CN111944764A - 一种表达猪塞内加谷病毒蛋白的细胞系、构建方法和应用 - Google Patents
一种表达猪塞内加谷病毒蛋白的细胞系、构建方法和应用 Download PDFInfo
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Abstract
本发明公开了一种表达猪塞内加谷病毒蛋白的细胞系、构建方法和应用,所述细胞系包含猪塞内加谷病毒3A基因,并且能够稳定表达猪塞内加谷病毒3A蛋白;所述构建方法包括3A基因的扩增、重组慢病毒载体pLV/3A的构建、293T细胞的培养和细胞系的构建步骤。本发明细胞系能够稳定高效表达猪塞内加谷病毒蛋白,可用于制备猪塞内加谷病毒疫苗。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种表达猪塞内加谷病毒蛋白的细胞系、构建方法和应用。
背景技术
猪塞内加谷病毒(SVV)属于小核糖核酸病毒科Senecavirus病毒属成员,可引起猪鼻镜及蹄冠处出现水疱、溃烂创面从而导致跛足甚至死亡。SVV为无囊膜的单股正链RNA病毒,基因组编码5个结构蛋白L、VP1、VP2、VP3和VP4,编码7个非结构蛋白2A、2B、2C、3A、3B、3C和3D。L、VP1、VP2、VP3和VP4在SVV结构组装中发挥重要作用,2A、2B、2C、3A、3B、3C和3D在SVV复制和转录中发挥重要作用。目前关于用慢病毒技术稳定表达3A蛋白仍未见报道。
公告号为CN110279855B的中国专利公开了一种免疫组合物,其包含:猪塞内卡病毒结构蛋白VP3和VP1蛋白,以及,猪塞内卡病毒结构蛋白VP2和/或VP4蛋白。进一步的,所述免疫组合物还可包括猪塞内卡病毒结构蛋白VP0。所述免疫组合物可用于制备猪塞内卡病毒新型基因工程亚单位疫苗,该疫苗的抗原性、免疫原性和功能与天然蛋白质相似,表达水平较高,免疫原性强,对动物没有致病性,并且该疫苗可以通过生物反应器大规模无血清悬浮培养制备,大大降低了疫苗生产成本。但是,该专利只是评价病毒结构蛋白的免疫原性,没有考虑病毒非结构蛋白在机体免疫反应中的重要作用。
公开号为CN110358741A的中国发明专利申请公开了一种表达猪塞内卡病毒VP2基因的重组杆状病毒及其制备方法与应用。该发明提供一种重组杆状病毒,其包含一个或多个拷贝的猪塞内卡病毒VP2蛋白的编码基因。该发明还提供猪塞内卡病毒亚单位疫苗,其包含采用所述重组杆状病毒表达的猪塞内卡病毒VP2蛋白。该发明通过人工密码子优化,实现了猪塞内卡病毒的VP2蛋白的高水平、高纯度表达,最大程度地保留了VP2蛋白的免疫原性。该发明提供的猪塞内卡病毒亚单位疫苗具有优异的免疫原性和高度的安全性,对猪塞内卡病毒的感染发挥理想的免疫保护效果。但是,该专利只能用一种特定细胞和特定培养基培养,增加了生产成本。
发明内容
为克服上述缺陷,本发明的目的在于提供表达猪塞内加谷病毒蛋白的细胞系、构建方法和应用。
为实现上述目的,本发明采用如下技术方案:
一种表达猪塞内加谷病毒蛋白的细胞系,所述细胞系包含猪塞内加谷病毒3A基因,并且能够稳定表达猪塞内加谷病毒3A蛋白。
优选地,所述3A基因的序列如SEQ ID NO.1所示;所述3A蛋白的氨基酸序列如SEQID NO.2所示。
一种上述的表达猪塞内加谷病毒蛋白的细胞系的构建方法,包括以下步骤
S1、3A基因的扩增;
S2、重组慢病毒载体pLV/3A的构建:
a.双酶切3A基因与pLV质粒
b.3A基因与pLV连接
反应体系为:10*Buffer:2μL;T4-DNA-Ligase:1μL;3A基因:1.05μL;pLV:3.14μL;ddH2O:12.81μL;反应条件为:PCR仪16℃连接6h;
c.转化感受态细胞
将步骤b得到的连接液加入到DH5α感受态细胞中,在不含Amp+抗性的LB液体培养基,37℃摇床培养1h,最后分离涂板;
d.挑菌
在超净台下用接种环挑取白色单个菌落,接种于含Amp+抗性的LB培养液中,37℃摇床培养12h;
e.从步骤d得到的培养液中提取重组质粒pLV/3A;
S3、293T细胞的培养
将293T细胞株置于含10%小牛血清的DMEM培养液中,37℃、5%CO2条件下培养箱中培养得到;
S4、细胞系的构建:
a.将步骤S3培养得到的293T细胞用10%的DMEM培养液培养至细胞长至75-85%,更换培养液为5%FBS的生长培养液,37℃、CO2培养箱中继续培养;
b.脂质体介导的转染:将pLV/3A与PMD2.G、psPAX2混合,并加入转染试剂中,混匀,室温静置,加入步骤a培养的293T细胞中,24h收集上清液,然后更换培养基为10%的DMEM培养液,继续培养24h后,第二次收集上清液,合并两次上清液,超速离心浓缩得到慢病毒;
c.取PK15细胞接种于24孔板,待细胞长至70-80%时,转导步骤b得到的慢病毒,培养箱中培养,得到细胞系。
优选地,步骤S1所述3A基因的扩增包括以下步骤:
(1)设计引物:
所述引物包括上游引物和下游引物,上游引物3A-F,序列为:AAGGAAAAAATGTACAAGGGAGGAGGCGGATCTGGAGGAGGCGGATCATGAGCCCTAACGAGAACGACGA;下游引物3A-R,序列:CGCGGATCCGCCTAGCTCCTAGGCGCTTTAGCAG;
(2)PCR扩增:将步骤(1)所得设计引物进行PCR扩增,扩增产物在琼脂凝胶上跑胶;
(3)基因的纯化与回收:将步骤(2)跑胶得到的含有目的条带的琼脂凝胶进行纯化和回收,得到DNA溶液。
优选地,步骤(3)中所述DNA溶液A260/A280比值大于1.80,且核酸浓度≥70μg/ml。
优选地,所述PCR扩增体系为:2X:25μL;dNTP:1μL;上游引物:2μL;下游引物:2μL;高保真酶:1μL;cDNA模板5μL;ddH2O:14μL;
反应步骤:首先94℃预变性10min,94℃变性30s,56℃退火30s,72℃延伸1min,总共循环35次,最后72℃延伸10min。
优选地,所述基因的纯化与回收包括以下步骤:
1)将PCR扩增后琼脂糖凝胶置于紫外灯下,然后切取目的片段,放于1.5ml的离心管中,称重;
2)根据胶块的重量和浓度,按每100mg琼脂糖加300-600μL Buffer B2的比例加入Buffer B2;
3)将离心管置于50℃水浴5-10min,间或混匀,直至胶块完全溶化;
4)将溶化好的溶液全部移入吸附柱,8000Xg离心30s,倒掉收集管中的液体,将吸附柱放入同一个收集管中;
5)向吸附柱中加入300μL Buffer B2,8000Xg离心30s,倒掉收集管中的液体,将吸附柱放入同一个收集管中;
6)向吸附柱中加入500μL Wash Solution,9000Xg离心30s,倒掉收集管中的液体,将吸附柱放入同一个收集管中;
7)重复步骤6)一次;
8)将空吸附柱和收集管放入离心机,9000Xg离心1min;
9)在吸附膜中央加入15-40μL Elution Buffer,室温静置1-2min,9000Xg离心1min,得到的DNA溶液。
优选地,所述重组质粒pLV/3A的提取包括以下步骤:
(1)取5mL步骤S2 d中挑菌步骤中的摇床振荡培养12h后的菌液分入五个1.5mLEP管中,8000xg离心2min,收集菌体,弃尽培养基;
(2)在一个EP管中加入250μL Buffer P1,将沉淀全部悬浮;
(3)加入250μL Buffer P2,立即温和颠倒离心管5-10次混匀,室温静置2-4min;
(4)加入350μL Buffer P3,立即温和颠倒离心管5-10次混匀;
(5)12000xg离心5-10min,将上清液移入吸附管,8,000xg离心30秒,倒掉收集管中液体;
(6)加入500μL Buffer DW1,9000xg离心30秒,倒掉收集管中液体;
(7)加入500μL Wash Solution,9000xg离心30秒,倒掉收集管中液体;
(8)重复步骤(7)一次;
(9)空吸附柱于9000xg离心1min;
(10)将吸附柱放入一个干净的1.5ml离心管中,在吸附膜中央加入50μL ElutionBuffer,室温静置1min后,离心1min,保存管中DNA溶液。
一种上述的细胞系在制备猪塞内加谷病毒疫苗中的应用。
优选地,所述的猪塞内加谷病毒疫苗由将所述细胞系分离纯化得到的3A蛋白与佐剂混合而成。
本发明的积极有益效果:
1.本发明首次以单独蛋白作为候选疫苗,更具有针对性,且慢病毒载体可以将外源基因有效地整合到宿主染色体上,从而达到持久性表达外源基因的目的,包装成功的慢病毒能够感染多种类型细胞并进行大规模纯化,表达量持久高效,克服了现有技术中培养病毒(例如培养杆状病毒)所需要特定细胞和血清的条件的缺陷,节省费用。
另外,本发明首次用慢病毒系统构建稳定表达SVV 3A细胞系,该细胞系能够稳定高效表达外源蛋白,便于后期蛋白纯化、亚单位疫苗研究和病毒蛋白致病机制研究,从而达到良好的的基因治疗效果和生产亚单位疫苗的目的,具有广阔的应用前景。
附图说明
图1为本发明3A基因扩增电泳图;
图2为本发明重组质粒pLV/3A的酶切验证电泳图;
图3为本发明细胞系结果图;
图4为本发明细胞系分离纯化得到的3A蛋白的鉴定图。
具体实施方式
下面结合一些具体实施例对本发明进一步说明。
一种表达猪塞内加谷病毒蛋白的细胞系,所述细胞系包含加谷病毒3A基因,并且能够稳定表达猪塞内加谷病毒3A蛋白。
所述3A基因的序列如SEQ ID NO.1所示;所述3A蛋白的氨基酸序列如SEQ ID NO.2所示。
实施例1 3A基因的扩增;
(1)设计引物:所述引物包括上游引物和下游引物,上游引物3A-F,序列为:CTAGCTAGCATGAGCCCTAACGAGAACGACGA,参见SEQ ID NO.3;下游引物3A-R,序列为:CGCGGATCCTTAAGCGTAATCTGGAACATCGTATGGGTAGCTCCTAGGCGCTTTAGCAG,参见SEQ ID NO.4;
(2)PCR扩增:将步骤(1)所得设计引物进行PCR扩增,扩增产物在琼脂凝胶上跑胶;
所述PCR扩增体系为:2X:25μL;dNTP:1μL;上游引物:2μL;下游引物:2μL;高保真酶:1μL;cDNA模板5μL;ddH2O:14μL;
cDNA模板是从猪病料(发病猪水泡液,河南丰源和普农牧有限公司)中分离的SVV中提取的核酸反转录成的cDNA,具体过程如下:
a.采集发病猪水泡液用滤膜过滤除菌,接种培养的293T细胞,待出现细胞病变后收集病毒液;
b.每1ml Buffer VRL加入4μl Carrier RNA(1ug/μl)配制Buffer VRL/CarrierRNA混合液,使用前,于60℃水浴3分钟让沉淀完全溶解,转移560μl Buffer VRL/CarrierRNA至1.5ml离心管中,转移140μl病毒液至装有Buffer VRL/Carrier RNA的离心管中,涡旋混匀20秒,室温25℃静置10min,加入560μl无水乙醇至裂解液中,涡旋混匀20秒;
c.把HiPure RNA Micro Column装在2ml收集管中,转移700μl混合掖至柱子中,10,000xg离心60秒,倒弃滤液,把柱子装在收集管中,转移剩余混合上清液至柱子中,10,000xg离心60秒,重复此步直到所有混合液都从柱子中过滤;把柱子装在新的收集管里,加入600μl Buffer VHB(已用乙醇稀释)至柱子中,10,000xg离心60秒,倒弃滤液,把柱子重新装回收集管中,加入600μl Buffer RW2(已用乙醇稀释)至柱子中,10,000x g离心60秒,倒弃滤液;把柱子重新装回收集管中,加入600μl Buffer RW2(已用乙醇稀释)至柱子中,10,000xg离心60秒,倒弃滤液,把柱子装回收集管中,13,000xg离心空柱3分钟甩干柱子,将柱子转移至新的1.5ml离心管,加入30μl RNase Free Water至柱子的膜中央,室温静置2分钟,13,000x g离心1分钟,弃去柱子,把病毒RNA保存于-80℃。
反转录体系为:5X:4μL;dNTP:1μL;Oligo(dT):1μL;MLV:1μL;RibonucleaseInhibitor:1μL;RNA模板5μL;ddH2O:7μL;反应步骤:42℃60min。
反应步骤:首先94℃预变性10min,94℃变性30s,56℃退火30s,72℃延伸1min,总共循环35次,最后72℃延伸10min;
其中琼脂糖凝胶浓度为1%,组成见表1,扩增电泳图参见图1;
表1琼脂凝胶的配方
反应体系 | 体积 |
琼脂糖 | 0.3g |
核酸染料 | 3μL |
TAE | 30mL |
(3)基因的纯化与回收:将步骤(2)跑胶得到的含有目的条带的琼脂凝胶进行纯化和回收,得到DNA溶液,DNA溶液的A260/A280比值大于1.80,且核酸浓度80.6μg/ml,具体包括以下步骤:
1)将PCR扩增后琼脂糖凝胶置于紫外灯下,然后切取目的片段,放于1.5ml的离心管中,称重;
2)根据胶块的重量和浓度,按每100mg琼脂糖加300-600μL Buffer B2的比例加入Buffer B2;
3)将离心管置于50℃水浴5-10min,间或混匀,直至胶块完全溶化;
4)将溶化好的溶液全部移入吸附柱,8000Xg离心30s,倒掉收集管中的液体,将吸附柱放入同一个收集管中;
5)向吸附柱中加入300μL Buffer B2,8000Xg离心30s,倒掉收集管中的液体,将吸附柱放入同一个收集管中;
6)向吸附柱中加入500μL Wash Solution,9000Xg离心30s,倒掉收集管中的液体,将吸附柱放入同一个收集管中;
7)重复步骤6)一次;
8)将空吸附柱和收集管放入离心机,9000Xg离心1min;
9)在吸附膜中央加入15-40μL Elution Buffer,室温静置1-2min,9000Xg离心1min,得到的DNA溶液,置于-20℃保存。
实施例2重组腺病毒载体pLV/3A的构建:
a.双酶切3A基因与pLV质粒
用BsrGI和BamHI分别双酶切步骤S1的3A基因和pLV质粒(哈尔滨兽医研究所),37℃水浴15min,回收纯化含有3A基因和pLV质粒的琼脂凝胶条带,得到3A基因与pLV质粒;
酶切体系一如下表2;
表2酶切体系一
pLV | 3A | |
10X | 5μL | 10μL |
BsrGI | 2.5μL | 2.4μL |
BamHI | 2.5μL | 2.4μL |
DNA | 25μL | 50μL |
dd H<sub>2</sub>O | 15μL | 35.2μL |
所得3A基因与pLV质粒的浓度如下表3;
表3 3A基因与PLV质粒的浓度
浓度 | A260/A280 | |
pLV | 15.9μg/μL | 2.10 |
3A基因 | 29.3μg/μL | 1.89 |
b.3A基因与pLV连接
反应体系为:10*Buffer:2μL;T4-DNA-Ligase:1μL;3A基因:1.05μL;pLV:3.14μL;ddH2O:12.81μL;反应条件为:PCR仪16℃连接6h;
c.转化感受态细胞
取DH5α感受态细胞(北京全式金生物技术有限公司)100μL于冰上融化5min,将步骤b得到的连接液加入到感受态细胞中,冰上放置30min,期间不能摇晃,接着42℃热激45s,然后冰上放置1min,接着加入900μL不含Amp+的LB液体培养基,37℃摇床培养1h,最后2500Xg离心3min,弃去上清,用300μL PBS重悬细胞后涂板,温箱过夜;
d.挑菌:在超净台下用接种环挑取白色单个菌落,接种于含Amp+抗性的LB培养液中,37℃摇床振荡培养12h;
e.重组质粒pLV/3A提取,包括以下步骤:
(1)步骤S2 d中挑菌步骤中的摇床振荡培养12h后的菌液分入五个1.5mLEP管中,8000xg离心2min,收集菌体,弃尽培养基;
(2)在每个EP管中加入250μL Buffer P1,将沉淀全部悬浮;
(3)加入250μL Buffer P2,立即温和颠倒离心管5-10次混匀,室温静置2-4min;
(4)加入350μL Buffer P3,立即温和颠倒离心管5-10次混匀;
(5)12000xg离心5-10min,将上清液移入吸附管,8000xg离心30秒,倒掉收集管中液体;
(6)加入500μL Buffer DW1,9000xg离心30秒,倒掉收集管中液体;
(7)加入500μL Wash Solution,9000xg离心30秒,倒掉收集管中液体;
(8)重复步骤(7)一次;
(9)空吸附柱于9000xg离心1min;
(10)将吸附柱放入一个干净的1.5ml离心管中,在吸附膜中央加入50μL ElutionBuffer,室温静置1min后,离心1min,保存管中DNA溶液;
f.重组质粒的酶切验证
将上述步骤提取的重组质粒进行酶切验证,酶切体系二见表4,酶切验证电泳图参见图2;
表4酶切体系二
总体积10μL | |
10*Buffer | 1μL |
BsrGI | 0.5μL |
BamHI | 0.5μL |
pLV/3A | 7μL |
ddH<sub>2</sub>O | 1μL |
37℃水浴15min,然后进行琼脂糖凝胶电泳,结果见图2;
由图2可知,3A基因正确连接到pLV质粒上。
实施例3 293T细胞的培养
将293T细胞株(哈尔滨兽医研究所)置于含10%小牛血清的DMEM培养液中,37℃、5%CO2条件下培养箱中培养,即得,具体包括以下步骤:
(1)配制50ml含10%小牛血清的DMEM培养液,准备38℃的水;
(2)用镊子将冻存的293T细胞株迅速放入38℃的水中,使其迅速融化;
(3)将冻存管放入离心管中,800rpm离心5min,注意要配平离心;
(4)离心结束后,弃上清,吸取1ml 10%培养液于离心管中,轻轻吹打混匀后,转入新的细胞瓶;再向冻存管中加入1ml 10%培养液,将残留细胞转至同一细胞瓶中,再加入4ml 10%培养液,置37℃、5%CO2培养箱培养24h后观察,若见细胞贴壁,表明复苏成功,并换液一次,每天在显微镜下观察细胞生长情况,每2天传代一次。
实施例4细胞系的构建
a.将步骤S3培养得到的293T细胞铺至培养皿,用10%的DMEM培养液培养至细胞长至80%,更换培养液为5%FBS的生长培养液,37℃、CO2培养箱中继续培养30min;
b.脂质体介导的转染:按照重量比pLV/3A:1.77ug;pMD2.G(哈尔滨兽医研究所):3.525ug:psPAX2(哈尔滨兽医研究所):0.705ug将三种质粒混合,并加入转染试剂LTX and PLUSTMReagents(Invitrogen,USA)中,轻柔混匀,室温静置30min,加入步骤a培养的293T细胞中,每碟所需转染混合液是600μL,轻轻晃动细胞碟使转染试剂和培养液混匀,共需收集两次病毒,即:转染后24h,将细胞培养的上清收集到无菌的离心管中,然后加入10%的DMEM培养液,继续培养24h后,第二次收集上清液,合并两次上清液,将收集的上清超速离心浓缩既得到所需要的慢病毒;
c.取生长良好的PK15细胞(哈尔滨兽医研究所)接种于24孔板,待细胞长至75%时转导步骤b浓缩的慢病毒,转导24h后更换成10%的DMEM培养液,在培养箱继续培养,同时设置不携带3A基因的空质粒pLV组作为对照,观察荧光显微镜和蛋白印迹鉴定SVV 3A在PK15细胞中的荧光强度和表达情况,细胞系结果图参见图3,将3A蛋白分离纯化鉴定图参见图4。
由图3可知,本发明转染空质粒pLV组(左图)和表达外源蛋白3A组(右图)的细胞系均包装成功;
由图4可知,本发明空质粒组pLV组(第一泳道)没有3A蛋白条带,而表达外源蛋白3A组(第二泳道)有特异性的3A条带,说明包装的细胞系能正确表达3A蛋白。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,本领域普通技术人员对本发明的技术方案所做的其他修改或者等同替换,只要不脱离本发明技术方案的精神和范围,均应涵盖在本发明的权利要求范围当中。
SEQUENCE LISTING
<110> 信阳农林学院
<120> 一种表达猪塞内加谷病毒蛋白的细胞系、构建方法和应用
<130> /
<160> 4
<170> PatentIn version 3.5
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agccctaacg agaacgacga cacccccgtc gacgaggcgt tgggtagagt tctcaccccc 60
gctgcggtcg acgaagcgct tgtcgacctc gctccagatg ccgacccggt tggccgcttg 120
gctattctcg ccaagctagg tcttgcccta gctgcggtca cccctggttt gataatcttg 180
gcagtgggac tctacaagta cttctctggc tctgatacag accgagaaga gacagaaagt 240
gaggagccta ctaaagcgcc taggagcgag 270
<210> 2
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<213> Artificial Sequence
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<400> 2
Ser Pro Asn Glu Asn Asp Asp Thr Pro Val Asp Glu Ala Leu Gly Arg
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Val Leu Thr Pro Ala Ala Val Asp Glu Ala Leu Val Asp Leu Ala Pro
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Asp Ala Asp Pro Val Gly Arg Leu Ala Ile Leu Ala Lys Leu Gly Leu
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Ala Leu Ala Ala Val Thr Pro Gly Leu Ile Ile Leu Ala Val Gly Leu
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Tyr Lys Tyr Phe Ser Gly Ser Asp Thr Asp Arg Glu Glu Thr Glu Ser
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Glu Glu Pro Thr Lys Ala Pro Arg Ser Glu
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gagaacgacg a 71
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cgcggatccg cctagctcct aggcgcttta gcag 34
Claims (10)
1.一种表达猪塞内加谷病毒蛋白的细胞系,其特征在于,所述细胞系包含猪塞内加谷病毒3A基因,并且能够稳定表达猪塞内加谷病毒3A蛋白。
2.根据权利要求1所述的表达猪塞内加谷病毒蛋白的细胞系,其特征在于,所述3A基因的序列如SEQ ID NO.1所示;所述3A蛋白的氨基酸序列如SEQ ID NO.2所示。
3.一种权利要求1或者2所述的表达猪塞内加谷病毒蛋白的细胞系的构建方法,其特征在于,包括以下步骤
S1、3A基因的扩增;
S2、重组慢病毒载体pLV/3A的构建:
a.双酶切3A基因与pLV质粒
b.3A基因与pLV连接
反应体系为:10*Buffer:2μL;T4-DNA-Ligase:1μL;3A基因:1.05μL;pLV:3.14μL;ddH2O:12.81μL;反应条件为:PCR仪16℃连接6h;
c.转化感受态细胞
将步骤b得到的连接液加入到DH5α感受态细胞中,在不含Amp+抗性的LB液体培养基,37℃摇床培养1h,最后分离涂板;
d.挑菌
在超净台下用接种环挑取白色单个菌落,接种于含Amp+抗性的LB培养液中,37℃摇床培养12h;
e.从步骤d得到的培养液中提取重组质粒pLV/3A;
S3、293T细胞的培养
将293T细胞株置于含10%小牛血清的DMEM培养液中,37℃、5%CO2条件下培养箱中培养得到;
S4、细胞系的构建:
a.将步骤S3培养得到的293T细胞用10%的DMEM培养液培养至细胞长至75-85%,更换培养液为5%FBS的生长培养液,37℃、CO2培养箱中继续培养;
b.脂质体介导的转染:将pLV/3A与PMD2.G、psPAX2混合,并加入转染试剂中,混匀,室温静置,加入步骤a培养的293T细胞中,24h收集上清液,然后更换培养基为10%的DMEM培养液,继续培养24h后,第二次收集上清液,合并两次上清液,超速离心浓缩得到慢病毒;
c.取PK15细胞接种于24孔板,待细胞长至70-80%时,转导步骤b得到的慢病毒,培养箱中培养,得到细胞系。
4.根据权利要求3所述的表达猪塞内加谷病毒蛋白的细胞系的构建方法,其特征在于,步骤S1所述3A基因的扩增包括以下步骤:
(1)设计引物:
所述引物包括上游引物和下游引物,上游引物3A-F,序列为:AAGGAAAAAATGTACAAGGGAGGAGGCGGATCTGGAGGAGGCGGATCAATGAGCCCTAACGAGAACGACGA;下游引物3A-R,序列:CGCGGATCCGCCTAGCTCCTAGGCGCTTTAGCAG;
(2)PCR扩增:将步骤(1)所得设计引物进行PCR扩增,扩增产物在琼脂凝胶上跑胶;
(3)基因的纯化与回收:将步骤(2)跑胶得到的含有目的条带的琼脂凝胶进行纯化和回收,得到DNA溶液。
5.根据权利要求4所述的表达猪塞内加谷病毒蛋白的细胞系的构建方法,其特征在于,步骤(3)中所述DNA溶液A260/A280比值大于1.80,且核酸浓度≥70μg/ml。
6.根据权利要求4所述的表达猪塞内加谷病毒蛋白的细胞系的构建方法,其特征在于,所述PCR扩增体系为:2X:25μL;dNTP:1μL;上游引物:2μL;下游引物:2μL;高保真酶:1μL;cDNA模板5μL;ddH2O:14μL;
反应步骤:首先94℃预变性10min,94℃变性30s,56℃退火30s,72℃延伸1min,总共循环35次,最后72℃延伸10min。
7.根据权利要求4所述的表达猪塞内加谷病毒蛋白的细胞系的构建方法,其特征在于,所述基因的纯化与回收包括以下步骤:
1)将PCR扩增后琼脂糖凝胶置于紫外灯下,然后切取目的片段,放于1.5ml的离心管中,称重;
2)根据胶块的重量和浓度,按每100mg琼脂糖加300-600μL Buffer B2的比例加入Buffer B2;
3)将离心管置于50℃水浴5-10min,间或混匀,直至胶块完全溶化;
4)将溶化好的溶液全部移入吸附柱,8000Xg离心30s,倒掉收集管中的液体,将吸附柱放入同一个收集管中;
5)向吸附柱中加入300μL Buffer B2,8000Xg离心30s,倒掉收集管中的液体,将吸附柱放入同一个收集管中;
6)向吸附柱中加入500μL Wash Solution,9000Xg离心30s,倒掉收集管中的液体,将吸附柱放入同一个收集管中;
7)重复步骤6)一次;
8)将空吸附柱和收集管放入离心机,9000Xg离心1min;
9)在吸附膜中央加入15-40μL Elution Buffer,室温静置1-2min,9000Xg离心1min,得到的DNA溶液。
8.根据权利要求3所述的表达猪塞内加谷病毒蛋白的细胞系的构建方法,其特征在于,所述重组质粒pLV/3A的提取包括以下步骤:
(1)取5mL步骤S2 d中挑菌步骤中的摇床振荡培养12h后的菌液分入五个1.5mLEP管中,8000xg离心2min,收集菌体,弃尽培养基;
(2)在EP管中加入250μL Buffer P1,将沉淀全部悬浮;
(3)加入250μL Buffer P2,立即温和颠倒离心管5-10次混匀,室温静置2-4min;
(4)加入350μL Buffer P3,立即温和颠倒离心管5-10次混匀;
(5)12000xg离心5-10min,将上清液移入吸附管,8,000xg离心30秒,倒掉收集管中液体;
(6)加入500μL Buffer DW1,9000xg离心30秒,倒掉收集管中液体;
(7)加入500μL Wash Solution,9000xg离心30秒,倒掉收集管中液体;
(8)重复步骤(7)一次;
(9)空吸附柱于9000xg离心1min;
(10)将吸附柱放入一个干净的1.5ml离心管中,在吸附膜中央加入50μL ElutionBuffer,室温静置1min后,离心1min,保存管中DNA溶液。
9.一种权利要求1或者2所述的细胞系在制备猪塞内加谷病毒疫苗中的应用。
10.根据权利要求9所述的应用,其特征在于,所述的猪塞内加谷病毒疫苗由将所述细胞系分离纯化得到的3A蛋白与佐剂混合而成。
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