酸和氨基酸序列分别如SEQ ID NO:17-18所示。
2.3重组hFSH-CTP融合蛋白和重组pFSH-CTP融合蛋白在哺乳动物细胞中的稳定表达
将上述2.1和2.2节中构建的表达质粒pUH3.0-hFSH-CTP和pUH3.0-pFSH-CTP转染入DHFR酶缺陷型CHO宿主细胞(CHO-DHFR-),图9显示了重组hFSH-CTP融合蛋白和重组pFSH-CTP融合蛋白单链结构的示意图。采用电穿孔方法进行转染,使用960μFd电容的GenePulser Electroporator(Bio-Rad Laboratories,Hercules,CA),将其电场设置为250V,在比色杯内的2×107~5×107个细胞中加入10μg用PvuI线性化的质粒DNA。在转染两天后,将培养基改成含100μg/mL卡那霉素抗性标记基因的生长培养基,获得经抗性初筛的转染子。采用ELISA的方法,用抗FSH抗体检测hFSH-CTP和pFSH-CTP的表达。利用DHFR扩增选择性标记基因提高重组二聚化蛋白的表达水平,为此在含有递增浓度MTX的生长培养基中,用DHFR基因共扩增转染的重组蛋白基因。用极限稀释法亚克隆能在高达6μM/mL MTX培养基中生长的转染子。对亚克隆细胞系的分泌率作进一步分析。筛选分泌水平超过约3(较佳地约5)μg/106(即百万)个细胞/24小时的细胞株,获得稳定高表达重组hFSH-CTP融合蛋白和重组pFSH-CTP融合蛋白的细胞株。
2.4重组hFSH-CTP融合蛋白和重组pFSH-CTP融合蛋白的生产和纯化
采用上述2.3节中得到的高产量细胞株,首先在培养皿中进行无血清驯化培养,然后转移到摇瓶中进行悬浮驯化培养,驯化过程中同时进行培养基的筛选,加入不同的成分观察细胞的生长状态、生长趋势,以及表达产物的活性和唾液酸等生化指标,优选的细胞培养条件为:基础培养基加入100μM Cu2+,feeding培养基加入2mM ManNAc(N-乙酰基-D-氨基甘露糖),该方法可使重组hFSH-CTP融合蛋白的糖基化程度增加,唾液酸含量提高约15%。驯化成功后,进行细胞扩增,扩增到足够量,进行7L生物反应器监测培养,在细胞密度超过1×107个/mL时开始降温至33℃培养,批次生长周期为20天。用ProteinA-HPLC方法测定重组hFSH-CTP融合蛋白和重组pFSH-CTP融合蛋白的表达量,结果显示,重组hFSH-CTP细胞株表达的累积产量达到300mg/L,重组pFSH-CTP细胞株表达的累积产量达到200mg/L。。
所述重组hFSH-CTP融合蛋白和重组pFSH-CTP融合蛋白的纯化包括以下步骤:
1)分段硫酸铵沉淀:将细胞液9000G离心,收集上清,缓慢加入硫酸铵固体颗粒并不断搅拌溶解,硫酸铵浓度达到15%时离心,收集上清,继续加入硫酸铵固体颗粒至35%,离心收集沉淀。
2)沉淀复溶及超滤换液:将上述沉淀样品复溶于20mmol,pH7.5的磷酸盐缓冲液(PBS),复溶过程缓慢搅拌,控制温度<10℃,防止样品变性。待固体完全溶解,样品溶液变得透明后,超滤换液,置换到20mmolPBS,10mmol NaCl,pH7.5的缓冲液中。
3)阴离子柱层析:将上述超滤缓冲液加样到20mmolPBS,10mmol NaCl,pH7.5平衡过的GE Q-Sepharose Fast Flow(货号:17-0510-01)层析柱,待目的蛋白结合后,使用平衡液继续洗涤,直至OD280低于0.01AU,再用20mmolPBS,100mmol NaCl,pH7.5的洗脱液洗脱目的蛋白,收集。
4)疏水柱层析:将上述阴离子Q柱活性收集液超滤更换为20mM Tris-HCl-1.8MNaCl(pH8.0)缓冲液,将该样品加样到用20mM Tris-HCl-1.8M NaCl(pH8.0)平衡过的GEPHENYL SEPHAROSE 6FF(货号:17-0973-05)层析柱,先用相同的平衡液淋洗,再用20mMTris-HCl-1.5M NaCl(pH8.0)淋洗,最后用20mM Tris-HCl-0.8M NaCl(pH8.0)缓冲液洗脱。
采用ELISA的方法,用抗FSH抗体和抗CTP抗体分别检测重组融合蛋白hFSH-CTP和pFSH-CTP在CHO细胞中的表达,如图12所示,在还原条件下进行SDS-PAGE胶电泳图谱,猪垂体FSH(商业产品)和本发明的重组hFSH-CTP融合蛋白、重组pFSH-CTP融合蛋白分别在43kDa和50kDa显示相对应的目标蛋白杂交条带,验证了本发明得到的重组hFSH-CTP融合蛋白和重组pFSH-CTP融合蛋白中含有FSH蛋白和CTP肽段。图13是利用上述纯化工艺获得的纯化hFSH-CTP融合蛋白和pFSH-CTP融合蛋白通过SEC-HPLC检测其纯度,结果显示,纯化的hFSH-CTP蛋白和pFSH-CTP蛋白纯度可分别达到98%以上。
实施例3重组FSH-Fc融合蛋白的药代动力学测定
将试验分为本发明提供的重组hFSH-vIgG2-hFc、hFSH-vIgG1-hFc、pFSH-vIgG2-hFc、pFSH-vIgG1-hFc、pFSH-pFc融合蛋白给药组和猪垂体FSH(商业产品)给药组,每组选用体重220±10g的雄性SD大鼠5只,分别按30IU/kg的剂量给予皮下注射。
猪垂体FSH组在给药后1h、2h、4h、6h、8h、12h、24h、36h、60h取血,重组hFSH-vIgG2-hFc、hFSH-vIgG1-hFc、pFSH-vIgG2-hFc、pFSH-vIgG1-hFc、pFSH-pFc融合蛋白分别在给药后0h、2h、6h、12h、24h、32h、48h、56h、72h、80h、96h、104h、120h取血,并在4℃3000rpm的条件下离心5min后,吸取血清,-20℃保存。采用ELISA试剂盒(BIOCHECK,美国)测定各时间点血浆中FSH免疫活性。使用kinetica4.4软件,通过统计矩法计算各组主要药代动力学参数。各组药代动力学曲线如图14所示,半衰期结果如表1所示。
结果显示,猪垂体FSH在大鼠体内的消除半衰期约为3.45h;而等剂量重组hFSH-vIgG2-hFc、hFSH-vIgG1-hFc、pFSH-vIgG2-hFc、pFSH-vIgG1-hFc、pFSH-pFc融合蛋白的消除半衰期分别约为56.22h、55.63h、53.98h、57.16h、56.73h,是猪垂体FSH半衰期的约15倍。
表1重组FSH-Fc融合蛋白和猪垂体FSH的半衰期
实施例4重组FSH-CTP融合蛋白的药代动力学测定
将试验分为本发明提供的重组hFSH-CTP融合蛋白给药组、重组pFSH-CTP融合蛋白给药组和猪垂体FSH(商业产品)给药组,每组选用体重220±10g的雄性SD大鼠5只,分别按30IU/kg的剂量给予皮下注射。
猪垂体FSH组在给药后1h、2h、4h、6h、8h、12h、24h、36h、60h取血,重组hFSH-CTP融合蛋白给药组和重组pFSH-CTP融合蛋白给药组在给药后0h、2h、4h、8h、12h、18h、24h、32h、48h、56h、72h、80h、96h、104h、120h取血,并在4℃3000rpm的条件下离心5min后,吸取血清,-20℃保存。采用ELISA试剂盒(BIOCHECK,美国)测定各时间点血浆中FSH免疫活性。使用kinetica4.4软件,通过统计矩法计算各组主要药代动力学参数。各组药代动力学曲线如图15所示,半衰期结果如表2所示。
结果显示,猪垂体FSH在大鼠体内的消除半衰期约为3.45h;而等剂量的重组hFSH-CTP融合蛋白和重组pFSH-CTP融合蛋白的消除半衰期分别约为18.58h和19.18h,两者的消除半衰期相近,它们的半衰期是猪垂体半衰期的5倍以上。
表2重组hFSH-Fc融合蛋白和猪垂体FSH的半衰期
实施例5重组hFSH-Fc融合蛋白在母猪批次化生产中的应用
选择1500头规模化母猪场,经过约6个月猪群和栏舍调整,由传统生产模式转变为3周批次化生产。具体来讲,将全群母猪分为7批基础母猪群,经产母猪采用定时输精技术,先同期断奶,断奶后通过FSH和PMSG等外源激素的处理后诱导同期发情,同时为了充分利用猪栏,不断更新基础母猪群,淘汰没有进入批次发情的经产母猪,补充后备母猪,后备母猪采用定时输精技术,先用烯丙孕素抑制其发情,再通过FSH和PMSG等外源激素的处理诱导同期发情,按上述方法操作保证同一批次的后备母猪与经产母猪同期发情、同期排卵、同期配种和同期分娩,使猪场操作流程化、标准化,大大提高生产和繁殖效率。
本实施中主要操作程序如下:
经产母猪定时输精:经产母猪400头,哺乳天数为27天,分为4组:空白对照组、重组hFSH-vIgG2-hFc给药组、重组hFSH-vIgG1-hFc给药组、PMSG给药组,每组各100头。
母猪在下午17:00断奶,在断奶后24h给药处理,重组hFSH-vIgG2-hFc给药组和重组hFSH-vIgG1-hFc给药组按照250单位/头、同时配合HCG 300单位/头肌肉注射给药;PMSG给药组按照1000单位/头肌肉注射给药;空白对照组不给药。给药后72h肌肉注射GnRH 100μg/头,之后分布于间隔24h和40h进行定时输精配种。重组hFSH-vIgG2-hFc给药组和重组hFSH-vIgG1-hFc给药组按照250单位/头、同时配合HCG 300单位/头肌肉注射给药;PMSG给药组按照1000单位/头肌肉注射给药;空白对照组不给药。给药后72h肌肉注射GnRH 100μg/头,之后分布于间隔24h和40h进行定时输精配种。
断奶后第2天开始观察和记录经产母猪一周内发情情况(出现静立反应即为发情),统计发情率;配种后25天至28天进行B超孕检,统计受胎率。
后备母猪定时输精:后备母猪120头,220-225日龄,随机分成四组:重组hFSH-vIgG2-hFc给药组、重组hFSH-vIgG1-hFc给药组、PMSG给药组和空白对照组,每组头数各为30头。
试验第1天,所有母猪开始饲喂烯丙孕素,连续饲喂18天,每天20mg,停喂后间隔42h,对三个给药组的母猪分别进行给药处理,重组hFSH-L-vIgG2-hFc和重组hFSH-L-vIgG1-hFc给药组分别按250单位/头、同时配合HCG 300单位/头肌肉注射给药;PMSG给药组按照1000单位/头肌肉注射给药,给药后间隔80h肌肉注射GnRH 100μg/头,分别于注射GnRH后24h和40h进行定时人工授精配种。空白对照组仅饲喂烯丙孕素,连续饲喂18天,每天20mg,不做其他给药处理。
停喂烯丙孕素后开始观察和记录后备母猪一周内发情情况(出现静立反应即为发情),统计发情率;配种后25天至28天进行B超孕检,统计受胎率。
整个批次化生产流程约21周(如例图1),按流程提前安排工作计划,经产母猪和后备母猪分别按上述定时输精方法,达到同一批次的后备和经产母猪配种同步化。配种后妊娠第114天,所有母猪注射氯前列醇钠进行同期分娩,分别记录以上各组分娩和产仔情况,统计母猪窝均产仔数。
结果分析如下:
经产母猪同期发情结果:观察各组发情情况,对照组一胎母猪断奶后第3天至7天,随机有母猪发情,每天都要查情和配种;重组hFSH-vIgG2-hFc和重组hFSH-vIgG1-hFc给药组、PMSG给药组的一胎母猪集中在断奶后第4天和5天发情,说明用重组hFSH-Fc融合蛋白给药处理后一胎经产母猪发情集中度显著提高,断奶到发情时间缩短到6天内。提示可在集中发情期进行配种,无需按常规每天查情和配种。
各组经产母猪批次化生产的结果见表3,结果表明,与空白对照组比较,重组hFSH-vIgG2-hFc和重组hFSH-vIgG1-hFc、PMSG给药组均能显著提高总受胎率(P<0.01),每头平均产仔数也均提高;其中重组hFSH-vIgG2-hFc的总受胎率和每头平均产仔数高于PMSG给药组,但无显著性差异。综合以上,说明本发明重组hFSH-Fc能够显著提高经产母猪发情率和受胎率,缩短断奶到发情时间间隔,窝均产仔数也有提高。
表3重组hFSH-Fc融合蛋白在经产母猪批次化生产上的应用效果
注:发情率、总受胎率、总分娩率采用χ2检验,不同肩标表示差异极显著(P<0.01),相同肩标表示差异不显著(P>0.05)。
后备母猪同期发情结果:对照组后备母猪发情随机分布于试验第22天至28天,每天都要查情和配种;重组hFSH-vIgG2-hFc和重组hFSH-vIgG1-hFc给药组的后备母猪发情集中分布在试验第22-23天;PMSG给药组的后备母猪发情分布在试验第21-23天。说明用重组hFSH-Fc融合蛋白给药处理后母猪发情集中度显著提高。提示可在集中发情期进行配种,无需按常规每天查情和配种。
各组后备母猪批次化生产的结果见表4,结果表明,与空白对照组比较,重组hFSH-vIgG2-hFc、重组hFSH-vIgG1-hFc和PMSG三个给药组的发情率、总受胎率明显提高(P<0.01),窝均产仔数无显著差异。其中重组hFSH-L-vIgG2-hFc给药组的发情率和受胎率比PMSG给药组高,但无显著差异。说明本发明重组hFSH-Fc能够显著提高后备母猪发情率、总受胎率,窝均产仔数也有提高。
表4重组hFSH-Fc融合蛋白在后备母猪批次化生产上的应用效果
注:总受胎率采用χ2检验,不同肩标表示差异极显著(P<0.01),相同肩标表示差异不显著(P>0.05)。
实施例6重组pFSH-Fc融合蛋白在母猪批次化生产中的应用
选择1500头规模猪场,采用3周批次生产,具体操作方式同实施例5。
主要操作程序如下:
经产母猪定时输精:经产母猪400头,哺乳天数为27天,分为5组:空白对照组、重组pFSH-vIgG2-hFc给药组、重组pFSH-vIgG1-hFc给药组、重组pFSH-pFc给药组、PMSG给药组,每组各80头。
母猪在下午17:00断奶,在断奶后24h给药处理,重组hFSH-vIgG2-hFc给药组和重组hFSH-vIgG1-hFc给药组按照250单位/头、同时配合HCG 300单位/头肌肉注射给药;PMSG给药组按照1000单位/头肌肉注射给药;空白对照组不给药。给药后72h肌肉注射GnRH 100μg/头,之后分布于间隔24h和40h进行定时输精配种。重组hFSH-vIgG2-hFc给药组和重组hFSH-vIgG1-hFc给药组按照250单位/头、同时配合HCG 300单位/头肌肉注射给药;PMSG给药组按照1000单位/头肌肉注射给药;空白对照组不给药。给药后72h肌肉注射GnRH 100μg/头,之后分布于间隔24h和40h进行定时输精配种。
断奶后第2天开始观察和记录经产母猪一周内发情情况(出现静立反应即为发情),统计发情率;配种后25天至28天进行B超孕检,统计受胎率。
后备母猪定时输精:后备母猪150头,220-225日龄,随机分成五组:重组pFSH-vIgG2-hFc给药组、重组pFSH-vIgG1-hFc给药组、重组pFSH-pFc、PMSG给药组、空白对照组,每组30头。
试验第1天,所有母猪开始饲喂烯丙孕素,连续饲喂18天,每天20mg,停喂后间隔42h,对三个给药组的母猪分别进行给药处理,重组hFSH-vIgG2-hFc和重组hFSH-vIgG1-hFc给药组分别按250单位/头、同时配合HCG 300单位/头肌肉注射给药;PMSG给药组按照1000单位/头肌肉注射给药,给药后间隔80h肌肉注射GnRH 100μg/头,分别于注射GnRH后24h和40h进行定时人工授精配种。空白对照组仅饲喂烯丙孕素,连续饲喂18天,每天20mg,不做其他给药处理。
停喂烯丙孕素后开始观察和记录后备母猪一周内发情情况(出现静立反应即为发情),统计发情率;配种后25天至28天进行B超孕检,统计受胎率。
整个批次化生产流程为21周(如例图1),按流程提前安排工作计划,经产母猪和后备母猪分别按上述定时输精方法,达到同一批次的后备和经产母猪配种同步化。配种后妊娠第114天,所有母猪注射氯前列醇钠进行同期分娩,分别记录以上各组分娩和产仔情况,统计母猪窝均产仔数。
结果分析如下:
经产母猪同期发情结果:观察各组发情情况,对照组一胎母猪断奶后第3天至7天,随机有母猪发情,每天都要查情和配种;重组pFSH-vIgG2-hFc给药组、重组pFSH-vIgG1-hFc给药组、重组pFSH-pFc给药组、PMSG给药组的一胎母猪集中在断奶后第4天和5天发情,说明用重组pFSH-Fc融合蛋白给药处理后一胎经产母猪发情集中度显著提高,断奶到发情时间缩短到6天内。提示可在集中发情期进行配种,无需按常规每天查情和配种。
各组经产母猪的定时输精的结果见表5,结果表明,与空白对照组比较,重组pFSH-vIgG2-hFc给药组和重组pFSH-vIgG1-hFc给药组、重组pFSH-pFc给药组、PMSG给药组均能够显著提高总受胎率(P<0.01),每头平均产仔数均有提高;其中重组pFSH-vIgG2-hFc给药组的总受胎率和每头平均产仔数高于PMSG给药组,但无显著性差异。综合以上,说明本发明重组pFSH-Fc能够显著提高后备母猪发情率和受胎率,缩短断奶到发情时间间隔,窝均产仔数也有提高。
表5重组pFSH-Fc融合蛋白在经产母猪批次化生产上的应用效果
注:母猪妊娠过程中无流产、淘汰和死亡。发情率、总受胎率、总分娩率采用χ2检验,不同肩标表示差异极显著(P<0.01),相同肩标表示差异不显著(P>0.05)。
后备母猪同期发情结果:对照组后备母猪发情随机分布于试验第22天至28天,每天都要查情和配种;重组pFSH-vIgG2-hFc给药组、重组pFSH-vIgG1-hFc给药组集中在22-23天发情;重组pFSH-pFc给药组、PMSG给药组集中在21-23天发情。说明用重组pFSH-Fc融合蛋白给药处理后母猪发情集中度显著提高。提示可在集中发情期进行配种,无需按常规每天查情和配种。
各组后备母猪的批次化生产结果见表6,结果表明,重组pFSH-vIgG2-hFc给药组和重组pFSH-vIgG1-hFc给药组、重组pFSH-pFc给药组和PMSG给药组的发情率和总受胎率均显著高于空白对照组(P<0.01),窝均产仔数无显著差异;其中重组pFSH-vIgG2-hFc给药组的发情率、总受胎率、窝均产仔数高于PMSG给药组,但无显著性差异。说明本发明重组pFSH-Fc能够显著提高后备母猪的发情率和受胎率,窝均产仔数也有提高。
表6重组pFSH-Fc融合蛋白在后备母猪批次化生产上的应用效果
注:母猪妊娠过程中无流产、淘汰和死亡。发情率、总受胎率、总分娩率采用χ2检验,不同肩标表示差异极显著(P<0.01),相同肩标表示差异不显著(P>0.05)。
实施例7重组FSH-CTP融合蛋白在母猪批次化生产中的应用
选择1100头规模化猪场,采用3周批次生产。具体来讲,将全群母猪分为7批基础母猪群,经产母猪采用定时输精技术,先同期断奶,断奶后通过FSH和PMSG等外源激素的处理后诱导同期发情,同时为了充分利用猪栏,不断更新基础母猪群,淘汰没有进入批次发情的经产母猪,补充后备母猪(年更新率30%左右),后备母猪采用定时输精技术,先用烯丙孕素抑制其发情,再通过FSH和PMSG等外源激素的处理诱导同期发情,按上述方法操作保证同一批次的后备母猪与经产母猪同期发情、同期排卵、同期配种和同期分娩,使猪场操作流程化、标准化,大大提高生产效率。
本实施中主要操作程序如下:
经产母猪定时输精:经产母猪300头,哺乳天数为27天,随机分成4组,空白对照组、重组hFSH-CTP给药组、重组pFSH-CTP给药组、PMSG给药组,每组各75头。
试验第1天,所有母猪均在下午17:00断奶,在断奶后24h给药处理,重组hFSH-CTP给药组和重组pFSH-CTP分别按照1200IU/头、同时配合HCG 300单位/头肌肉注射给药;PMSG给药组按照1000单位/头肌肉注射给药。随后在上述给药时间后72h肌肉注射GnRH 100μg/头,之后分别于间隔24h和40h进行定时输精配种。空白对照组不做任何给药处理,按照常规人工输精方案每天进行查情和输精配种。
断奶后第2天(试验第2天)开始观察和记录母猪一周内发情情况(出现静立反应即为发情),统计发情率;配种后25天至28天进行B超孕检,统计受胎率;114天后记录分娩和产仔情况,统计窝均产仔数。
后备母猪定时输精:后备母猪100头,220-225日龄,所有母猪均完成疫苗免疫,转至大栏饲养,给药和输精配种前转至定位栏饲养。随机分成4组,重组hFSH-CTP给药组、重组pFSH-CTP给药组、PMSG给药组和空白对照组,每组各25头。
试验第1天,后备母猪饲喂烯丙孕素,连续饲喂18天,每天20mg,停喂后间隔42h进行给药处理,重组hFSH-CTP给药组和重组pFSH-CTP给药组分别按照1200IU/头、同时配合HCG 300单位/头肌肉注射给药;PMSG给药组按照1000单位/头肌肉注射给药;空白对照组不给药。随后在给药后间隔80h肌肉注射GnRH 100μg,分别于注射GnRH后24h和40h进行定时人工授精配种。空白对照组仅饲喂烯丙孕素,连续饲喂18天,每天20mg,不做其他给药处理。
停喂烯丙孕素后(试验第19天)开始观察和记录母猪一周内发情情况(出现静立反应即为发情),统计发情率;配种后25天至28天进行B超孕检,统计受胎率;114天后记录分娩和产仔情况,统计窝均产仔数。
整个批次化生产流程约21周(如例图1),按流程提前安排工作计划,经产母猪和后备母猪分别按上述定时输精方法,达到同一批次的后备和经产母猪配种同步化。配种后妊娠第114天,所有母猪注射氯前列醇钠进行同期分娩,分别记录以上各组分娩和产仔情况,统计母猪窝均产仔数。
结果分析如下:
经产母猪同期发情结果:观察各组发情情况,对照组一胎母猪从断奶后第3天至7天,随机有母猪发情,每天都要查情和配种;重组hFSH-CTP给药组、PMSG给药组的一胎母猪集中在第4天和5天发情,说明用重组hFSH-CTP融合蛋白给药处理后一胎经产母猪发情集中度显著提高,断奶到发情时间缩短到6天内。提示可在集中发情期进行配种,无需按常规每天查情和配种,可节省劳力和输精成本。
各组经产母猪的定时输精的结果见表7,结果表明,与空白对照组比较,本发明重组hFSH-CTP给药组能够显著提高发情率和受胎率(P<0.01),窝均产仔数也有提高,但无显著差异。其中重组hFSH-CTP给药组的发情率和受胎率、窝均产仔数高于PMSG给药组,但无显著差异(P>0.05)。综合以上,说明本发明重组hFSH-CTP能够显著提高后备母猪发情率、总受胎率和总分娩率,窝均产仔数也有提高。
表7重组FSH-CTP融合蛋白在经产母猪定时输精上应用效果
注:母猪在妊娠过程中无流产、死亡和淘汰。总受胎率采用χ2检验,不同肩标表示差异极显著(P<0.01),相同肩标表示差异不显著(P>0.05)。
后备母猪同期发情结果:对照组后备母猪发情随机分布于试验第22天至28天,每天都要查情和配种;重组hFSH-CTP给药组的后备母猪发情集中分布在试验第22-23天;PMSG给药组的后备母猪发情分布在试验第21-23天。说明用重组hFSH-CTP融合蛋白给药处理后母猪发情集中度显著提高。提示可在集中发情期进行配种,无需按常规每天查情和配种,可节省劳力和输精成本。
各组后备母猪的定时输精的结果见表8,结果表明,重组hFSH-CTP给药组和PMSG给药组的发情率和总受胎率均显著高于空白对照组(P<0.01),窝均产仔数无显著性差异。其中重组hFSH-CTP给药组的发情率和受胎率、窝均产仔数高于PMSG组,但无显著性差异。说明本发明重组hFSH-CTP能够显著提高后备母猪发情率和总受胎率,窝均产仔数也有提高。
表8重组FSH-CTP融合蛋白在后备母猪定时输精上应用效果
注:母猪在妊娠过程中无流产、死亡和淘汰。总受胎率采用χ2检验,不同肩标表示差异极显著(P<0.01),相同肩标表示差异不显著(P>0.05)。
序列表
<110> 广州威生医药科技有限公司
<120> 长效重组FSH融合蛋白在母猪批次化生产中的应用
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1485
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgaggagcc tcggggccct gctcttgctg ctgagcgcct gcctggcggt gagcgctaac 60
tcatgtgagc tgactaatat caccattgcc atcgaaaaag aggaatgcag gttctgtatt 120
agtatcaaca ctacctggtg cgctggctac tgttatacaa gggatctggt gtataaggac 180
ccagcacggc ccaaaatcca gaagacatgc actttcaaag aactggtgta cgagactgtg 240
agggtccctg gctgtgccca ccatgctgat tccctgtaca cttatccagt ggccacccag 300
tgccactgtg gaaagtgcga tagtgactca acagactgta ctgtgcgagg cctgggacct 360
tcttactgca gttttggcga aatgaaggag ccccgtttcc aggattccag ctctagtaaa 420
gctccccctc cttccctgcc ctcaccctca agactgcctg gaccttccga cactcccatc 480
ctgccacagg cccccgatgt gcaggactgc cctgaatgta ctctgcagga gaaccccttc 540
ttttctcagc ccggcgctcc tatcctgcag tgtatgggat gctgttttag tagagcatat 600
cctaccccac tgcgctcaaa gaaaacaatg ctggtccaga agaatgtgac aagcgaatct 660
acttgctgtg tggctaaatc ctacaaccgc gtgaccgtga tgggcggctt caaggtggag 720
aatcacacag catgccattg ttctacttgc tactaccata agagtggatc cggtggcggt 780
tccggtggag gcggaagcgg cggtggagga tcagtggagt gccctccatg tccagcaccc 840
cctgtcgcag gtccatctgt gttcctgttt ccacccaagc ctaaagacac tctgatgatc 900
tcccgcaccc cagaagtcac ctgtgtggtc gtggatgtga gccatgaaga ccccgaggtc 960
cagttcaatt ggtacgtgga tggcgtcgag gtgcacaacg ctaagacaaa acctagagaa 1020
gagcagttca actctacctt tcgcgtcgtg agtgtgctga cagtcgtgca ccaggactgg 1080
ctgaatggca aggagtataa gtgcaaagtg agcaacaaag gactgcctgc ctcaatcgaa 1140
aagactattt ccaagaccaa aggacagcca agagagcccc aggtgtacac cctgcctcca 1200
agccgcgaag agatgactaa aaatcaggtc tctctgacct gtctggtgaa ggggttttat 1260
cctagtgata tcgccgtgga atgggagtca aacggtcagc cagagaacaa ttacaagacc 1320
acacccccta tgctggacag cgatgggtct ttctttctgt atagcaaact gacagtggac 1380
aagtctcggt ggcagcaggg taacgtcttc tcttgcagtg tgatgcacga agcactgcac 1440
aatcattaca cccagaagtc actgtcactg agcccaggaa aatga 1485
<210> 2
<211> 494
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Arg Ser Leu Gly Ala Leu Leu Leu Leu Leu Ser Ala Cys Leu Ala
1 5 10 15
Val Ser Ala Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Ala Ile Glu
20 25 30
Lys Glu Glu Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala
35 40 45
Gly Tyr Cys Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro
50 55 60
Lys Ile Gln Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val
65 70 75 80
Arg Val Pro Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro
85 90 95
Val Ala Thr Gln Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp
100 105 110
Cys Thr Val Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met
115 120 125
Lys Glu Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro
130 135 140
Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile
145 150 155 160
Leu Pro Gln Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln
165 170 175
Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met
180 185 190
Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys
195 200 205
Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val
210 215 220
Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu
225 230 235 240
Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser Gly
245 250 255
Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val
260 265 270
Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
275 280 285
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
290 295 300
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
305 310 315 320
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
325 330 335
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val
340 345 350
Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
355 360 365
Lys Val Ser Asn Lys Gly Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser
370 375 380
Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
385 390 395 400
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
405 410 415
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
420 425 430
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp
435 440 445
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
450 455 460
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
465 470 475 480
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
485 490
<210> 3
<211> 1497
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgaggagcc tcggggccct gctcttgctg ctgagcgcct gcctggcggt gagcgctaac 60
tcatgtgagc tgactaatat caccattgcc atcgaaaaag aggaatgcag gttctgtatt 120
agtatcaaca ctacctggtg cgctggctac tgttatacaa gggatctggt gtataaggac 180
ccagcacggc ccaaaatcca gaagacatgc actttcaaag aactggtgta cgagactgtg 240
agggtccctg gctgtgccca ccatgctgat tccctgtaca cttatccagt ggccacccag 300
tgccactgtg gaaagtgcga tagtgactca acagactgta ctgtgcgagg cctgggacct 360
tcttactgca gttttggcga aatgaaggag ccccgtttcc aggattccag ctctagtaaa 420
gctccccctc cttccctgcc ctcaccctca agactgcctg gaccttccga cactcccatc 480
ctgccacagg cccccgatgt gcaggactgc cctgaatgta ctctgcagga gaaccccttc 540
ttttctcagc ccggcgctcc tatcctgcag tgtatgggat gctgttttag tagagcatat 600
cctaccccac tgcgctcaaa gaaaacaatg ctggtccaga agaatgtgac aagcgaatct 660
acttgctgtg tggctaaatc ctacaaccgc gtgaccgtga tgggcggctt caaggtggag 720
aatcacacag catgccattg ttctacttgc tactaccata agagtggatc cggtggcggt 780
tccggtggag gcggaagcgg cggtggagga tcagacaaaa ctcacacatg cccaccgtgc 840
ccagcacctg aagtcgcggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 900
accctcatga tctcccggac acctgaggtc acatgcgtgg tggtggacgt gagccacgaa 960
gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 1020
aagccgcggg aggagcagta caacagcacg taccgggtgg tcagcgtcct caccgtcctg 1080
caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1140
gcctccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1200
accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 1260
aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 1320
aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 1380
ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1440
gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaatga 1497
<210> 4
<211> 498
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Arg Ser Leu Gly Ala Leu Leu Leu Leu Leu Ser Ala Cys Leu Ala
1 5 10 15
Val Ser Ala Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Ala Ile Glu
20 25 30
Lys Glu Glu Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala
35 40 45
Gly Tyr Cys Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro
50 55 60
Lys Ile Gln Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val
65 70 75 80
Arg Val Pro Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro
85 90 95
Val Ala Thr Gln Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp
100 105 110
Cys Thr Val Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met
115 120 125
Lys Glu Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro
130 135 140
Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile
145 150 155 160
Leu Pro Gln Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln
165 170 175
Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met
180 185 190
Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys
195 200 205
Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val
210 215 220
Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu
225 230 235 240
Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser Gly
245 250 255
Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
260 265 270
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Val Ala Gly Gly
275 280 285
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
290 295 300
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
305 310 315 320
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
325 330 335
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
340 345 350
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
355 360 365
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu
370 375 380
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
385 390 395 400
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
405 410 415
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
420 425 430
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
435 440 445
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
450 455 460
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
465 470 475 480
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
485 490 495
Gly Lys
<210> 5
<211> 1509
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgaagtccc tgcagttctg ttttctgttt tgttgctgga aggccatctg ttgtaattct 60
tgcgagctga ccaatatcac tatcaccgtg gagaaggagg aatgcaactt ttgtatctcc 120
attaatacca catggtgcgc cggctactgt tatacacgag acctggtgta caaagatcca 180
gctcgtccca acatccagaa aacctgcaca ttcaaggagc tggtctatga aactgtgaag 240
gtccctggct gtgcccacca tgctgacagc ctgtacacat atccagtggc cactgagtgc 300
cactgtggaa agtgcgactc agattccaca gattgtactg tcaggggcct gggaccctct 360
tactgcagct tttctgagat gaaagaaccc cgtttccagg attccagctc tagtaaagct 420
ccccctcctt ccctgccctc accctcaaga ctgcctggac cttccgacac tcccatcctg 480
ccacagtttc ctgatgggga gttcaccatg cagggttgcc cagagtgtaa actgaaggaa 540
aacaaatact tctccaagct gggggccccc atctatcagt gtatgggttg ctgtttctcc 600
agagcctacc ccacacctgc tcgcagtaag aaaactatgc tggtgcctaa gaatattact 660
agcgaggcta cctgctgtgt cgctaaagcc ttcaccaagg ccacagtgat gggaaacgcc 720
cgagtcgaga atcacaccga atgccattgt agtacatgct actatcacaa atcaggatcc 780
ggtggcggtt ccggtggagg cggaagcggc ggtggaggat caaagaccaa accaccatgt 840
cccatatgcc caggctgtga agtggccggg ccctcggtct tcatcttccc tccaaaaccc 900
aaggacaccc tcatgatctc ccagaccccc gaggtcacgt gcgtggtggt ggacgtcagc 960
aaggagcacg ccgaggtcca gttctcctgg tacgtggacg gcgtagaggt gcacacggcc 1020
gagacgagac caaaggagga gcagttcaac agcacctacc gtgtggtcag cgtcctgccc 1080
atccagcacc aggactggct gaaggggaag gagttcaagt gcaaggtcaa caacgtagac 1140
ctcccagccc ccatcacgag gaccatctcc aaggctatag ggcagagccg ggagccgcag 1200
gtgtacaccc tgcccccacc cgccgaggag ctgtccagga gcaaagtcac cgtaacctgc 1260
ctggtcattg gcttctaccc acctgacatc catgttgagt ggaagagcaa cggacagccg 1320
gagccagagg gcaattaccg caccaccccg ccccagcagg acgtggacgg gaccttcttc 1380
ctgtacagca agctcgcggt ggacaaggca agatgggacc atggagaaac atttgagtgt 1440
gcggtgatgc acgaggctct gcacaaccac tacacccaga agtccatctc caagactcag 1500
ggtaaatga 1509
<210> 6
<211> 502
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Lys Ser Leu Gln Phe Cys Phe Leu Phe Cys Cys Trp Lys Ala Ile
1 5 10 15
Cys Cys Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Thr Val Glu Lys
20 25 30
Glu Glu Cys Asn Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly
35 40 45
Tyr Cys Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Asn
50 55 60
Ile Gln Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Lys
65 70 75 80
Val Pro Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val
85 90 95
Ala Thr Glu Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys
100 105 110
Thr Val Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Ser Glu Met Lys
115 120 125
Glu Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser
130 135 140
Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu
145 150 155 160
Pro Gln Phe Pro Asp Gly Glu Phe Thr Met Gln Gly Cys Pro Glu Cys
165 170 175
Lys Leu Lys Glu Asn Lys Tyr Phe Ser Lys Leu Gly Ala Pro Ile Tyr
180 185 190
Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Ala Arg
195 200 205
Ser Lys Lys Thr Met Leu Val Pro Lys Asn Ile Thr Ser Glu Ala Thr
210 215 220
Cys Cys Val Ala Lys Ala Phe Thr Lys Ala Thr Val Met Gly Asn Ala
225 230 235 240
Arg Val Glu Asn His Thr Glu Cys His Cys Ser Thr Cys Tyr Tyr His
245 250 255
Lys Ser Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
260 265 270
Gly Ser Lys Thr Lys Pro Pro Cys Pro Ile Cys Pro Gly Cys Glu Val
275 280 285
Ala Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu
290 295 300
Met Ile Ser Gln Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
305 310 315 320
Lys Glu His Ala Glu Val Gln Phe Ser Trp Tyr Val Asp Gly Val Glu
325 330 335
Val His Thr Ala Glu Thr Arg Pro Lys Glu Glu Gln Phe Asn Ser Thr
340 345 350
Tyr Arg Val Val Ser Val Leu Pro Ile Gln His Gln Asp Trp Leu Lys
355 360 365
Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Val Asp Leu Pro Ala Pro
370 375 380
Ile Thr Arg Thr Ile Ser Lys Ala Ile Gly Gln Ser Arg Glu Pro Gln
385 390 395 400
Val Tyr Thr Leu Pro Pro Pro Ala Glu Glu Leu Ser Arg Ser Lys Val
405 410 415
Thr Val Thr Cys Leu Val Ile Gly Phe Tyr Pro Pro Asp Ile His Val
420 425 430
Glu Trp Lys Ser Asn Gly Gln Pro Glu Pro Glu Gly Asn Tyr Arg Thr
435 440 445
Thr Pro Pro Gln Gln Asp Val Asp Gly Thr Phe Phe Leu Tyr Ser Lys
450 455 460
Leu Ala Val Asp Lys Ala Arg Trp Asp His Gly Glu Thr Phe Glu Cys
465 470 475 480
Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile
485 490 495
Ser Lys Thr Gln Gly Lys
500
<210> 7
<211> 1494
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atgaagtccc tgcagttctg ttttctgttt tgttgctgga aggccatctg ttgtaattct 60
tgcgagctga ccaatatcac tatcaccgtg gagaaggagg aatgcaactt ttgtatctcc 120
attaatacca catggtgcgc cggctactgt tatacacgag acctggtgta caaagatcca 180
gctcgtccca acatccagaa aacctgcaca ttcaaggagc tggtctatga aactgtgaag 240
gtccctggct gtgcccacca tgctgacagc ctgtacacat atccagtggc cactgagtgc 300
cactgtggaa agtgcgactc agattccaca gattgtactg tcaggggcct gggaccctct 360
tactgcagct tttctgagat gaaagaaccc cgtttccagg attccagctc tagtaaagct 420
ccccctcctt ccctgccctc accctcaaga ctgcctggac cttccgacac tcccatcctg 480
ccacagtttc ctgatgggga gttcaccatg cagggttgcc cagagtgtaa actgaaggaa 540
aacaaatact tctccaagct gggggccccc atctatcagt gtatgggttg ctgtttctcc 600
agagcctacc ccacacctgc tcgcagtaag aaaactatgc tggtgcctaa gaatattact 660
agcgaggcta cctgctgtgt cgctaaagcc ttcaccaagg ccacagtgat gggaaacgcc 720
cgagtcgaga atcacaccga atgccattgt agtacatgct actatcacaa atcaggatcc 780
ggtggcggtt ccggtggagg cggaagcggc ggtggaggat cagtggagtg ccctccatgt 840
ccagcacccc ctgtcgcagg tccatctgtg ttcctgtttc cacccaagcc taaagacact 900
ctgatgatct cccgcacccc agaagtcacc tgtgtggtcg tggatgtgag ccatgaagac 960
cccgaggtcc agttcaattg gtacgtggat ggcgtcgagg tgcacaacgc taagacaaaa 1020
cctagagaag agcagttcaa ctctaccttt cgcgtcgtga gtgtgctgac agtcgtgcac 1080
caggactggc tgaatggcaa ggagtataag tgcaaagtga gcaacaaagg actgcctgcc 1140
tcaatcgaaa agactatttc caagaccaaa ggacagccaa gagagcccca ggtgtacacc 1200
ctgcctccaa gccgcgaaga gatgactaaa aatcaggtct ctctgacctg tctggtgaag 1260
gggttttatc ctagtgatat cgccgtggaa tgggagtcaa acggtcagcc agagaacaat 1320
tacaagacca caccccctat gctggacagc gatgggtctt tctttctgta tagcaaactg 1380
acagtggaca agtctcggtg gcagcagggt aacgtcttct cttgcagtgt gatgcacgaa 1440
gcactgcaca atcattacac ccagaagtca ctgtcactga gcccaggaaa atga 1494
<210> 8
<211> 497
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Lys Ser Leu Gln Phe Cys Phe Leu Phe Cys Cys Trp Lys Ala Ile
1 5 10 15
Cys Cys Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Thr Val Glu Lys
20 25 30
Glu Glu Cys Asn Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly
35 40 45
Tyr Cys Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Asn
50 55 60
Ile Gln Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Lys
65 70 75 80
Val Pro Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val
85 90 95
Ala Thr Glu Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys
100 105 110
Thr Val Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Ser Glu Met Lys
115 120 125
Glu Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser
130 135 140
Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu
145 150 155 160
Pro Gln Phe Pro Asp Gly Glu Phe Thr Met Gln Gly Cys Pro Glu Cys
165 170 175
Lys Leu Lys Glu Asn Lys Tyr Phe Ser Lys Leu Gly Ala Pro Ile Tyr
180 185 190
Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Ala Arg
195 200 205
Ser Lys Lys Thr Met Leu Val Pro Lys Asn Ile Thr Ser Glu Ala Thr
210 215 220
Cys Cys Val Ala Lys Ala Phe Thr Lys Ala Thr Val Met Gly Asn Ala
225 230 235 240
Arg Val Glu Asn His Thr Glu Cys His Cys Ser Thr Cys Tyr Tyr His
245 250 255
Lys Ser Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
260 265 270
Gly Ser Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro
275 280 285
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
290 295 300
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
305 310 315 320
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
325 330 335
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val
340 345 350
Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu
355 360 365
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Ser Ile Glu Lys
370 375 380
Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
385 390 395 400
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
405 410 415
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
420 425 430
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu
435 440 445
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
450 455 460
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
465 470 475 480
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
485 490 495
Lys
<210> 9
<211> 1506
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atgaagtccc tgcagttctg ttttctgttt tgttgctgga aggccatctg ttgtaattct 60
tgcgagctga ccaatatcac tatcaccgtg gagaaggagg aatgcaactt ttgtatctcc 120
attaatacca catggtgcgc cggctactgt tatacacgag acctggtgta caaagatcca 180
gctcgtccca acatccagaa aacctgcaca ttcaaggagc tggtctatga aactgtgaag 240
gtccctggct gtgcccacca tgctgacagc ctgtacacat atccagtggc cactgagtgc 300
cactgtggaa agtgcgactc agattccaca gattgtactg tcaggggcct gggaccctct 360
tactgcagct tttctgagat gaaagaaccc cgtttccagg attccagctc tagtaaagct 420
ccccctcctt ccctgccctc accctcaaga ctgcctggac cttccgacac tcccatcctg 480
ccacagtttc ctgatgggga gttcaccatg cagggttgcc cagagtgtaa actgaaggaa 540
aacaaatact tctccaagct gggggccccc atctatcagt gtatgggttg ctgtttctcc 600
agagcctacc ccacacctgc tcgcagtaag aaaactatgc tggtgcctaa gaatattact 660
agcgaggcta cctgctgtgt cgctaaagcc ttcaccaagg ccacagtgat gggaaacgcc 720
cgagtcgaga atcacaccga atgccattgt agtacatgct actatcacaa atcaggatcc 780
ggtggcggtt ccggtggagg cggaagcggc ggtggaggat cagacaaaac tcacacatgc 840
ccaccgtgcc cagcacctga agtcgcgggg ggaccgtcag tcttcctctt ccccccaaaa 900
cccaaggaca ccctcatgat ctcccggaca cctgaggtca catgcgtggt ggtggacgtg 960
agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 1020
gccaagacaa agccgcggga ggagcagtac aacagcacgt accgggtggt cagcgtcctc 1080
accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa 1140
gccctcccag cctccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 1200
caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 1260
tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 1320
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1380
tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1440
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1500
aaatga 1506
<210> 10
<211> 501
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Lys Ser Leu Gln Phe Cys Phe Leu Phe Cys Cys Trp Lys Ala Ile
1 5 10 15
Cys Cys Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Thr Val Glu Lys
20 25 30
Glu Glu Cys Asn Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly
35 40 45
Tyr Cys Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Asn
50 55 60
Ile Gln Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Lys
65 70 75 80
Val Pro Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val
85 90 95
Ala Thr Glu Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys
100 105 110
Thr Val Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Ser Glu Met Lys
115 120 125
Glu Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser
130 135 140
Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu
145 150 155 160
Pro Gln Phe Pro Asp Gly Glu Phe Thr Met Gln Gly Cys Pro Glu Cys
165 170 175
Lys Leu Lys Glu Asn Lys Tyr Phe Ser Lys Leu Gly Ala Pro Ile Tyr
180 185 190
Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Ala Arg
195 200 205
Ser Lys Lys Thr Met Leu Val Pro Lys Asn Ile Thr Ser Glu Ala Thr
210 215 220
Cys Cys Val Ala Lys Ala Phe Thr Lys Ala Thr Val Met Gly Asn Ala
225 230 235 240
Arg Val Glu Asn His Thr Glu Cys His Cys Ser Thr Cys Tyr Tyr His
245 250 255
Lys Ser Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
260 265 270
Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Val
275 280 285
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
290 295 300
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
305 310 315 320
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
325 330 335
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
340 345 350
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
355 360 365
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
370 375 380
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
385 390 395 400
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
405 410 415
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
420 425 430
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
435 440 445
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
450 455 460
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
465 470 475 480
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
485 490 495
Leu Ser Pro Gly Lys
500
<210> 11
<211> 492
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atgaggagcc tcggggccct gctcttgctg ctgagcgcct gcctggcggt gagcgctaac 60
tcatgtgagc tgactaatat caccattgcc atcgaaaaag aggaatgcag gttctgtatt 120
agtatcaaca ctacctggtg cgctggctac tgttatacaa gggatctggt gtataaggac 180
ccagcacggc ccaaaatcca gaagacatgc actttcaaag aactggtgta cgagactgtg 240
agggtccctg gctgtgccca ccatgctgat tccctgtaca cttatccagt ggccacccag 300
tgccactgtg gaaagtgcga tagtgactca acagactgta ctgtgcgagg cctgggacct 360
tcttactgca gttttggcga aatgaaggag ccccgtttcc aggattccag ctctagtaaa 420
gctccccctc cttccctgcc ctcaccctca agactgcctg gaccttccga cactcccatc 480
ctgccacagt ga 492
<210> 12
<211> 163
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Arg Ser Leu Gly Ala Leu Leu Leu Leu Leu Ser Ala Cys Leu Ala
1 5 10 15
Val Ser Ala Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Ala Ile Glu
20 25 30
Lys Glu Glu Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala
35 40 45
Gly Tyr Cys Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro
50 55 60
Lys Ile Gln Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val
65 70 75 80
Arg Val Pro Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro
85 90 95
Val Ala Thr Gln Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp
100 105 110
Cys Thr Val Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met
115 120 125
Lys Glu Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro
130 135 140
Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile
145 150 155 160
Leu Pro Gln
<210> 13
<211> 336
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
atgaggagcc tcggggccct gctcttgctg ctgagcgcct gcctggcggt gagcgctgcc 60
cccgatgtgc aggactgccc tgaatgtact ctgcaggaga accccttctt ttctcagccc 120
ggcgctccta tcctgcagtg tatgggatgc tgttttagta gagcatatcc taccccactg 180
cgctcaaaga aaacaatgct ggtccagaag aatgtgacaa gcgaatctac ttgctgtgtg 240
gctaaatcct acaaccgcgt gaccgtgatg ggcggcttca aggtggagaa tcacacagca 300
tgccattgtt ctacttgcta ctaccataag agttag 336
<210> 14
<211> 111
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Met Arg Ser Leu Gly Ala Leu Leu Leu Leu Leu Ser Ala Cys Leu Ala
1 5 10 15
Val Ser Ala Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln
20 25 30
Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met
35 40 45
Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys
50 55 60
Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val
65 70 75 80
Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu
85 90 95
Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser
100 105 110
<210> 15
<211> 489
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
atgaagtccc tgcagttctg ttttctgttt tgttgctgga aggccatctg ttgtaattct 60
tgcgagctga ccaatatcac tatcaccgtg gagaaggagg aatgcaactt ttgtatctcc 120
attaatacca catggtgcgc cggctactgt tatacacgag acctggtgta caaagatcca 180
gctcgtccca acatccagaa aacctgcaca ttcaaggagc tggtctatga aactgtgaag 240
gtccctggct gtgcccacca tgctgacagc ctgtacacat atccagtggc cactgagtgc 300
cactgtggaa agtgcgactc agattccaca gattgtactg tcaggggcct gggaccctct 360
tactgcagct tttctgagat gaaagaaccc cgtttccagg attccagctc tagtaaagct 420
ccccctcctt ccctgccctc accctcaaga ctgcctggac cttccgacac tcccatcctg 480
ccacagtga 489
<210> 16
<211> 162
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Met Lys Ser Leu Gln Phe Cys Phe Leu Phe Cys Cys Trp Lys Ala Ile
1 5 10 15
Cys Cys Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Thr Val Glu Lys
20 25 30
Glu Glu Cys Asn Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly
35 40 45
Tyr Cys Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Asn
50 55 60
Ile Gln Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Lys
65 70 75 80
Val Pro Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val
85 90 95
Ala Thr Glu Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys
100 105 110
Thr Val Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Ser Glu Met Lys
115 120 125
Glu Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser
130 135 140
Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu
145 150 155 160
Pro Gln
<210> 17
<211> 363
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
atggactact atcggaagta tgctgcagtg atcctggcta ttctgtccgt cttcctgcag 60
attctgcata gctttcctga tggggagttc accatgcagg gttgcccaga gtgtaaactg 120
aaggaaaaca aatacttctc caagctgggg gcccccatct atcagtgtat gggttgctgt 180
ttctccagag cctaccccac acctgctcgc agtaagaaaa ctatgctggt gcctaagaat 240
attactagcg aggctacctg ctgtgtcgct aaagccttca ccaaggccac agtgatggga 300
aacgcccgag tcgagaatca caccgaatgc cattgtagta catgctacta tcacaaatca 360
tag 363
<210> 18
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Met Asp Tyr Tyr Arg Lys Tyr Ala Ala Val Ile Leu Ala Ile Leu Ser
1 5 10 15
Val Phe Leu Gln Ile Leu His Ser Phe Pro Asp Gly Glu Phe Thr Met
20 25 30
Gln Gly Cys Pro Glu Cys Lys Leu Lys Glu Asn Lys Tyr Phe Ser Lys
35 40 45
Leu Gly Ala Pro Ile Tyr Gln Cys Met Gly Cys Cys Phe Ser Arg Ala
50 55 60
Tyr Pro Thr Pro Ala Arg Ser Lys Lys Thr Met Leu Val Pro Lys Asn
65 70 75 80
Ile Thr Ser Glu Ala Thr Cys Cys Val Ala Lys Ala Phe Thr Lys Ala
85 90 95
Thr Val Met Gly Asn Ala Arg Val Glu Asn His Thr Glu Cys His Cys
100 105 110
Ser Thr Cys Tyr Tyr His Lys Ser
115 120
<210> 19
<211> 99
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
ccccgtttcc aggattccag ctctagtaaa gctccccctc cttccctgcc ctcaccctca 60
agactgcctg gaccttccga cactcccatc ctgccacag 99
<210> 20
<211> 33
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu
1 5 10 15
Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro
20 25 30
Gln
Claims (6)
1.重组FSH融合蛋白在母猪批次化生产中的应用,其特征在于,所述融合蛋白任选自:
(1)重组hFSH-CTP融合蛋白由hFSHα亚基和hFSHβ-CTP亚基组成,其中hFSHβ-CTP亚基的氨基酸序列从N端到C端包含hFSHβ亚基和CTP;或,
(2)重组pFSH-CTP融合蛋白由pFSHα亚基和pFSHβ-CTP亚基组成,其中pFSHβ-CTP亚基的氨基酸序列从N端到C端依次包含pFSHβ亚基和CTP。
2.根据权利要求1所述的应用,其中(1)和(2)中所述的CTP的氨基酸序列为来自hCGβ链羧基末端的33个氨基酸残基,如SEQ ID NO:20所示。
3.根据权利要求1~2中任一项所述的应用,其中(1)中所述融合蛋白中包含的hFSHβ-CTP亚基的氨基酸序列如SEQ ID NO:12所示,所述融合蛋白中包含的hFSHα亚基的氨基酸序列如SEQ ID NO:14所示;其中(2)中所述融合蛋白中包含的pFSHβ-CTP亚基的氨基酸序列如SEQ ID NO:16所示,所述融合蛋白中包含的pFSHα亚基的氨基酸序列如SEQ ID NO:18所示。
4.根据权利要求1~3中任一项所述的应用,其中所述母猪为经产母猪或后备母猪。
5.根据权利要求1~4中任一项所述的应用,其中所述重组FSH融合蛋白的制备方法包括以下步骤:
a)构建编码重组hFSH-CTP或pFSH-CTP融合蛋白的基因表达载体
采用人工合成方法获得编码人hFSH-CTP或pFSH-CTP融合蛋白的基因,插入到哺乳动物细胞表达载体,获得含有hFSH-CTP或pFSH-CTP融合蛋白基因的表达质粒;
b)重组hFSH-CTP或pFSH-CTP融合蛋白在哺乳动物宿主细胞中的稳定表达
将含有hFSH-CTP或pFSH-CTP融合蛋白的表达载体转染到哺乳动物宿主细胞,筛选稳定表达hFSH-CTP或pFSH-CTP融合的细胞株;
c)高密度细胞培养重组hFSH-CTP或pFSH-CTP融合蛋白;
d)重组hFSH-CTP或pFSH-CTP融合蛋白的纯化:包括使用分段硫酸铵沉淀、沉淀复溶及超滤换液、阴离子柱层析和疏水柱层析进行纯化。
6.根据权利要求5所述的应用,其中所述的基因表达载体为pCDNA3、pCMV/ZEO、pIRES、pDR、pBK、pSPORT或pCMV-DHFR,优选为pCDNA3,更优选为经过基因改造后的pCDNA3;其中所述的细胞转染方法包括电穿孔转染方法、磷酸钙转染、脂质体转染和原生质融合,优选为电穿孔转染方法;其中所述的哺乳动物宿主细胞包括CHO、HEK293、BHK、NS0和Sp2/0细胞,优选为CHO细胞,更优选为DHFR酶缺陷型CHO悬浮细胞(CHO DHFR-)。
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