CN112390861A - 表达猪塞内加谷病毒vp1蛋白的细胞系、构建方法和应用 - Google Patents
表达猪塞内加谷病毒vp1蛋白的细胞系、构建方法和应用 Download PDFInfo
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Abstract
本发明公开了表达猪塞内加谷病毒VP1蛋白的细胞系、构建方法和应用,所述细胞系含有猪塞内加谷病毒VP1基因,并且能够稳定表达猪塞内加谷病毒VP1蛋白,所述构件方法包括设计引物,扩增VP1基因并纯化回收;构建pLV/VP1重组质粒;扩增S2获得重组质粒;将S3获得的重组质粒进行病毒包装,获得含有重组质粒的慢病毒;将S4获得的慢病毒转染PK15细胞系,得到表达猪塞内加谷病毒VP1蛋白的细胞系;该细胞系能够精准的产生大量的VP1蛋白,用于制备猪塞内加谷病毒亚单位疫苗,避免了产生许多无关抗原诱发的抗体,疫苗的副反应小且不易引起相关疾病,具有广阔的应用前景。
Description
技术领域
本发明属于基因工程技术领域,具体涉及表达猪塞内加谷病毒VP1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、VP1、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、VP1、3B、3C和 3D在SVV复制和转录中发挥重要作用。
公开号为CN109679927A的中国发明专利申请公开了一种猪塞内加谷病毒灭活疫苗的制备方法,其特征在于,包括以下步骤:1)用细胞培养液对PK-15 细胞进行培养,当所述PK-15细胞长成单层时,弃去细胞培养液,更换为DMEM 营养液后培养48~60h,得到培养物;所述DMEM营养液中含有猪塞内加谷病毒,所述猪塞内加谷病毒的体积浓度为0.1~0.2%;2)将所述步骤1)得到的培养物进行过滤,调节得到的滤液的pH值为7.6~7.8后,再与二乙烯亚胺混合,将得到的混合物进行灭活,得到灭活病毒液;所述混合物中二乙烯亚胺的浓度为 1.5~2.5mmol/L;3)将所述步骤2)得到的灭活病毒液与佐剂混合、乳化,得到猪塞内加谷病毒灭活疫苗,该发明得到的是灭活病毒而不是VP1蛋白,只能制作普通疫苗,无法避免产生许多无关抗原诱发的抗体,疫苗的副反应大,容易引起相关疾病。
发明内容
有鉴于此,本发明的目的是针对现有技术的不足,提供表达猪塞内加谷病毒VP1蛋白的细胞系和构建方法,构建的细胞系准确的产生大量VP1蛋白,能够应用于制作猪塞内加谷病毒亚单位疫苗,避免了产生许多无关抗原诱发的抗体,疫苗的副反应小且不易引起相关疾病,具有广阔的应用前景。
为达到上述目的,本发明采用以下技术方案:
表达猪塞内加谷病毒VP1蛋白的细胞系,所述细胞系含有猪塞内加谷病毒 VP1基因,并且能够稳定表达猪塞内加谷病毒VP1蛋白。
进一步的,所述VP1基因的序列如SEQ ID NO.1所示,所述VP1蛋白的氨基酸序列如SEQ ID NO.2所示。
进一步的,包括以下步骤:
S1、设计引物,扩增VP1基因并纯化回收;
S2、构建pLV/VP1重组质粒;
S3、扩增S2获得的pLV/VP1重组质粒;
S4、将S3获得的pLV/VP1重组质粒进行病毒包装,获得含有pLV/VP1重组质粒的慢病毒;
S5、将S4获得的慢病毒转染PK15细胞系,得到表达猪塞内加谷病毒VP1蛋白的细胞系。
进一步的,所述引物包括上游引物和下游引物,所述上游引物为VP1-F,参见SEQID NO.3,所述下游引物为VP1-R,序列为SEQ ID NO.4。
进一步的,为在制备猪塞内加谷病毒亚单位疫苗中的应用。
进一步的,所述猪塞内加谷病毒亚单位疫苗由所述细胞系表达的经分离纯化后的VP1蛋白与佐剂混合而成。
本发明的有益效果是:
1、本发明包装成功的慢病毒能够高效迅速的感染PK15细胞并进行大规模纯化,表达量持久高效,能够产生高纯度的VP1蛋白,便于后期蛋白纯化。
2、本发明中细胞系表达的VP1蛋白和佐剂混合能够用于制备亚单位疫苗,该疫苗能诱发猪体内产生抗体,由于其内部不含有核酸,避免了产生许多无关抗原诱发的抗体,疫苗的副反应小且不易引起相关疾病,具有广阔的应用前景。
附图说明
图1为本发明VP1基因扩增电泳图;
图2为重组质粒pLV/VP1的PCR验证电泳图;
图3为重组质粒pLV/VP1的酶切验证电泳图;
图4为细胞系外源蛋白鉴定图;
图5为VP1蛋白的鉴定图。
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。
表达猪塞内加谷病毒VP1蛋白的细胞系,其特征在于:所述细胞系含有猪塞内加谷病毒VP1基因,并且能够稳定表达猪塞内加谷病毒VP1蛋白。
所述VP1基因的序列如SEQ ID NO.1所示;所述VP1蛋白的氨基酸序列如 SEQ IDNO.2所示。
表达猪塞内加谷病毒VP1蛋白细胞系的构件方法,包括以下步骤:
S1、设计引物,扩增VP1基因并纯化回收;
1)设计引物
引物包括上游引物和下游引物,上游引物VP1-F,序列为:
AAGGAAAAAATGTACAAGGGAGGAGGCGGATCTGGAGGAGGCGGATCAATGTCCACCGACAACGCCGAGAC,参见SEQ ID NO.3;下游引物 VP1-R,序列为:CGCGGATCCGCCTATTGCATCAGCATCTTTT,参见SEQ ID NO.4;
2)扩增VP1基因
PCR扩增体系为:2X Buffer:25μL;dNTP:1μL;上游引物:2μL;下游引物:2μL;高保真酶:1μL;cDNA模板5μL;ddH2O:14μL;
其中,cDNA模板是从猪病料(发病猪水泡液)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BufferVRL/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600μ l 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 Buffer:4μL;dNTP:1μL;Oligo(dT):1μL;MLV: 1μL;Ribonuclease Inhibitor:1μL;RNA模板5μL;ddH2O:7μL;反应步骤:42℃60min。
将PCR扩增体系加入到PCR仪中,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,完成 VP1基因的扩增。
3)纯化和回收VP1基因
A:配置琼脂凝胶,琼脂凝胶的组成见表1,其中琼脂糖凝胶浓度为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 |
B:跑胶将步骤2)中得到的PCR扩增后的产物在步骤a配置好的琼脂凝胶上跑胶,跑胶电泳图参见图1;
C:纯化和回收目的基因,具体步骤为:
a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;
b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BufferB2;
c将离心管置于50℃水浴5-10min,间或混匀,直至胶块完全溶化;
d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;
e向吸附柱中加入300μL BufferB2,8000Xg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;
f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;
g重复步骤f一次;
h将空吸附柱和收集管放入离心机,9000Xg离心1min;
i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,完成基因的纯化和回收,此时,DNA溶液的A260/A280 比值大于1.80,且核酸浓度80.6μg/ml,置于-20℃保存。
S2、构建pLV/VP1重组质粒:
a.双酶切VP1基因与pLV质粒并分别纯化回收
用Nhel和BamHI分别双酶切步骤S1的VP1基因和pLV质粒(哈尔滨兽医研究所),VP1基因和pLV质粒的酶切体系如表2所示,每个体系均37℃水浴15min,之后分别回收双酶切后的VP1基因和pLV质粒,回收纯化的步骤和 S1中的步骤3)相同,得到VP1基因与pLV质粒;
表2 VP1基因和pLV质粒的酶切体系
pLV | VP1 | |
10X | 5μL | 10μL |
BamHI | 5μL | 3.36μL |
Nhel | 5μL | 5μL |
DNA | 25μL | 50μL |
ddH<sub>2</sub>O | 10μL | 33.28μL |
所得VP1基因与pLV质粒的浓度如下表3;
表3 VP1基因与pLV质粒的浓度
浓度 | A260/A280 | |
pLV | 15.9μg/μL | 2.10 |
VP1基因 | 47.8μg/μL | 1.92 |
b.连接VP1基因与pLV质粒
连接体系: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,得到含有pLV/VP1重组质粒的连接液;
S3、扩增S2获得的pLV/VP1重组质粒.
a转化感受态细胞
取DH5α感受态细胞100μL于冰上融化5min,将步骤S2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不含氨苄青霉素的LB液体培养基,37℃摇床培养1h,最后2500Xg离心3min,弃去上清,用300μLPBS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,接种于含氨苄青霉素抗性的LB培养液中,37℃摇床振荡培养12h;
e.提取重组质粒pLV/VP1,包括以下步骤:
(1)步骤S3d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250μLBufferP1,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350μLBufferP3,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500μLBuffer DW1,9000xg离心30秒,倒掉收集管中液体;
(7)加入500μLWash 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50μLElutionBuffer,室温静置1min后,离心1min,保存管中溶液,溶液内含有pLV/VP1 重组质粒;
f.鉴定pLV/VP1重组质粒
(1)PCR验证
取步骤S3e中获得的部分含有pLV/VP1重组质粒的溶液进行PCR扩增,扩增体系为:2X:25μL;dNTP:1μL;上游引物:2μL;下游引物:2μL;高保真酶:1μL;重组质粒溶液:1μL;ddH2O:18μL;
扩增后进行琼脂糖凝胶电泳,电泳图如图2所示,由图2可知,VP1基因正确连接到pLV质粒上。
(2)酶切验证
将步骤S3e提取的含有pLV/VP1重组质粒的溶液进行酶切验证,酶切体系如表4所示:
表4 pLV/VP1重组质粒酶切体系
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3;
由图3可知,VP1基因正确连接到pLV质粒上。
S4、将S3e获得的pLV/VP1重组质粒进行病毒包装,获得含有pLV/VP1重组质粒的慢病毒,步骤如下:
a.培养AD293细胞
(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AD293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1ml10%培养液,将残留细胞转至同一细胞瓶中,再加入4ml10%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;
b慢病毒包装,获得含有pLV/VP1重组质粒的慢病毒,包括以下步骤:
(1)将步骤S4a培养得到的AD293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;
(2)脂质体介导的转染:
按照质粒重量比pLV/3A:1.77ug;pMD2.G:3.525ug:psPAX2:0.705ug 将三种质粒混合,之后将DNA体系加入到转染试剂体系中,颠倒混匀,室温下静置30min,加入步骤S4.b.(1)培养的293T细胞中,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,得到所需要的含有pLV/VP1重组质粒的慢病毒;
S5、将S4获得的慢病毒转染PK15细胞系,得到表达猪塞内加谷病毒VP1 蛋白的细胞系,步骤为:
取生长良好的PK15细胞接种于24孔板,待细胞长至75%时转导步骤S4 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VP1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猪塞内加谷病毒VP1在PK15细胞中的荧光强度和表达情况,细胞系结果图参见图4,表明外源蛋白正常表达。其中,表达外源蛋白VP1组如图4右图所示,转染空质粒pLV组如图4左图所示。
弃掉培养细胞的上清液,用PBS洗涤3遍,加入1ml裂解液于培养的细胞中裂解细胞,收集于EP管中,10000转离心3min,收集上清加入蛋白上样缓冲液,沸水中煮5min,之后对处理过的溶液进行电泳,电泳结果如图5所示,本发明空质粒组pLV组(第一泳道)没有VP1蛋白条带,而表达外源蛋白VP1 组(第二泳道)有特异性的VP1条带,说明细胞系表达的外源蛋白是VP1蛋白,得到了稳定、大量表达猪塞内加谷病毒VP1蛋白的细胞系,由图5可知,外源蛋白成功大量表达。
步骤S5获得了表达猪塞内加谷病毒VP1蛋白的细胞系,该细胞系表达出的 VP1蛋白能够制备亚单位疫苗,制备亚单位疫苗的步骤为:
1、培养步骤S5中获得的PK15细胞系,使其表达大量的VP1蛋白;
2、将上述步骤培养的PK15细胞系超声破碎10000rpm/min离心8min取裂解上清,使用亲和层析介质Ni-NTA对VP1蛋白进行纯化回收,得到纯化VP1蛋白;
3、水相制备:将纯化VP1蛋白和吐温-80以质量分数25:1比例混匀制成水相;
4、油相制备:将白油、司本-80和硬脂酸铝以47:3:1比例混匀制成油相;
5、乳化:取4份质量的油相放入搅拌机中,同时逐滴加入2份质量的水相,加完后以1000转/min乳化10分钟,得到亚单位疫苗。
亚单位疫苗的检验
1.外观和剂型:制作的疫苗是乳白色油包水乳剂。加入少量制备的疫苗于冷水中不扩散。
2.稳定性:加入10ml制备的疫苗于离心管中,以3000转/min离心15分钟,管底析出0.1ml的水相,符合规定。
3.黏度:制作的疫苗黏度值为46cP,符合《中国兽药典》的规定。
4.无菌检验:制作的疫苗按照《中国兽药典》的规定进行,无细菌的生长。
亚单位疫苗的效力检验
将4周龄SPF仔猪30头,其中15头各颈部肌肉注射疫苗2ml,15头注射猪塞内加谷病毒灭活病毒疫苗2ml,另外10只免疫生理盐水作为对照,接种后 21天,每头猪分别采血,分离血清,ELISA检测VP1抗体水平,数据如下:
表5实验组和对照组体内抗体浓度对照表
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> 表达猪塞内加谷病毒VP1蛋白的细胞系、构建方法和应用
<130> /
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<170> PatentIn version 3.5
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tccaccgaca acgctgagac tggggttatt gaggcaggta acactgacac cgatttctct 60
ggcgaactgg cggctcctgg ctctaaccat accaatgtca aattcctgtt tgaccgatct 120
cgactactga atgtaattaa ggtactggag aaggacgccg tcttcccccg tcctttcccc 180
acagcaacag gtgcacagca ggacgatggt tacttttgtc ttctgacacc ccgcccaaca 240
gtcgcttccc ggcccgccac tcgtttcggc ctgtacgtca acccatctga cagtggcgtt 300
cttgctaaca cttcactgga tttcaatttt tacagtttag cctgtttcac ttacttcaga 360
tctgaccttg aagtcacggt ggtctcgctg gagccagatt tggaatttgc cgtggggtgg 420
ttcccctctg gcagtgagta ccaggcttct agcttcgtct acgaccaact gcatgtaccc 480
taccacttta ctgggcgcac tccccgcgct ttcaccagca agggtggaaa ggtatctttc 540
gtgctccctt ggaactctgt ctcttccgtg cttcccgtgc gctggggggg cgcttccaag 600
ctttcttctg ccacgcgggg tctgccggct catgctgact gggggaccat ttacgccttc 660
atcccccgtc ctaacgagaa gaaaagcacc gctgtaaagc acgtggcggt gtacgttcgg 720
tacaagaacg cgcgtgcttg gtgccccaac atgcttccct tccgcagcta caagcaaaag 780
atgctgatgc aa 792
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Ser Thr Asp Asn Ala Glu Thr Gly Val Ile Glu Ala Gly Asn Thr Asp
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Thr Asp Phe Ser Gly Glu Leu Ala Ala Pro Gly Ser Asn His Thr Asn
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Val Lys Phe Leu Phe Asp Arg Ser Arg Leu Leu Asn Val Ile Lys Val
35 40 45
Leu Glu Lys Asp Ala Val Phe Pro Arg Pro Phe Pro Thr Ala Thr Gly
50 55 60
Ala Gln Gln Asp Asp Gly Tyr Phe Cys Leu Leu Thr Pro Arg Pro Thr
65 70 75 80
Val Ala Ser Arg Pro Ala Thr Arg Phe Gly Leu Tyr Val Asn Pro Ser
85 90 95
Asp Ser Gly Val Leu Ala Asn Thr Ser Leu Asp Phe Asn Phe Tyr Ser
100 105 110
Leu Ala Cys Phe Thr Tyr Phe Arg Ser Asp Leu Glu Val Thr Val Val
115 120 125
Ser Leu Glu Pro Asp Leu Glu Phe Ala Val Gly Trp Phe Pro Ser Gly
130 135 140
Ser Glu Tyr Gln Ala Ser Ser Phe Val Tyr Asp Gln Leu His Val Pro
145 150 155 160
Tyr His Phe Thr Gly Arg Thr Pro Arg Ala Phe Thr Ser Lys Gly Gly
165 170 175
Lys Val Ser Phe Val Leu Pro Trp Asn Ser Val Ser Ser Val Leu Pro
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Val Arg Trp Gly Gly Ala Ser Lys Leu Ser Ser Ala Thr Arg Gly Leu
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Pro Ala His Ala Asp Trp Gly Thr Ile Tyr Ala Phe Ile Pro Arg Pro
210 215 220
Asn Glu Lys Lys Ser Thr Ala Val Lys His Val Ala Val Tyr Val Arg
225 230 235 240
Tyr Lys Asn Ala Arg Ala Trp Cys Pro Asn Met Leu Pro Phe Arg Ser
245 250 255
Tyr Lys Gln Lys Met Leu Met Gln
260
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aaggaaaaaa tgtacaaggg aggaggcgga tctggaggag gcggatcaat gtccaccgac 60
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cgcggatccg cctattgcat cagcatcttt t 31
Claims (5)
1.表达猪塞内加谷病毒VP1蛋白的细胞系,其特征在于:所述细胞系含有猪塞内加谷病毒VP1基因,并且能够稳定表达猪塞内加谷病毒VP1蛋白。
2.根据权利要求1所述的表达猪塞内加谷病毒VP1蛋白的细胞系,其特征在于,所述VP1基因的序列如SEQ ID NO.1所示,所述VP1蛋白的氨基酸序列如SEQ ID NO.2所示。
3.如权利要求1或2所述的表达猪塞内加谷病毒VP1蛋白细胞系的构件方法,其特征在于,包括以下步骤:
S1、设计引物,扩增VP1基因并纯化回收;
S2、构建pLV/VP1重组质粒;
S3、扩增S2获得的pLV/VP1重组质粒;
S4、将S3获得的pLV/VP1重组质粒进行病毒包装,获得含有pLV/VP1重组质粒的慢病毒;
S5、将S4获得的慢病毒转染PK15细胞系,得到表达猪塞内加谷病毒VP1蛋白的细胞系。
4. 如权利要求3所述的表达猪塞内加谷病毒VP1蛋白细胞系的构建方法,其特征在于,所述引物包括上游引物和下游引物,所述上游引物为VP1-F,参见SEQ ID NO.3,所述下游引物为VP1-R,序列为SEQ ID NO.4。
5.如权利要求1或2所述的表达猪塞内加谷病毒VP1蛋白的细胞系的应用,其特征在于,为在制备猪塞内加谷病毒亚单位疫苗中的应用。
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