CN108611371A - 乳汁中表达gp5-m蛋白的奶山羊乳腺生物反应器的制备方法 - Google Patents
乳汁中表达gp5-m蛋白的奶山羊乳腺生物反应器的制备方法 Download PDFInfo
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Abstract
本发明公开了乳汁中表达GP5‑M融合蛋白的羊乳腺生物反应器的制备方法,制备GP5‑M融合表达的基因打靶载体,敲入奶山羊β‑乳球蛋白第一外显子上,筛选中靶细胞,利用体细胞核移植技术制备转基因奶山羊,通过乳腺生物反应器生产含有GP5‑M融合蛋白的乳汁,从乳汁中分离纯化GP5‑M融合蛋白,添加佐剂制备基因工程亚单位疫苗,达到防控PRRS的目的。
Description
技术领域
本发明涉及动物基因工程技术领域,特别是涉及乳汁中表达GP5-M融合蛋白的羊乳腺生物反应器的制备方法。
背景技术
猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS),俗称为猪蓝耳病,是由猪繁殖与呼吸综合征病毒(PRRSV)引起的一种高度接触性的传染病,临床上表现为母猪晚期流产、早产、死胎和木乃伊胎等繁殖障碍,仔猪严重的呼吸道症状,死亡率高。该病在全世界大范围流行,给养猪业造成了巨大的经济损失。根据核苷酸序列差异,病毒可分欧洲和美洲两个基因型。我国分离的一系列PRRSV毒株大多是美洲型,对我国造成巨大经济损失,2008年该病被列为一类动物疫病。
疫苗接种是防治该病的主要手段。目前,已经获得批准上市的PRRSV疫苗包括灭活疫苗和弱毒疫苗。PRRS弱毒疫苗具有多种优势,包括诱导的免疫力强、保护期长、抗体产生速度快,而且母猪免疫接种可以使幼仔猪获得被动免疫。当前广泛使用的弱毒疫苗主要有PRRSATP(Boehringer Ingelheim)、PRRS MLV、PRRS CH-1R株弱毒苗、Porcilis PRRS V(Netherland)、PrimePacPRRS(Schering-Plough),以及HP-PPRS V变异株HuN4弱毒苗等。虽然PRRS弱毒疫苗有很多优点,但是在安全性方面存在一定的问题。研究发现,在接种弱毒疫苗后,导致母猪繁殖性能障碍,有的出现死胎和木乃伊胎,还可能造成疫苗毒的传播,引起PRRSV的持续感染,出现典型的PRRS症状。因此,人们在使用弱毒疫苗时极为慎重。在未发生PRRS的猪场中避免使用弱毒疫苗,不用弱毒疫苗免疫种猪,在欧洲一些国家禁止用弱毒疫苗。灭活疫苗在防治PRRS中最早应用的疫苗,有多株商品化的安全的PRRS灭活苗,其灭活疫苗的使用不存在安全性问题,便于贮存和运输。目前常使用的灭活苗有加拿大的北美洲型PRRSV(IAF-klop)灭活苗,法国的欧洲型PRRSV灭活苗,德国PRRomiSe及我国的CH-la株灭活苗。用PRRS灭活疫苗免疫猪后,可刺激猪体产生特异性抗体,起到免疫保护作用。但是灭活苗也存在一些缺点,灭活过程中可能破坏抗原表位,一次免疫不能产生足够的免疫效力,需要重复接种,免疫剂量大,成本增加。
当前,商品化的PRRS疫苗主要是灭活苗与弱毒苗,二者都存在一定的缺陷。为了预防和控制PRRS发生,急需要研发出安全、高效和廉价的新型疫苗。最新的研究表明,使用DNA重组生物技术,将病毒保护性抗原基因***细菌、酵母菌或哺乳动物细胞中,使之充分表达,经纯化后可制备基因工程亚单位疫苗。开发高效的基因工程疫苗成为当前PRRSV疫苗研究的一个热点,但是目前尚未取得突破,没有形成商品化的基因工程疫苗。有研究表明,PRRSV基因组大小约为15kb,编码至少9个开放阅读框(openreading frames,ORFs),编码主要结构蛋白GP5、M和N蛋白,次要结构蛋白GP2、GP3和GP4,以及一些与病毒转录和复制有关的酶。近年来,随着对PRRSV致病机制研究的不断深入,己经阐明该病毒的结构蛋白GP3、GP5和M蛋白是其主要的保护性抗原。其中GP5具有主要的保护性抗原位点,在体液免疫中有显著优势,诱导机体产生中和性抗体,M蛋白能诱导机体产生较强的细胞免疫。因此,PRRS基因工程疫苗的研究中,往往以这两个基因为主要的靶基因。Jiang Y(Vaccine,2006b,24(3):2869-2879)等构建了单独表达GP5和M的DNA疫苗,小鼠和猪免疫试验表明,用GP5和M载体共同免疫的小鼠和猪体,产生了显著增强的抗体水平和淋巴细胞增殖反应,独用GP5或M载体免疫,体内中和抗体水平较低,甚至不产生中和抗体。Jiang W(Vet ImmunolImmunopathol,2006a,113(1-2):169-180)等用重组腺病毒构建的含有GPS、M及其融合基因GPS+M的疫苗,免疫小鼠显示,含GPS+M的重组腺病毒疫苗能诱导较高的淋巴细胞增殖和一定的病毒特异性反应。用表达截断的GPS和M蛋白的BCG株免疫猪,随后在PRRSV强毒攻击中,免疫猪对阻止PRRSV感染表现出部分保护(Bastos et al.,Vaccine,2002,21:21-29)。由此推测GP5和M形成的二硫键结构可能与GP5的转运和过翻译有关,GP5和M共表达形成的二硫键结构增加了免疫效果,可以作为构建新的DNA疫苗的一种策略。
利用细菌和酵母等生产的药用蛋白虽然成本较低,但是由于这些微生物不能进行精确地转录后加工,不能合成众多的活性蛋白,而哺乳动物细胞体外生成的活性物质的价格极高。因此,全球众多生物技术公司利用转基因动物生物反应器生产药用蛋白和生物分子。一些转基因羊生产的蛋白产品已经进入临床实验阶段。2006年,GTC-Biotherapeutics生产的抗凝血酶(antithrombin III,ATIII)三期临床完成,正式被EuropeanMedicinesAgency(EMEA)批准,作为药物进行生产。该蛋白是首个作为药物的转基因动物产品,在心肺转流术中,用于肝素抵抗的病人。此外,利用乳腺生物反应器研制的重要蛋白有α-葡萄糖苷酶(enzymeα-glucosidase)、人血红蛋白(human hemoglobin)、红细胞生成素(erythropoietin(EPO))和人凝血因子(human clotting factor VIII)等。
因此,制备一种PRRS亚单位疫苗,既可以有效防控PRRS的发生,也可以避免传统灭活疫苗和弱毒疫苗的缺陷是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了乳汁中表达GP5-M融合蛋白的羊乳腺生物反应器的制备方法,利用构建GP5-M融合表达的基因打靶载体,并将其敲入奶山羊β-乳球蛋白第一外显子上,筛选中靶细胞,利用体细胞核移植技术制备转基因奶山羊,通过乳腺生物反应器生产含有GP5-M融合蛋白的乳汁,从乳汁中分离纯化GP5-M融合蛋白,添加佐剂制备基因工程亚单位疫苗,达到防控PRRS的目的。利用奶山羊乳腺生物反应器制备GP5-M融合蛋白,经过纯化后制备PRRS亚单位疫苗,既可以有效防控PRRS的发生,也可以避免传统灭活疫苗和弱毒疫苗的缺陷。
为了实现上述目的,本发明采用如下技术方案:
乳汁中表达GP5-M融合蛋白的羊乳腺生物反应器的制备方法,具体步骤如下:
(1)构建基因打靶载体pBT-GP5-M
a、参照GenBank中猪繁殖与呼吸综合征病毒PRRSV美洲型代表株VR2332的GP5和M蛋白基因序列,设计并合成引物;
用RT-PCR方法分别扩增出PRRSV野毒株GP5和M蛋白基因片段,利用GP linker将两个基因连接起来,同时添加his标签和BGH pA组成GP5-M基因;
b、以克隆山羊β乳球蛋白基因CDS区上游810bp同源序列SEQ ID NO:10和下游715bp同源序列SEQ ID NO:11,分别作为5’端和3’端同源臂,构建基因打靶载体pBT-GP5-M;
(2)打靶载体pBT-GP5-M转染山羊胎儿成纤维细胞
将1-3代的山羊胎儿成纤维细胞,培养至细胞达到80%汇合时,将打靶载体pBT-GP5-M和TALEN载体共同导入山羊胎儿成纤维细胞,筛选出中靶细胞进行接种、扩大培养;
其中,中靶细胞为目的基因整合到奶山羊β-乳球蛋白第一外显子上的细胞;
优选地,TALEN载体的构建方法为:利用TAL Effector靶向预测工具“TALEffector-Nucleotide Targeter(TALE-NT)2.0,根据山羊BLG基因序列(GenBank:Z33881),设计针对BLG exon1的靶序列,根据靶位点的DNA序列,利用单元组装法构建TALEs模块,通过不断的酶切和链接构建与靶位点序列对应的多元模块pTBE1-L和pTBE1-R,然后将模块TBE1-L和TBE1-R分别链接到真核表达载体pCS2-PERR和pCS2-PEAS中,从而构建识别并切割BLG第二外显子位点的TALE表达载体pTALEN-E1-L和pTALEN-E1-R;
(3)中靶细胞的PCR鉴定
对中靶细胞进行PCR鉴定,确认中靶细胞克隆;对中靶细胞克隆进行染色体分析,将染色体数正常比例大于70%的中靶细胞克隆用于核移植,制备转基因奶山羊;
(4)转GP5-M基因克隆奶山羊的制备
将步骤(3)得到的所述中靶细胞100%汇合后,核移植到去核后的***中;放入融合液,再进行融合形成重组胚胎;将所述重组胚胎植入同步发情的受体羊输卵管中,得到转基因克隆羊;
优选地,中靶细胞中靶细胞100%汇合1~3d后用于核移植;
优选地,将所述中靶细胞注入去核***的透明带下,进行融合形成重组胚胎;
(5)转基因克隆奶山羊的检测
提取出生小羊血液基因组DNA,进行PCR检测,结果表达打靶载体正确***山羊β-乳球蛋白基因座第一外显子上,读码框正确;
本发明构建的GP5-M融合蛋白基因打靶载体,敲入奶山羊β-乳球蛋白基因座位点,能够利用内源β-乳球蛋白调控区指导目的基因在山羊的乳腺中特异的高效表达,获得乳汁中表达GP5-M融合蛋白的羊乳腺生物反应器,为GP5-M融合蛋白的制备奠定基础。
进一步地,步骤(1)中所述引物如下:
GP5基因扩增引物:
Forward:TGTATCGTGCCGTTCTATCTTG;SEQ ID NO:1
Reverse:GCAGTTCTTCGCAAGCCTAAT;SEQ ID NO:2
M基因扩增引物:
Forward:TCGGGTACATGACATTCGTGC;SEQ ID NO:3
Reverse:TGCCGCAATCGGATGAAA;SEQ ID NO:4。
进一步地,步骤(3)中所述PCR鉴定的引物如下:
5’junction鉴定引物:
Forward:TAGCAACGCCCAAGTGGATTC;SEQ ID NO:5;
Reverse:AAGCAACTGGCTGGTGGTGAG;SEQ ID NO:6;
3’junction鉴定引物
Forward:CCACCAAGCGAAACATCG;SEQ ID NO:7;
Reverse:TGACCACACACAGGCACC;SEQ ID NO:8。
本发明利用PCR扩增打靶载体与基因组连接处的序列,确定打靶载体正确整合到打靶位点上,扩增序列跨越同源臂,5’junction鉴定上游引物设计在5′端同源臂外侧的基因组上,下游引物位于目的基因GP5上;3’junction鉴定上游引物设计在neo基因上,下游引物位于3′端同源臂外侧基因组上,设计鉴定引物序列。
进一步地,步骤(1)中所述基因打靶载体pBT-GP5-M包括目的基因、his标签、GP5基因序列、GP linker、M基因序列、BGH pA,两个loxP序列和neo基因。其中BGH pA为牛生长激素多聚腺苷加尾信号;neo基因为抗生素筛选基因。本发明构建的基因打靶载体能够利用山羊β乳球蛋白编码区的上游转录调控元件,使所述GP5-M基因进行特异性高效表达。
进一步地,步骤(2)中打靶载体pBT-GP5-M和TALEN载体共同导入山羊胎儿成纤维细胞的方法为电穿孔技术。
本发明利用利用电穿孔技术将靶载体pBT-GP5-M和TALEN载体共同导入山羊胎儿成纤维细胞,其中TALEN打靶载体在所述山羊β乳球蛋白基因第一外显子区产生双链断裂,启动DNA双链断裂修复;同时引入连接有组装所述GP5-M基因的同源臂载体,使所述GP5-M基因特异的***山羊β乳球蛋白基因,利用山羊β乳球蛋白编码区的上游转录调控元件,使所述GP5-M基因得以表达。
进一步地,步骤(4)所述融合液的组成为,0.3mol/L甘露醇、0.05mol/L氯化钙、0.1mol/L硫酸镁、0.27mol/L组氨酸、0.1%BSA。
进一步地,步骤(4)所述融合方法为微电极挤压融合法。
进一步地,步骤(5)中所述PCR检测的引物、反应体系、反应条件与所述步骤(3)中靶细胞鉴定一致。
一种制备GP5-M融合蛋白的方法,所述的乳汁中表达GP5-M融合蛋白的奶山羊乳腺生物反应器制备GP5-M融合蛋白,具体步骤如下:
(1)待基因打靶羊分娩后采集乳样;
(2)乳汁中GP5-M融合蛋白纯化;
a、取转基因羊乳汁,离心脱脂,弃去上层脂肪和下层沉淀杂质,吸取中间层清液,调整pH值到4-6,静置10-25min,离心去除酪蛋白,收集上层乳清;
b、采用Ni层析柱纯化his标签-GP5-M融合蛋白,并进一步纯化蛋白,始终保持蛋白溶液处于低温4℃;
优选地,步骤a中利用乙酸调整PH值;步骤b中利用AKTA purifier蛋白纯化仪进一步纯化蛋白
(3)将纯化后GP5-M蛋白进行Westernblotting鉴定。
本发明公开了利用基因奶山羊乳腺生物反应器生产含有GP5-M融合蛋白的乳汁,从乳汁中分离纯化GP5-M融合蛋白,利用奶山羊乳腺生物反应器生产GP5-M蛋白产量大,成本低,具有重要的经济和社会价值。
GP5-M融合蛋白经纯化后在制备猪繁殖与呼吸综合征疫苗中应用。
通过奶山羊乳腺生物反应器生产的PRRSV主要结构蛋白GP5-M融合蛋白,具有免疫活性,既可以有效防控PRRS的发生,也可以避免传统灭活疫苗和弱毒疫苗的缺陷。
经由上述的技术方案可知,与现有技术相比,本发明提供了乳汁中表达GP5-M蛋白的奶山羊乳腺生物反应器的制备方法,具有如下技术优点:
(1)本发明利用TALEN基因编辑技术,将GP5-M融合蛋白基因打靶载体,敲入奶山羊β-乳球蛋白基因座位点,能够利用内源β-乳球蛋白调控区指导目的基因在山羊的乳腺中特异的高效表达。
(2)奶山羊泌乳期280-300天,产奶量800-1000kg,繁殖周期短,饲养费用低,是研制乳腺生物反应器的重要动物品种,利用奶山羊乳腺生物反应器生产GP5-M融合蛋白,产量大,成本低。
(3)PRRS病毒在动物细胞中复制,结构蛋白在细胞中合成,组装病毒,乳腺细胞表达的GP5-M融合蛋白能够进行正确的化学修饰和加工,相比原核表达的蛋白,更能保持其生物学活性。
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,下面描述中的附图仅仅是本发明的实施例,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,还可以根据提供的附图获得其他的附图。
图1附图为本发明提供的TALEN识别位点的设计。
图2附图为本发明提供的GP5-M基因打靶载体图谱。
图3附图为本发明提供的中靶细胞PCR鉴定引物设计方案。
图4附图为本发明提供的中靶细胞5’端整合位点PCR鉴定结果。
图5附图为本发明提供的中靶细胞克隆3’端整合位点PCR检测结果。
图6附图为本发明提供的中靶细胞。
图7附图为本发明提供的转基因克隆胚胎。
图8附图为本发明提供的受体山羊。
图9附图为本发明提供的转基因羊妊娠B超检查。
图10附图为本发明提供的转基因克隆奶山羊上游整合位点PCR鉴定。
图11附图为本发明提供的转基因克隆奶山羊下游整合位点PCR鉴定。
图12附图为本发明提供的蛋白纯化SDS-PAGE电泳。
图13附图为本发明提供的Westernblotting鉴定融合蛋白表达。
图14附图为本发明提供的小鼠免疫后抗体水平检测。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
基因打靶载体的构建
(1)GP5-M基因的克隆
参照GenBank中猪繁殖与呼吸综合征病毒(PRRSV)美洲型代表株VR2332的GP5和M蛋白基因序列,设计并合成2对引物SEQ ID NO:1-SEQ ID NO:4,引物序列如下:
GP5基因扩增引物:
Forward:TGTATCGTGCCGTTCTATCTTG;SEQ ID NO:1
Reverse:GCAGTTCTTCGCAAGCCTAAT;SEQ ID NO:2
M基因扩增引物:
Forward:TCGGGTACATGACATTCGTGC;SEQ ID NO:3
Reverse:TGCCGCAATCGGATGAAA;SEQ ID NO:4
提取病毒基因组RNA,利用随机引物反转录合成cDNA,以此cDNA为模板利用上述引物,用RT-PCR方法即反应条件:98℃3min;98℃10sec,54℃30sec,72℃50sec(30Cycles);72℃10min;4℃forever。
反应体系:反应体系按TaKaRa EmeraldAmp PCR MasterMix说明书配制(20μl):
分别扩增出PRRSV野毒株GP5和M蛋白基因片段603bp和522bp,并利用GP linker将两个基因连接起来,同时添加表达和纯化相关元件即his标签和BGH pA,组装成GPS-M基因表达序列SEQ ID NO:9。
(2)基因打靶载体pBT-GP5-M图谱
利用TAL Effector靶向预测工具“TAL Effector-Nucleotide Targeter(TALE-NT)2.0,根据山羊BLG基因序列(GenBank:Z33881),设计针对BLG exon1的靶序列,如图1所示,根据靶位点的DNA序列,利用单元组装法构建TALEs模块,通过不断的酶切和链接构建与靶位点序列对应的多元模块pTBE1-L和pTBE1-R,然后将模块TBE1-L和TBE1-R分别链接到真核表达载体pCS2-PERR和pCS2-PEAS中,从而构建识别并切割BLG第二外显子位点的TALE表达载体pTALEN-E1-L和pTALEN-E1-R。
根据上述TALEN打靶载体,其在山羊β乳球蛋白(C.hircus beta-lactoglobulin)基因第一外显子区产生双链断裂,启动DNA双链断裂修复,同时引入连接有组装目的基因的同源臂载体,利用DNA双链断裂引发的DNA重组修复过程,使组装的目的DNA序列特异的***山羊β乳球蛋白基因,利用山羊β乳球蛋白编码区的上游转录调控元件,使目的基因得以表达。克隆山羊β乳球蛋白基因CDS区上游810bp同源序列SEQ ID NO:10,其中包含β-乳球蛋白信号肽序列和下游715bp同源序列SEQ ID NO:11,分别作为5’端和3’端同源臂,构建基因打靶载体pBT-GP5-M,如图2。
实施例2
打靶载体pBT-GP5-M转染山羊胎儿成纤维细胞
山羊胎儿成纤维细胞分离于怀孕40天的胎儿组织,即将山羊胎儿剖腹产采集,去除四肢、头和内脏,组织块剪碎培养,细胞汇合后冻存。从液氮中取原代山羊胎儿成纤维细胞于38℃解冻,离心,弃上清,加细胞培养液DMEM/F12+10%FBS重悬,取细胞悬浮液接种于6孔板中,置于培养箱中38℃,5%CO2条件下培养。待1-3代山羊胎儿成纤维细胞达到80%汇合时,吸弃培养液,用无Ca2+、Mg2+的PBS冲洗细胞,加入胰酶消化液,消化细胞。在倒置显微镜下观察细胞,待大多数细胞回缩、变圆、细胞间隙扩大时,用DMEM/F12+10%FBS的细胞培养液终止消化,用移液器吹打后,离心收集,悬浮,按1∶3的比例接种于6孔板,放入CO2培养箱中培养。将打靶载体pBT-GP5-M和TALEN载体利用电穿孔技术共同导入山羊胎儿成纤维细胞,即,以TALEN载体5μg和打靶载体质粒10μg加入400μL细胞电转染液(Cyto Salts:Opti-MEM=3:1,Cyto Salts:KCl 120mM,CaCl2·2H2O 0.15mM,K2HPO410mM,MgCl2·6H2O 5mM),并使用上述混合液重新悬浮细胞,将细胞悬液转移到用细胞电转染液润洗过的BTX电穿孔杯,使用BTX ECM2001电穿孔仪进行电转染,即电压500V持续1ms,3次电击,并将电转染后的胎儿成纤维细胞悬液,转移到4个100mm细胞培养皿中培养。最后利用药物筛选7-10天,其中,本发明以G418作为筛选药物,由于表达载体pBT-GP5-M的骨架上有G418抗性基因neo,pBT-GP5-M成功转染的山羊胎儿成纤维细胞能够在含有一定浓度G418的培养液中存活下来,而未转染成功的正常细胞会在该浓度下死亡。当平皿中出现细胞克隆,挑取细胞克隆分别接种于24孔板中,扩大培养,一部分细胞冻存,另一部分细胞用于鉴定。
实施例3
中靶细胞的PCR鉴定
设计鉴定引物如图3所示,利用PCR扩增打靶载体与基因组连接处的序列,确定打靶载体正确整合到打靶位点上,扩增序列跨越同源臂,5’junction鉴定上游引物设计在5′端同源臂外侧的基因组上,下游引物位于目的基因GP5上;3’junction鉴定上游引物设计在neo基因上,下游引物位于3′端同源臂外侧基因组上;
上述鉴定引物序列如下:
5’junction鉴定引物(PCR产物1226bp):
Forward:TAGCAACGCCCAAGTGGATTC;SEQ ID NO:5
Reverse:AAGCAACTGGCTGGTGGTGAG;SEQ ID NO:6
3’junction鉴定引物(PCR产物2388bp)
Forward:CCACCAAGCGAAACATCG;SEQ ID NO:7
Reverse:TGACCACACACAGGCACC;SEQ ID NO:8
其中,PCR扩增反应条件如下:
5’junction:98℃3min;98℃10sec,60℃30sec,72℃1min20sec(30Cycles);72℃10min;12℃forever。
3’junction:98℃3min;98℃10sec,60℃30sec,72℃2min30sec(30Cycles);72℃10min;12℃forever。
PCR反应体系:反应体系按TaKaRaEmeraldAmp PCRMasterMix说明书配制(20μl):
PCR产物进行1%琼脂糖凝胶电泳检测,检测结果如图4和5所示:实验筛选细胞克隆48个,利用PCR进行鉴定,结果如图所示,将扩增获得的5′端连接序列(1228bp)和3′端连接序列(2388bp)进行胶回收,然后连接、转化和测序,通过比对最终确认中靶的细胞株有9株,分别为1、5、14、15、37、38、44、47和48号细胞克隆。对细胞克隆进行染色体分析,染色体数正常比例大于70%的细胞克隆(5和44号)用于核移植,制备转基因奶山羊。
实施例4
转GP5-M基因克隆奶山羊的制备
转基因细胞,100%汇合后1~3d用于核移植。从屠宰场采集的卵巢处理干净,用灭菌手术刀片将卵巢表面2~6mm的卵泡切开,体式显微镜下采集卵丘***复合物(Cumulus-oocyte complexes,COCs),按本实验室建立的成熟条件成熟培养22~24h,用0.1%透明质酸酶吹打消化去除颗粒细胞,选择排出第一极体的优质***去核。在显微镜下吸出第1极体及部分胞质,供体细胞注入到去核***的透明带下。注射后的细胞-卵胞质重组体以微电极挤压融合法用两次电脉冲即32V,每次20μs的条件下进行融合。融合前先将重组体放入融合液中预平衡4~6min。其中融合液的组成为0.3mol/L甘露醇、0.05mol/L氯化钙、0.1mol/L硫酸镁、0.27mol/L组氨酸、0.1%BSA。将融合后的胚胎放入10%FBS的M199中,添加7.5ug/mL的CB,38.5℃、5%CO2、饱合湿度下培养,2~3h后观察融合情况。融合的重组胚用含5μmol/L的Ionomycin处理4min,然后在含2mmol/L 6-Dimethylaminopurine(6-DMAP)的培养液内培养3h,洗3次后转移到mSOFaa或G1/G2培养液中进行培养。培养7d后检查囊胚发育率,或将培养20~24h的克隆胚胎移植入同步发情的受体羊输卵管中,每月用B超检查妊娠情况。
如图6-9所示,利用中靶细胞,构建转基因克隆胚胎,本发明共移植转GP5-M基因克隆胚胎418枚到19头同期发情的受体奶山羊体内,一个月后有7头建立妊娠,3个月后有5头维持妊娠并继续被监测,共生产转基因克隆羊6只,3只存活到成年,命名为TG-GP5-M-2,TG-GP5-M-3和TG-GP5-M-6。
实施例5
转基因克隆奶山羊的检测
提取出生小羊血液基因组DNA,进行PCR检测,引物、反应体系、反应条件与中靶细胞鉴定一致,见图10-11。将PCR产物进行胶回收,然后连接、转化和测序,结果表达打靶载体正确***山羊β-乳球蛋白基因座第一外显子上,读码框正确。待基因打靶羊生长到15-16月龄时,自然发情,与普通公羊配种,检测妊娠情况。分娩后不同时间段采集乳样,用于蛋白纯化。
实施例6
乳汁中GP5-M蛋白纯化
取100ml转基因羊乳汁,4℃10000g离心30min脱脂,弃去上层脂肪和下层沉淀杂质,吸取中间层清液,用乙酸调整PH值到4.6,静置15min,4℃10000g离心30min去除酪蛋白,收集上层乳清备用。本方法采用Ni层析柱纯化his标签-GP5-M融合蛋白,使用AKTApurifier蛋白纯化仪纯化,始终保持蛋白溶液处于低温4℃,并快速操作,具体操作步骤为:(1)利用0.45μm滤膜过滤乳清,加入1/10体积的Binding Buffer;(2)开机后,用20%乙醇洗泵,自动停止后将柱子连入***,流速设定为1mL/min,用20%乙醇洗泵30mL;(3)用ddH20洗探头,将A探头放入Binding Buffer,B探头放入Elution Buffer,洗泵;(4)自动用BindingBuffer平衡柱子;(5)待蓝线到最高且水平时,可以上样。将A探头放入样品中,流速调为0.5mL/min,红线最高水平时刻接一管样;(6)上样完后暂停,用ddH20洗探头,放回BindingBuffer,继续平衡柱子。待红线开始下降时将流速设定为1mL/min;(7)待红线下降至最低且水平时,即可洗脱。调整B(Elution Buffer)的百分量,可从10%开始,红线有峰出现就收集样品,一般50%之前洗一个峰,再直接50%洗脱,最后100%洗脱,每次洗20mL左右。(8)洗脱完成后用ddH20洗探头,两个探头都放回20%乙醇中,洗泵,再用20%乙醇过柱,直至红线和蓝线均下降到最低且水平,纯化即结束。(9)样品处理后SDS-PAGE检测,如图12所示,不同阶段收集样品蛋白纯度分析,第5~6泳道蛋白含量和纯度较高,将纯度较高的样品测定蛋白浓度,分装,-80℃保存。
实施例7
转基因羊乳汁中GP5-M融合蛋白的Westernblotting鉴定
取20μL转基因羊乳汁样品,加入2×蛋白上样缓冲液,煮沸5min制备样品。取10μL上述制备的样品加入到12%的SDS-PAGE蛋白胶胶槽中,进行电泳。将胶卸下,按由上至下的顺序,将滤纸、凝胶、NC膜和滤纸依次叠放在一起,然后用玻璃管赶走各层之间的气泡。注意上下层滤纸不能接触否则短路,在半干转膜仪上以15V电压,转印45min。用TBST洗涤NC膜3次,3min/次,转印的NC膜在5%脱脂乳中封闭过夜。TBST洗涤3次,5min/次,用5000倍稀释的临床分离猪阳性血清作为一抗,室温孵育1h。TBST洗涤NC膜3次,5min/次,用HRP标记的兔抗猪二抗室温孵育1h。TBST洗涤NC膜3次,5min/次,增强型化学发光试剂盒的A液和B液混合后加入NC膜上,室温孵育5min,曝光显影,水洗终止。如图13所示,图中显示前三个泳道转基因羊乳样显示出条带,第四个泳道为普通羊乳样照组没有条带;下图为乳汁中β-酪蛋白检测,转基因羊和普通羊乳样均有条带,证明转基因羊乳样中含有GP5-M融合蛋白。
实施例8
纯化的GP5-M蛋白免疫小鼠后产生PRRSV特异性抗体水平监测
Balb/c小鼠40只,雌性,清洁级,6-8周龄,随机分成2组,一组利用20μg纯化的GP5-M融合蛋白加100μL佐剂乳化后免疫小鼠,另一组注射100μL佐剂(对照组)。以相同方法免疫3次,每免疫间隔2周,首次免疫后0、3、5、7、9和11周于小鼠的眼眶静脉丛采血,分离血清,ELISA法检测小鼠血清中抗体水平。具体操作:包被抗原为灭活的PRRSV,每孔100μL于4℃包被过夜,PBST洗涤3次,每次3min;5%小牛血清置37℃封闭40min,PBST洗涤3次,每次3min;小鼠血清稀释100倍,每孔100μL,37℃作用1h,PBST洗涤3次,每次3min;辣根过氧化物酶标记的羊抗鼠IgG抗体,稀释倍数为1:3000,每孔100μL,37℃作用40min,PBST洗涤3次,每次3min;加入TMB和过氧化氢尿素溶液每孔各50μL,室温作用10min,每孔再加入终止液(2mol/LH2SO4溶液),酶标仪下读取450nm波长OD值并记录。
结果如图14所示,首免后3周实验组与对照组抗体水平差异不显著,5-7周注射GP5-M融合蛋白组小鼠抗体水平显著升高,9-11周实验组小鼠抗体水平极显著升高;对照组小鼠抗体水平不变。实验结果说明,山羊乳腺表达的GP5-M融合蛋白能作为免疫原刺激小鼠产生特异性抗体。
本发明提供了乳汁中表达GP5-M融合蛋白的羊乳腺生物反应器的制备方法,利用构建GP5-M融合表达的基因打靶载体,并将其敲入奶山羊β-乳球蛋白第一外显子上,筛选中靶细胞,利用体细胞核移植技术制备转基因奶山羊,通过乳腺生物反应器生产含有GP5-M融合蛋白的乳汁,从乳汁中分离纯化GP5-M融合蛋白,添加佐剂制备基因工程亚单位疫苗,达到防控PRRS的目的。利用奶山羊乳腺生物反应器制备GP5-M融合蛋白,经过纯化后制备PRRS亚单位疫苗,既可以有效防控PRRS的发生,也可以避免传统灭活疫苗和弱毒疫苗的缺陷。
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。对于实施例公开的装置而言,由于其与实施例公开的方法相对应,所以描述的比较简单,相关之处参见方法部分说明即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
序列表
<110> 西北农林科技大学
<120> 乳汁中表达GP5-M蛋白的奶山羊乳腺生物反应器的制备方法
<160> 11
<170> SIPOSequenceListing 1.0
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<212> DNA
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tgtatcgtgc cgttctatct tg 22
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<213> Artificial Sequence
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gcagttcttc gcaagcctaa t 21
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<212> DNA
<213> Artificial Sequence
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tcgggtacat gacattcgtg c 21
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<211> 18
<212> DNA
<213> Artificial Sequence
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tgccgcaatc ggatgaaa 18
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<211> 21
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<213> Artificial Sequence
<400> 5
tagcaacgcc caagtggatt c 21
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 6
aagcaactgg ctggtggtga g 21
<210> 7
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 7
ccaccaagcg aaacatcg 18
<210> 8
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 8
tgaccacaca caggcacc 18
<210> 9
<211> 1377
<212> DNA
<213> Artificial Sequence
<400> 9
atgttgggga agtgcttgac cgcgtgctgt tgctcgcgat tgcttttttt gtggtgtatc 60
gtgccgttct atcttgctgt gctcgtcaac gccagcaaca acaacagctc tcatattcag 120
ttgatttata acttaacgct atgtgagctg aatggcacag attggctggc acaaaaattt 180
gactgggcag tggagacttt tgtcatcttc cccgtgttga ctcacattgt ttcctatggg 240
gcactcacca ccagccattt ccttgacaca gttggtctgg ccactgtgtc caccgccgga 300
tattatcacg ggcggtatgt cttgagtagc atttacgcag tctgtgctct ggctgcgctg 360
atttgctttg tcattaggct tgcgaagaac tgcatgtcct ggcgctactc ttgtaccaga 420
tataccaact tccttttgga cactaaaggc agactctatc gttggcggtc gcccgtcatt 480
gtggagaaag ggggtaaggt tgaggtcgaa ggtcacctga tcgacctcaa gagagttgtg 540
cttgatggtt ccgcggcaac ccctttaacc agagtttcag cggaacaatg gggtcgtctc 600
gggcccggac ctatggggtc gtctctagac gacttctgca atgatagcac agctccacag 660
aaggtgcttt tggcgttttc cattacctac acgccagtga tgatatatgc tctaaaggta 720
agtcgcggcc gactgctagg gcttctgcac cttttgattt ttctgaattg tgcttttacc 780
ttcgggtaca tgacattcgt gcactttgag agcacaaata gggtcgcgct cactatggga 840
gcagtagttg cacttctttg gggagtgtac tcagccatag aaacctggaa attcatcacc 900
tccagatgcc gtctgtgctt gctaggccgc aagtacattc tggcccctgc ccaccacgtc 960
gaaagtgccg cgggctttca tccgattgcg gcaaatgata accacgcatt tgtcgtccgg 1020
cgtcccggct ccactacggt caacggcaca ttggtgcccg ggttggaaag cctcgtgttg 1080
ggtggcagaa aagctgttaa gcagggagtg gtaaaccttg ttaaatatgc caaatgactc 1140
gaccgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct 1200
tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc 1260
attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg 1320
aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gctcgag 1377
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<212> DNA
<213> Artificial Sequence
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gtcctgggag acttaaaatg tgggaggtgg gaggtgggag gttgggccct gtgggcctgc 60
ccatcccgtg tgcctgcatg gagccccagt gctgctcagc cgtgcccccg ccgcagggtc 120
aggtcacttt cccgtcctgg gggttattat gaccgttgtc attttcattg ccatttttgc 180
taccctaact gggcagcagg tgcttgcaga gccctcgata ccgaccaggt cccccctcgg 240
agctccacct gaaccccgtg tcacccttgc cccagcctgc agaggatggg gtcactgcag 300
agatcccttc acccaaggcc acggtcacat ggtttggagg agctggtgcc caaggcagag 360
gccaccctct aggacacacc tgtccccagt gctggctctg acctgccctt gtctaagagg 420
ctgaccccgg aagtgttcct ggcactggca gccagcctga cccagagtcc agacacccac 480
ctgtgccccc acttctgggg tctaccagga accgtctagg cccagagggg gacttcctgc 540
ttggccccgg atggaagaag gcctcctatt gtcctcgtag aggaagccac cccggggccc 600
ggggatgagc caagtaggat tccgggaacc tcgtggctgg gggcccggcc cgggctggct 660
ggctggcacg cctcctgtat aaggccccga gcccgctgtc tcagccctcc actccctgca 720
gagctcagaa gcacgacccc agctgcagcc atgaagtgcc tcctgcttgc cctgggcctg 780
gccctcgcct gtggcatcca ggccatcatc catcatcacc atcaccatgc tagc 834
<210> 11
<211> 727
<212> DNA
<213> Artificial Sequence
<400> 11
gtcgacggcc tggacatcca gaaggttcga gggtggccgg gtgggtggta ggttgcaggg 60
cgggcagggg agctgggcct cagagaccaa gagaggctgt gacgtcgggt tcgcatcagt 120
cagctagggc agcctgacaa atcccccgct ggggcagctt ggaccaggcg ttcactgcct 180
tgcattctgg aggctggaag ccaagatcca ggtgtcggcc aggctggctt ctcctgcggc 240
cgctctccgc ggagagacgg ccgtcttctc catgtcctct gcgagccctg atttcctctt 300
cctgtgaggc cgccaggcct gctggaaaca cgcctgcctg cacagcctca cacgaccttt 360
gtcatctctt taaaggccgt gtctccagag tcatgtgttg aagttctggg ggttagtggg 420
acacagttca gcccctaaaa gagtctctct gcccctcaaa ttttccccac ctccagccat 480
gtctccccaa gatccaaatg ttgccacgtg tgggggggct catctgggtc cctctttggg 540
ctcagagtga gtctggggag agcattcccc agggtgcaga gttgggggga gtatctcagg 600
gctgcccagg ccggggtggg acagagagcc cactgtgggg ctgggggccc cttcccaccc 660
ccggagtgca actcaaggtc cctctccagg tggcggggac ttggtactcc ttggctatgg 720
catcgat 727
Claims (10)
1.乳汁中表达GP5-M融合蛋白的奶山羊乳腺生物反应器的制备方法,其特征在于,具体步骤如下:
(1)构建基因打靶载体pBT-GP5-M
a、参照GenBank中猪繁殖与呼吸综合征病毒PRRSV美洲型代表株VR2332的GP5和M蛋白基因序列,设计并合成引物;
用RT-PCR方法分别扩增出PRRSV野毒株GP5和M蛋白基因片段,利用GP linker将两个基因连接起来,同时添加his标签和BGHpA组成GP5-M基因;
b、以克隆山羊β乳球蛋白基因CDS区上游810bp同源序列SEQ ID NO:10和下游715bp同源序列SEQ ID NO:11,分别作为5’端和3’端同源臂,构建基因打靶载体pBT-GP5-M;
(2)打靶载体pBT-GP5-M转染山羊胎儿成纤维细胞
将1-3代的山羊胎儿成纤维细胞,培养至细胞达到80%汇合时,将打靶载体pBT-GP5-M和TALEN载体共同导入山羊胎儿成纤维细胞,筛选出中靶细胞进行接种并扩大培养;
(3)中靶细胞的PCR鉴定
对中靶细胞进行PCR鉴定,确认中靶细胞克隆;对中靶细胞克隆进行染色体分析,将染色体数正常比例大于70%的中靶细胞克隆用于核移植,制备转基因奶山羊;
(4)转GP5-M基因克隆奶山羊的制备
将步骤(3)得到的所述中靶细胞100%汇合后,核移植到去核后的***中;放入融合液,再进行融合形成重组胚胎;将所述重组胚胎植入同步发情的受体羊输卵管中,得到转基因克隆羊;
(5)转基因克隆奶山羊的检测
提取出生小羊血液基因组DNA,进行PCR检测,结果表达打靶载体正确***山羊β-乳球蛋白基因座第一外显子上,且读码框正确。
2.根据权利要求1所述的乳汁中表达GP5-M蛋白的奶山羊乳腺生物反应器的制备方法,其特征在于,步骤(1)中所述引物如下:
GP5基因扩增引物:
Forward:TGTATCGTGCCGTTCTATCTTG;SEQ ID NO:1
Reverse:GCAGTTCTTCGCAAGCCTAAT;SEQ ID NO:2
M基因扩增引物:
Forward:TCGGGTACATGACATTCGTGC;SEQ ID NO:3
Reverse:TGCCGCAATCGGATGAAA;SEQ ID NO:4。
3.根据权利要求1所述的乳汁中表达GP5-M蛋白的奶山羊乳腺生物反应器的制备方法,其特征在于,步骤(3)中所述PCR鉴定的引物如下:
5’junction鉴定引物:
Forward:TAGCAACGCCCAAGTGGATTC;SEQ ID NO:5;
Reverse:AAGCAACTGGCTGGTGGTGAG;SEQ ID NO:6;
3’junction鉴定引物
Forward:CCACCAAGCGAAACATCG;SEQ ID NO:7;
Reverse:TGACCACACACAGGCACC;SEQ ID NO:8。
4.根据权利要求1所述的乳汁中表达GP5-M蛋白的奶山羊乳腺生物反应器的制备方法,其特征在于,步骤(1)中所述基因打靶载体pBT-GP5-M包括包含目的基因、his标签、GP5基因序列、GP linker、M基因序列、BGH pA,两个loxP序列和neo基因。
5.根据权利要求1所述的乳汁中表达GP5-M融合蛋白的奶山羊乳腺生物反应器的制备方法,其特征在于,步骤(2)中打靶载体pBT-GP5-M和TALEN载体共同导入山羊胎儿成纤维细胞的方法为电穿孔技术。
6.根据权利要求1所述的乳汁中表达GP5-M融合蛋白的奶山羊乳腺生物反应器的制备方法,其特征在于,步骤(4)所述融合液的组成为0.3mol/L甘露醇、0.05mol/L氯化钙、0.1mol/L硫酸镁、0.27mol/L组氨酸和0.1%BSA。
7.根据权利要求1所述的乳汁中表达GP5-M融合蛋白的奶山羊乳腺生物反应器的制备方法,其特征在于,步骤(4)所述融合方法为微电极挤压融合法。
8.根据权利要求1所述的乳汁中表达GP5-M融合蛋白的奶山羊乳腺生物反应器的制备方法,其特征在于,步骤(5)中所述PCR检测的引物、反应体系、反应条件与所述步骤(3)中靶细胞鉴定一致。
9.一种制备GP5-M融合蛋白的方法,其特征在于,根据权利要求1所述乳汁中表达GP5-M融合蛋白的奶山羊乳腺生物反应器制备GP5-M融合蛋白,具体步骤如下:
(1)待基因打靶羊分娩后采集乳样;
(2)乳汁中GP5-M融合蛋白纯化;
a、取转基因羊乳汁,离心脱脂,弃去上层脂肪和下层沉淀杂质,吸取中间层清液,调整pH值到4-6,静置10-25min,离心去除酪蛋白,收集上层乳清;
b、采用Ni层析柱纯化his标签-GP5-M融合蛋白,并进一步纯化蛋白,始终保持蛋白溶液处于低温4℃;
(3)将纯化后GP5-M蛋白进行Westernblotting鉴定。
10.根据权利要求9所述的GP5-M融合蛋白经纯化后在制备猪繁殖与呼吸综合征疫苗中的应用。
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---|---|---|---|---|
CN104726495A (zh) * | 2015-03-25 | 2015-06-24 | 西北农林科技大学 | 一种基于talen介导的基因打靶敲除山羊blg的载体及重组细胞 |
-
2018
- 2018-05-14 CN CN201810457000.2A patent/CN108611371A/zh active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104726495A (zh) * | 2015-03-25 | 2015-06-24 | 西北农林科技大学 | 一种基于talen介导的基因打靶敲除山羊blg的载体及重组细胞 |
Non-Patent Citations (4)
Title |
---|
WENMING JIANG等: "Recombinant adenovirus expressing GP5 and M fusion proteins of porcine reproductive and respiratory syndrome virus induce both humoral and cell-mediated immune responses in mice", 《VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY》 * |
云涛等: "表达猪繁殖与呼吸综合征病毒GP5和M融合蛋白的重组腺病毒的构建及其对小鼠的免疫原性", 《生物工程学报》 * |
兰辉等: "猪繁殖与呼吸综合症病毒结构蛋白GP5-M腺病毒载体的构建及其在山羊乳腺中的表达", 《西北农业学报》 * |
葛恒涛: "利用山羊乳腺生物反应器制备具有免疫原性病毒结构蛋白的研究", 《中国博士学位论文全文数据库 基础科学辑》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111349653A (zh) * | 2020-05-26 | 2020-06-30 | 中国农业大学 | 一种人α-乳清蛋白基因定点整合转基因奶牛的制备方法 |
CN115896227A (zh) * | 2022-12-20 | 2023-04-04 | 西北农林科技大学 | 一种鉴定外源基因整合位点以增强转基因表达的方法 |
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