CN115073557A - 一种重组高纯度的非洲猪瘟病毒pK205R亚单位蛋白及其制备方法和应用 - Google Patents
一种重组高纯度的非洲猪瘟病毒pK205R亚单位蛋白及其制备方法和应用 Download PDFInfo
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- CN115073557A CN115073557A CN202110261349.0A CN202110261349A CN115073557A CN 115073557 A CN115073557 A CN 115073557A CN 202110261349 A CN202110261349 A CN 202110261349A CN 115073557 A CN115073557 A CN 115073557A
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- swine fever
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Abstract
本发明公开了一种重组高纯度的非洲猪瘟病毒pK205R亚单位蛋白及其制备方法和应用,其氨基酸序列如SEQ ID NO.3所示,所述制备方法如下:1)重组杆状病毒获得:将密码子优化后的非洲猪瘟病毒pK205R蛋白的核苷酸序列如SEQ ID NO.1所示的表达基因克隆到转移载体pBacPAK8中,然后与线性化的DNA质粒共转染昆虫细胞,得到含有非洲猪瘟病毒pK205R亚单位蛋白表达基因的重组杆状病毒;2)发酵培养:再将所述重组杆状病毒进行扩大培养后进行发酵,收集培养液;3)亚单位蛋白纯化:对步骤2)中所述培养液进行纯化,纯化后得到重组的非洲猪瘟病毒pK205R亚单位可溶性蛋白。本发明能够提供一种可大规模工业化生产的非洲猪瘟蛋白pK205R亚单位蛋白且制备方法简单、成本低能够达到国家现有标准。
Description
技术领域
本发明属于兽用生物制品技术领域。涉及一种高纯度非洲猪瘟pK205R蛋白大规模制备方法和应用。
背景技术
非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fevervirus,ASFV)引起的猪的急性、热性、高度接触性传染病,发病率及死亡率高达100%。猪感染非洲猪瘟病毒后出现皮肤充血,内脏器官出血,高热为特征临床症状,猪是ASFV自然感染的唯一哺乳动物宿主,包括家猪和野猪,尤其是家猪,易感性极高,对畜牧业影响巨大。世界动物卫生组织将其列为A类疫病,我国也将其列为一类动物传染病。
该病最早在1921年于非洲的肯尼亚国家确认发生,对非洲乃至全球多个国家的养猪业造成极大打击。2018年8月份以来,在我国多个省份爆发,给我国养猪业带来了严重的经济损失。虽然,国内外学者对非洲猪瘟做了大量的研究工作,但研究发现:常规制备的非洲猪瘟灭活苗效果不明显,而弱毒苗保护效果也不好,且安全性差,易造成散毒。目前,世界上尚未有有效预防非洲猪瘟的疫苗和治疗该病的药物发现,迫切需要新型疫苗的研发和生产来预防非洲猪瘟。
ASFV病毒是具有囊膜的虫媒DNA病毒。病毒颗粒呈二十面体对称结构,平均直径200nm,表面有含糖脂类的囊膜覆盖。其病毒基因组为双股线性DNA,大小为170-190kb,整个基因组大约有150个ORF,编码150-200中蛋白质。K205R基因是一早期转录基因,目前对该基因研究较少,推测该基因可能参与病毒的组装。pK205R蛋白由K205R基因编码的病毒蛋白,pK205R蛋白能较早刺激机体产生抗体,对病毒感染早期的诊断和治疗具有重要作用。世界动物卫生组织(OIE)推荐的诊断ASFV的方法主要是病原鉴定和血清学试验,病原鉴定存在操作繁琐或者仪器昂贵、存在生物安全的风险等一系列缺点;在血清学检测技术中,ELISA是OIE规定的国际贸易检验方法,但需要制备检测抗原,当前的技术存在的缺陷是不能在原核表达***中大规模表达,且纯化后的蛋白纯度较低,本发明成功构建一种能高表达pK205R蛋白的重组杆状病毒,并结合简单快速的纯化方法,得到大量高纯度的pK205R蛋白,且得到的pK205R蛋白不论是在空间结构,还是在翻译后修饰等方面与天然蛋白更为接近,能提高检测方法的特异性,为非洲猪瘟诊断和预防提供重要帮助。
发明内容
为解决现有技术的上述技术问题,本发明提供本发明提供非洲猪瘟pK205R亚单位蛋白。本发明还提供一种非洲猪瘟pK205R亚单位蛋白的制备方法及利用该蛋白制备一种重组的高纯度非洲猪瘟病毒pK205R亚单位蛋白总抗体检测方法。
为实现上述目的,本发明提供了一种重组高纯度的非洲猪瘟病毒pK205R亚单位蛋白,所述重组高纯度的非洲猪瘟病毒pK205R亚单位蛋白为非洲猪瘟病毒蛋白,其氨基酸序列如SEQ ID NO.3所示。
在本发明的优选技术方案中,密码子优化后的所述重组高纯度的非洲猪瘟病毒pK205R亚单位蛋白的核苷酸序列如SEQ ID NO.1所示。密码子优化前的所述重组高纯度的非洲猪瘟病毒pK205R亚单位蛋白的核苷酸序列如SEQ ID NO.2所示。
根据本发明的另一个方面,本发明提供了一种重组高纯度的非洲猪瘟pK205R亚单位蛋白的制备方法,所述制备方法包括如下步骤:
1)重组杆状病毒获得:将密码子优化后的非洲猪瘟病毒pK205R蛋白的核苷酸序列如SEQ ID NO.1所示的表达基因克隆到转移载体pBacPAK8中,然后与线性化的DNA质粒共转染昆虫细胞,得到含有非洲猪瘟病毒pK205R亚单位蛋白表达基因的重组杆状病毒;
2)发酵培养:再将所述重组杆状病毒进行扩大培养后进行发酵,收集培养液;
3)亚单位蛋白纯化:对步骤2)中所述培养液进行纯化,纯化后得到重组的非洲猪瘟病毒pK205R亚单位可溶性蛋白。
在本发明的优选技术方案中,步骤1)中,所述昆虫细胞为sf9细胞和/或HighFive5细胞。
在本发明的优选技术方案中,步骤2)中,所述发酵培养的细胞为sf9细胞和/或HighFive5细胞。
在本发明的优选技术方案中,步骤2)中,将所述重组杆状病毒进行扩大培养的方法包括以下步骤:
2-1)将所述重组杆状病毒转染sf9细胞和/或HighFive5细胞,培养72h,得到P1代重组杆状病毒;
2-2)将所述第一代重组杆状病毒转染sf9细胞和/或HighFive5细胞,培养72h,得到P2代重组杆状病毒;
2-3)重复转染sf9细胞和/或HighFive5细胞的步骤,获得Pn代重组杆状病毒。
在本发明的优选技术方案中,步骤2-3)中,所述n为自然数。
在本发明的优选技术方案中,步骤3)中,使用镍柱对所述培养液进行纯化。
根据本发明的又一个方面,本发明提供了所述重组高纯度的非洲猪瘟病毒pK205R亚单位蛋白在制备用于预防、诊断和治疗非洲猪瘟病毒感染的疫苗中的应用。
根据本发明的再一个方面,本发明提供了一种重组的高纯度非洲猪瘟病毒pK205R亚单位蛋白的总抗体检测方法,所述总抗体检测方法包括将非洲猪瘟病毒pK205R蛋白包被在抗原包被板检测区内,可与待检样品中的特异性抗体反应,形成抗原抗体复合物,再加入羊抗猪IgG二抗的酶结合物,形成“抗原-抗体-二抗-酶”复合物,最后通过加入TMB显示液进行显色,并读取光吸收值(OD450)。
实验有效性判定:只有当满足以下条件时检测才能有效:阳性对照OD值-阴性对照OD值>0.5;阴性对照OD值<0.15。
结果计算与判定:S/P=(样品OD值-阴性对照OD值)/(阳性对照OD值-阴性对照OD值);S/P值≥0.35,判定为ASFV抗体阳性;S/P值<0.35,判定为ASFV抗体阴性。
在本发明的优选技术方案中,所述总抗体检测方法包括如下步骤:
(1)将待检样品用样品稀释液在样品稀释板中稀释100倍(如取297μL样品稀释液再加入3μL待检样品),将其分别加入抗原包被反应板,每孔加100μL,同时设置阳性对照组、阴性对照组,其中,阳性对照组加入相应的100μL阳性对照液,阴性对照组加入100μL阴性对照液,每个样品和对照平行加样2个孔;
(2)加样完成后,将抗原包被反应板置37℃孵育1h后,洗涤液(提前将25×浓缩洗涤液稀释成1×,即为洗涤液,稀释时取10ml 25×浓缩洗涤液加到240ml超纯水中,混匀即得)洗板3-5次,甩干;
(3)每孔再加入100μL酶结合物,37℃孵育0.5h,洗涤液洗板3-5次,甩干;
(4)每孔加入100μL TMB显色液,室温避光显色10-15min,每孔加入100μL终止液;
(5)将抗原包被反应板置于酶标仪中,在450nm测定其光吸收值OD450;
(6)结果分析:实验有效性判定:只有当满足以下条件时检测才能有效:阳性对照OD值-阴性对照OD值>0.5;阴性对照OD值<0.15;结果计算与判定:S/P=(样品OD值—阴性对照OD值)/(阳性对照OD值—阴性对照OD值);S/P值≥0.35,判定为ASFV抗体阳性;S/P值<0.35,判定为ASFV抗体阴性。
在本发明的优选技术方案中,采用一试剂盒进行检测,所述试剂盒有一个盒体1,盒体1内设有抗原包被反应板存放槽2、样品稀释板存放槽3、非洲猪瘟病毒pK205R亚单位蛋白阳性对照液孔槽4、非洲猪瘟病毒pK205R亚单位蛋白阴性对照液孔槽5、样品稀释液孔槽6、酶结合物孔槽7、浓缩洗涤液孔槽8、TMB显色液9、终止液孔槽10和操作说明书存放槽11;抗原包被反应板存放槽2内放置抗原包被反应板,样品稀释板存放槽3内放置样品稀释板,各孔槽中4-10放置装有相应溶液的试剂瓶,操作说明书存放槽11内放置操作说明书。
其包被酶标板的抗原为重组杆状病毒表达和纯化的非洲猪瘟病毒pK205R亚单位蛋白。
所述非洲猪瘟病毒pK205R亚单位蛋白阳性对照液为:用样品稀释液100倍稀释的非洲猪瘟病毒pK205R亚单位蛋白猪阳性血清,分别按2mL/瓶分装。
所述非洲猪瘟病毒pK205R亚单位蛋白抗体阴性对照液为:用样品稀释液100倍稀释的不含有非洲猪瘟病毒pK205R亚单位蛋白抗体的猪阴性血清,按4ml/瓶分装。
所述样品稀释液为:1%BSA溶液,按照250ml/瓶分装。
所述酶结合物为:用样品稀释液5,000倍稀释的商品化羊抗猪IgG酶标二抗,按60mL/瓶分装。
所述浓缩洗涤液为:25×PBST溶液,按照150ml/瓶分装。
所述TMB显色液为:商品化的显示液,按60mL/瓶分装,所述的终止液为2M H2SO4溶液,按60ml/瓶分装。
与现有技术相比,本发明公开的表达序列、表达载体以及相应的纯化制备方法克服了现有技术中的缺陷,解决了大肠杆菌表达pK205R蛋白产量低、纯度差,与天然蛋白差距大,导致用表达的pK205R蛋白制备的试剂盒检测抗体特异性差等的问题。本发明能够在重组杆状病毒中直接可溶性表达pK205R,克服了现有技术中的诸多问题,且制备方法简单、表达量高、纯度高、成本低。
附图说明
图1表示pK205R基因序列优化前后比对结果。
图2表示pBacPAK8-OPTI-K205R质粒图谱。
图3表示pBacPAK8-OPTI-K205R双酶切鉴定结果:M是DNA Marker:DL10000Marker;1-2号质粒Mlu I/HindIII双酶切,条带大小分别为4117bp,1933bp,酶切结果正确。
图4表示重组pK205R的SDS-PAGE纯化检测结果:1是蛋白Marker,2是纯化后的pK205R蛋白。
图5表示pK205R蛋白纯化后Western-blot检测结果:1是蛋白Marker,2是pK205R蛋白。
具体实施方式
以下将结合附图和实施例对本发明做进一步说明,本发明的实施例仅用于说明本发明的技术方案,并非限定本发明。
本发明实施例中所使用的细胞、质粒和试剂均为市售产品,其中:
酶标板和样品稀释板均购自NUNC公司;
酶结合物购自美国Earthox公司;
TMB显示液购自北京索莱宝科技有限公司。
实施例1 pK205R蛋白表达及制备
1.1非洲猪瘟pK205R蛋白的选择
非洲猪瘟蛋白pK205R是由K205R基因编码的一段多肽,目前研究指出,pK205R蛋白能较早刺激机体产生抗体,对病毒感染早期的诊断和治疗具有重要作用。因此,利用pK205R蛋白对非洲猪瘟病毒感染早期诊断和治疗存在重要作用,尽管pK205R蛋白报道在原核表达***中存在表达,但是存在的缺点是表达量低,纯化后的蛋白纯度较低,蛋白与天然蛋白差距较大,导致制备的检试剂盒特异性差等缺点。
目前还没有重组杆状病毒表达***中能大规模可溶性表达及纯化得到高纯度pK205R蛋白,并将该蛋白用于非洲猪瘟病毒pK205R亚单位蛋白总抗体检测的报道。
1.2非洲猪瘟pK205R蛋白密码子优化
本实验室以2018年在中国报道流行的非洲猪瘟毒株亚型,参考Georgia 2007/1全基因序列(GenBank:FR682468.1)为模板,对编码非洲猪瘟病毒pK205R蛋白的核苷酸序列如SEQ ID NO.2所示(非洲猪瘟病毒pK205R蛋白的氨基酸序列如SEQ ID NO.3所示)进行密码子优化,得到OPTI-K205R序列,如SEQ ID NO.1所示,该序列合成工作委托南京金斯瑞生物科技有限公司完成。如图1所示,优化前后有20.3%的核苷酸序列不同。并克隆至转移载体pBacPAK8质粒,得到命名为pBacPAK8-OPTI-K205R的转移质粒。图2展示了转移质粒的谱图,图3展示了转移质粒的酶切鉴定结果,表明构建的pBacPAK8-OPTI-K205R质粒中包含密码子优化后的K205R基因。
1.3重组转移pBacPAK8-OPTI-K205R质粒与线性化DNA质粒共转染sf9细胞,制备P1代毒
(1)准备步骤:生物安全柜紫外灭菌30min;BacPAK Grace’s基础培养基恢复至室温,预先准备好悬浮培养的sf9细胞,细胞处于对数生长期,密度为1.5-2.5×106cell/ml(通常在做转染的前1-2天控制细胞密度,以确保转染当天有合适的细胞)。
(2)从28℃摇床中取出sf9细胞(125mL培养瓶),铺六孔板(孔直径35mm),每孔8-9×105个细胞,室温静置30-45min。
(3)弃掉培养基,加2ml/孔BacPAK Grace’s基础培养基,轻轻混匀之后弃掉,再加入2ml/孔基础培养基,室温孵育10-30min,期间可以准备转染质粒混合液:
(4)用TE缓冲液稀释质粒至100ng/μl的浓度。
(5)转染质粒混合液体系如下表:
(6)分别加入4μl转染试剂至上述准备好的质粒样本中,轻轻混合,室温孵育15min。
(7)弃去单层细胞中的培养基,加入1.5ml BacPAK Grace’s基础培养基。
(8)将步骤(6)中的混合质粒逐滴加入单层细胞,并轻轻晃动6孔板,加完之后置于27℃培养5h。之后更换为BacPAK Grace’s基础培养基。
(9)共转染完成之后培养约5天,收集P1代毒:在500g条件下对六孔板进行离心5min后将实验组的孔中上清转移至另一干净的EP管中,用锡箔纸包裹,4℃避光保存,得到P1代病毒。
1.4、制备P2代毒
P1代病毒的滴度经测定约1×106-1×107(pfu)/ml。其中pfu(plaque formingunits)表示空斑形成单位。
扩毒的MOI应控制在0.05-0.1。其中MOI(multiplicity of infection)表示感染时病毒与细胞数量的比值,如下式1.4.1所示。
P1扩P2病毒所需用的P1病毒量用上面公式1.4.1计算。
P1扩P2病毒步骤如下:铺细胞sf9,细胞量根据需要决定,单层细胞密度高些。算出所需P1体积后加入P1代病毒,27℃培养箱孵育72h后收毒。收毒方法同P1,在500g条件下离心5min后转移上清4℃避光保存,获得P2代毒。例如1.4步骤中采用10cm盘铺1×107个细胞,P1代毒加入1ml,收获P2代病毒,病毒滴度约为1×108(pfu)/ml。
1.5、制备P3代毒
P2扩P3病毒所需用的P2病毒量用上述公式1.4.1计算。
该步骤例如采用125ml摇瓶,细胞密度为1.5-2.5×106细胞/ml的25ml悬浮sf9细胞,扩毒的MOI=0.1,加入P2代毒。
P2扩P3病毒步骤如下:取处于对数生长期的,细胞密度为1.5-2.5×106细胞/ml的25ml悬浮sf9细胞,细胞量根据需要决定。计算出所需P2代毒体积后加入病毒,27℃培养箱摇床培养72h进行收毒。收毒方法同P1,在500g条件下离心5min后转移上清避光保存,获得P3代毒,其病毒滴度为1×109(pfu)/ml。
1.6非洲猪瘟pK205R蛋白发酵表达
在0.5-10之间选择适宜的MOI值,例如该实施例中选择MOI值为1时的表达情况时,将P3代毒按照发酵体积1:100接种于生长至对数期的High5细胞(Gibco)密度为1.5-2.5×106细胞/ml中进行发酵表达培养27℃培养箱摇床培养。每隔24小时收集样品如24、48、72h后收集细胞培养液,根据细胞生长状态选择适当的蛋白收获时间,细胞活率为50%左右。
1.7、非洲猪瘟pK205R蛋白纯化
(1)收集细胞培养液并透析纯化:上述步骤1.6中发酵培养72h后于室温、4000rpm条件下离心20min收集细胞培养液上清。上清通过滤器过0.8μm膜,再按照GE公司AKTA仪器操作说明书使用AKTA仪器进行蛋白透析纯化操作,得到纯化后的pK205R蛋白。同时预留80μL纯化前和纯化后的上清样品加入20μL的5×SDS-样品缓冲液,用于SDS-PAGE检测。
(2)除菌过滤:在生物安全柜中,过0.2μm低蛋白结合滤器,无菌的溶液存放于4℃冰箱,经上述步骤(1)透析纯化后的pK205R蛋白如图4所示,其中1表示蛋白marker;2表示纯化后的pK205R蛋白。SDS-PAGE检测结果表明:未纯化前,细胞培养上清中的目的蛋白纯度均能达到70%以上,而经过镍柱纯化(指用AKTA仪器纯化)后的目的蛋白经SDS-PAGE检测纯度可达到85%以上。
1.8非洲猪瘟pK205R蛋白的鉴定
1.8.1 SDS-PAGE检测
将实施例1纯化后的蛋白进行SDS-PAGE检测,所用样品中pK205R蛋白浓度为2μg/孔,结果如图4所示:从图中可以计算出,纯化后的pK205R蛋白SDS-PAGE纯度为85%,分子量约为24kD。
1.8.2 WESTERN-BLOT检测
将实例1纯化后的蛋白进行WESTERN-BLOT检测,转膜时间为1h,所用抗体为AntiHis-Tag Mouse,稀释比为1:2000,孵育时间为1h,结果如图5所示:从图中结果可以看出,纯化后的pK205R蛋白能与抗体发生有效结合。
实施例2:非洲猪瘟病毒pK205R亚单位蛋白总抗体检测试剂盒
2.1试剂盒组成
一种高纯度非洲猪瘟病毒pK205R亚单位蛋白总抗体检测试剂盒:包括1、盒体内设有抗原包被反应板存放槽;2、样品稀释板存放槽;3、非洲猪瘟病毒pK205R亚单位蛋白阳性对照液孔槽;4、非洲猪瘟病毒pK205R亚单位蛋白阴性对照液孔槽;5、样品稀释液孔槽;6、酶结合物孔槽;7、浓缩洗涤液孔槽;8、TMB显色液9、终止液孔槽10和操作说明书存放槽11;抗原包被反应板存放槽2内放置抗原包被反应板,样品稀释板存放槽3内放置样品稀释板,各孔槽中4-10放置装有相应溶液的试剂瓶,操作说明书存放槽11内放置操作说明书。抗原包被板包被非洲猪瘟病毒pK205R亚单位蛋白抗原。
其中,抗原包被反应板的规格为96孔,每个试剂盒共有5块包被板。样品稀释板的规格为96孔,每个试剂盒共有5块样品稀释板。抗原包被反应板和样品稀释板都是可以拆卸的96孔板,每块板都可以拆卸为12排,每排8孔。
2.2试剂盒制备
(1)抗原包被反应板制备:检测区在酶标板孔中均匀包被非洲猪瘟pK120R蛋白抗原,包被量为0.5-2μg/孔,包被缓冲液为0.05mol/L pH 9.6的碳酸盐缓冲液(即1L碳酸盐缓冲液中含1.59g Na2CO3和2.93g NaHCO3),2~8℃过夜后,使用PBST洗板3次;洗板后加入封闭液,封闭液为质量浓度为1-5%的BSA或脱脂牛奶的任一种或其组合,200μL/孔,37℃封闭2h,干燥后密封保存。
(3)酶结合物:用样品稀释液将商品化羊抗猪IgG酶标二抗按照1:5,000稀释而成,按60mL/瓶分装。
(5)TMB显色液:为商品化的TMB显示液,按60mL/瓶分装。
(6)终止液:为2M H2SO4溶液,配制时在178.26ml的超纯水中加入21.76ml的浓硫酸,混匀即得,按60ml/瓶分装。
(7)阳性对照液:用样品稀释液100倍稀释的非洲猪瘟病毒pK205R亚单位蛋白猪阳性血清,分别按2mL/瓶分装。
(8)阴性对照液:用样品稀释液100倍稀释的不含有非洲猪瘟病毒pK205R亚单位蛋白d的猪阴性血清,按4ml/瓶分装。
(9)组装:按每一个试剂盒配抗原包被反应板5块、样品稀释板5块、非洲猪瘟病毒pK205R亚单位蛋白猪阳性对照液1瓶、非洲猪瘟病毒pK205R亚单位蛋白猪阴性对照液1瓶、样品稀释液1瓶、酶结合物1瓶、浓缩洗涤液1瓶、TMB显色液1瓶、终止液1瓶和操作说明书1份进行装盒。
2.3试剂盒检测方法
(1)将待检样品用样品稀释液在样品稀释板中稀释100倍(如取297μL样品稀释液再加入3μL待检样品),将其分别加入抗原包被反应板,每孔加100μL,同时设置阳性对照组、阴性对照组,其中,阳性对照组加入相应的100μL阳性对照液,阴性对照组加入100μL阴性对照液,每个样品和对照平行加样2个孔;
(2)加样完成后,将抗原包被反应板置37℃孵育1h后,洗涤液(提前将25×浓缩洗涤液稀释成1×,即为洗涤液,稀释时取10ml 25×浓缩洗涤液加到240ml超纯水中,混匀即得)洗板3-5次,甩干;
(3)每孔再加入100μL酶结合物,37℃孵育0.5h,洗涤液洗板3-5次,甩干;
(4)每孔加入100μL TMB显色液,室温避光显色10-15min,每孔加入100μL终止液;
(5)将抗原包被反应板置于酶标仪中,在450nm测定其光吸收值OD450;
(6)结果分析:
实验有效性判定:只有当满足以下条件时检测才能有效:阳性对照OD值-阴性对照OD值>0.5;阴性对照OD值<0.15。
结果计算与判定:S/P=(样品OD值—阴性对照OD值)/(阳性对照OD值—阴性对照OD值);S/P值≥0.35,判定为ASFV抗体阳性;S/P值<0.35,判定为ASFV抗体阴性。
2.4试剂盒批内重复性试验
选取3块同一批次抗原包被的酶标板,在相同试验条件下分别对10份阴性、5份弱阳性和5份强阳性血清进行平行试验,结果显示抗原包被酶标板的批内变异系数小于4.35%(表1)。
2.5试剂盒批间重复性试验
选取3块不同批次包被的微量板,室温平衡后在相同试验条件下,分别对10份阴性、5份弱阳性和5份强阳性血清进行平行试验,结果显示抗原包被酶标板的批间变异系数小于7%(表2)。
2.6不同样本抗体检测
采用本发明方法与西班牙Ingenasa公司ELISA试剂盒(INGEZIM PPA COMPAC)进行比对分析,样品为200份随机血清。结果以阴阳性进行判断,下表3示出了参考方法比对结果。
表3:200份临床血清样品的方法比对
根据上表3数据可知,当分别用两种方法检测200份临床血清样品时,其符合率结果为:
阴性符合率=168/171*100%=98.2%;
阳性符合率=19/22*100%=86.4%;
总符合率=(168+19)/200*100%=93.5%。
由以上结果可知,本发明方法和参考方法(INGEZIM PPA COMPAC)的总符合率达到93.5%,其灵敏度均符合OIE要求,且包被抗原表达量高,纯度好,特异性好,生产成本低。
本发明通过上面的实施例进行举例说明,但是,应当理解,本发明并不限于这里所描述的特殊实例和实施方案。在这里包含这些特殊实例和实施方案的目的在于帮助本领域中的技术人员实践本发明。任何本领域中的技术人员很容易在不脱离本发明精神和范围的情况下进行进一步的改进和完善,因此本发明只受到本发明权利要求的内容和范围的限制,其意图涵盖所有包括在由附录权利要求所限定的本发明精神和范围内的备选方案和等同方案。
序列表
<110> 浙江海隆生物科技有限公司
<120> 一种重组高纯度的非洲猪瘟病毒pK205R亚单位蛋白及其制备方法和应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 4
<211> 636
<212> DNA
<213> 密码子优化后的pK205R蛋白核苷酸序列(DNA)
<400> 4
atggtggagc cacgcgaaca gttcttccag gacctgctgt ccgccgtgga ccagcagatg 60
gacaccgtca agaacgacat caaggacatc atgaaggaaa agacttcttt catggtgtca 120
ttcgagaact tcatcgaacg ttacgacacc atggagaaga acatccagga cctgcagaac 180
aagtacgagg aaatggctgc caacctgatg actgtcatga ccgacactaa gatccagctg 240
ggagctatca tcgcccagct ggagatcctg atgatcaacg gtactccact gcctgctaag 300
aagaccacta tcaaggaggc catgcccctg ccatccagca acaccaacaa cgaacagact 360
tcccctcccg ctagcggcaa gacctctgag actcctaaga agaaccccac caacgctatg 420
ttcttcactc gcagcgaatg ggcctcttca aacaccttcc gtgagaagtt cctgactcct 480
gaaatccagg ctatcctgga cgagcagttc gccaacaaga ccggtatcga gaggctgcac 540
gccgaaggcc tgtacatgtg gagaactcag ttctctgacg agcagaagaa gatggtcaag 600
gaaatgatga agaagcacca ccaccaccac cactaa 636
<210> 4
<211> 636
<212> DNA
<213> 密码子优化前的pK205R蛋白核苷酸序列(DNA)
<400> 4
atggttgagc cacgcgaaca gttttttcaa gatctgcttt cagcagtgga tcaacaaatg 60
gacactgtaa aaaatgacat aaaagacatt atgaaagaaa aaacgtcttt tatggtatca 120
ttcgaaaact ttatagaacg ttacgatacc atggaaaaaa atattcaaga ccttcagaat 180
aagtacgaag aaatggcggc caaccttatg accgtcatga cggatacaaa aattcagctt 240
ggagccatta tcgcccaact tgagattcta atgataaatg gcactccact tccggcaaaa 300
aagacaacaa ttaaggaggc tatgccctta ccttcatcaa acacgaataa tgaacaaacg 360
agtcctcccg cctcaggcaa aacaagtgaa acacctaaaa aaaatcccac gaatgcgatg 420
ttcttcacgc gtagcgaatg ggcatcctcg aatacttttc gagaaaagtt tttaacacca 480
gaaattcaag ccatattgga tgagcagttt gcaaacaaga ccgggatcga aagattgcat 540
gccgagggtc tttacatgtg gagaacccaa ttctctgacg aacagaagaa aatggtcaaa 600
gagatgatga agaagcacca ccaccaccac cactaa 636
<210> 4
<211> 211
<212> PRT
<213> pK205R蛋白的氨基酸序列(PRT)
<400> 4
Met Val Glu Pro Arg Glu Gln Phe Phe Gln Asp Leu Leu Ser Ala Val
1 5 10 15
Asp Gln Gln Met Asp Thr Val Lys Asn Asp Ile Lys Asp Ile Met Lys
20 25 30
Glu Lys Thr Ser Phe Met Val Ser Phe Glu Asn Phe Ile Glu Arg Tyr
35 40 45
Asp Thr Met Glu Lys Asn Ile Gln Asp Leu Gln Asn Lys Tyr Glu Glu
50 55 60
Met Ala Ala Asn Leu Met Thr Val Met Thr Asp Thr Lys Ile Gln Leu
65 70 75 80
Gly Ala Ile Ile Ala Gln Leu Glu Ile Leu Met Ile Asn Gly Thr Pro
85 90 95
Leu Pro Ala Lys Lys Thr Thr Ile Lys Glu Ala Met Pro Leu Pro Ser
100 105 110
Ser Asn Thr Asn Asn Glu Gln Thr Ser Pro Pro Ala Ser Gly Lys Thr
115 120 125
Ser Glu Thr Pro Lys Lys Asn Pro Thr Asn Ala Met Phe Phe Thr Arg
130 135 140
Ser Glu Trp Ala Ser Ser Asn Thr Phe Arg Glu Lys Phe Leu Thr Pro
145 150 155 160
Glu Ile Gln Ala Ile Leu Asp Glu Gln Phe Ala Asn Lys Thr Gly Ile
165 170 175
Glu Arg Leu His Ala Glu Gly Leu Tyr Met Trp Arg Thr Gln Phe Ser
180 185 190
Asp Glu Gln Lys Lys Met Val Lys Glu Met Met Lys Lys His His His
195 200 205
His His His
210
Claims (9)
1.一种重组高纯度的非洲猪瘟病毒pK205R亚单位蛋白,其特征在于,所述重组高纯度的非洲猪瘟病毒pK205R亚单位蛋白为非洲猪瘟病毒蛋白,其氨基酸序列如SEQ ID NO.3所示。
2.如权利要求1所述的重组高纯度的非洲猪瘟病毒pK205R亚单位蛋白,其特征在于,密码子优化后的所述重组高纯度的非洲猪瘟病毒pK205R亚单位蛋白的核苷酸序列如SEQ IDNO.1所示。
3.一种制备如权利要求1~2任一所述的重组高纯度的非洲猪瘟病毒pK205R亚单位蛋白的方法,其特征在于,所述方法包括以下步骤:
1)重组杆状病毒获得:将密码子优化后的非洲猪瘟病毒pK205R蛋白的核苷酸序列如SEQ ID NO.1所示的表达基因克隆到转移载体pBacPAK8中,然后与线性化的DNA质粒共转染昆虫细胞,得到含有非洲猪瘟病毒pK205R亚单位蛋白表达基因的重组杆状病毒;
2)发酵培养:再将所述重组杆状病毒进行扩大培养后进行发酵,收集培养液;
3)亚单位蛋白纯化:对步骤2)中所述培养液进行纯化,纯化后得到重组的非洲猪瘟病毒pK205R亚单位可溶性蛋白。
4.如权利要求3所述的制备方法,其特征在于,步骤1)中,所述昆虫细胞为sf9细胞和/或HighFive5细胞。
5.如权利要求3所述的制备方法,其特征在于,步骤2)中,所述发酵培养的细胞为sf9细胞和/或HighFive5细胞。
6.如权利要求5所述的制备方法,其特征在于,步骤2)中,将所述重组杆状病毒进行扩大培养的方法包括以下步骤:
2-1)将所述重组杆状病毒转染sf9细胞和/或HighFive5细胞,培养72h,得到P1代重组杆状病毒;
2-2)将所述第一代重组杆状病毒转染sf9细胞和/或HighFive5细胞,培养72h,得到P2代重组杆状病毒;
2-3)重复转染sf9细胞和/或HighFive5细胞的步骤,获得Pn代重组杆状病毒。
7.如权利要求6所述的制备方法,其特征在于,步骤2-3)中,所述n为自然数。
8.如权利要求3所述的制备方法,其特征在于,步骤3)中,使用镍柱对所述培养液进行纯化。
9.一种如权利要求1-2任一权利要求所述的重组高纯度的非洲猪瘟病毒pK205R亚单位蛋白在制备用于预防、诊断和治疗非洲猪瘟病毒感染的疫苗中的应用。
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CN116903709A (zh) * | 2023-07-03 | 2023-10-20 | 华中农业大学 | 一种非洲猪瘟病毒pK205R蛋白抗原表位肽及其单克隆抗体与应用 |
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CN116082524A (zh) * | 2023-01-10 | 2023-05-09 | 华南生物医药研究院 | 一种非洲猪瘟病毒p30-p54重组融合蛋白及其构建方法和应用 |
CN116082524B (zh) * | 2023-01-10 | 2023-08-08 | 华南生物医药研究院 | 一种非洲猪瘟病毒p30-p54重组融合蛋白及其构建方法和应用 |
CN116903709A (zh) * | 2023-07-03 | 2023-10-20 | 华中农业大学 | 一种非洲猪瘟病毒pK205R蛋白抗原表位肽及其单克隆抗体与应用 |
CN116903709B (zh) * | 2023-07-03 | 2024-05-07 | 华中农业大学 | 一种非洲猪瘟病毒pK205R蛋白抗原表位肽及其单克隆抗体与应用 |
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