CN113755513A - 一种基于非洲猪瘟病毒p30、p72蛋白的病毒样颗粒的制备与应用 - Google Patents
一种基于非洲猪瘟病毒p30、p72蛋白的病毒样颗粒的制备与应用 Download PDFInfo
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Abstract
本发明公开了一种表面展示非洲猪瘟病毒P30、P72蛋白的重组病毒样颗粒的制备与应用，通过T7噬菌体展示技术，将SpyTag标签蛋白展示于T7噬菌体表面，从而构建了一种工程化噬菌体(T7‑ST)。同时，通过大肠杆菌原核表达系统，表达纯化了P30‑Spycatcher和P72‑Spycatcher融合蛋白。在适当的缓冲体系中，将T7‑ST与P30‑SC、P72‑SC两种融合蛋白分别混合孵育，最终得到表面展示P30‑SC、P72‑SC蛋白的重组VLP颗粒。通过VLP制备的新型基因工程疫苗，具有免疫保护效果突出、安全性好、抗原纯度高等特点。还能为机体提供多重保护。
Description
技术领域
本发明属于生物技术领域，涉及一种表面展示非洲猪瘟病毒P30、P72蛋白的病毒样颗粒(VLP)颗粒的制备及其应用。
背景技术
非洲猪瘟(Infection with African swine fever virus，ASF)是由非洲猪瘟病毒(African Swine fever virus，ASFV)感染家猪和野猪而引起的一种急性、出血性的烈性传染病。急性型非洲猪瘟起病快，病程短，以高热、食欲不振、发紫、内脏大出血、病死率近100％为特征[1]。世界动物卫生组织(OIE)将其列为法定报告动物疫病，我国将其列为一类动物疫病和重点防范的外来动物疫病之一。我国自2018年8月暴发首例ASF以来，截止2019年6月6日，已有32个省份累计暴发137起疫情(OIE和农业农村部统计数据)，几乎席卷我国所有省份和地区。我国作为世界猪肉产量最大国(约占世界总产量的50％)，这场突如其来的ASF疫情对我国的养猪业构成巨大威胁[2]。
ASFV为有囊膜的双链DNA病毒，是非洲猪瘟相关病毒科(Asfarviridae)非洲猪瘟病毒属(Asfivirus)的唯一成员，也是迄今发现的唯一DNA虫媒病毒。胞外病毒粒子直径约为200nm，由内核(Internal core)、内核心壳(Core shell)、内膜(Inner envelope)、衣壳(Capsid)、囊膜(External envelope)形成的同心多层20面体对称结构组成[3]。ASFV基因组为170-190kb的线性双链DNA，含有约150-181个开放阅读框，编码约150-200种蛋白[4]。内核心壳由PP220、PP62等多聚蛋白构成。内膜在电子显微镜下为单脂膜[5]，主要来源于内质网膜，包含p54、p17、p12蛋白。衣壳由2 000多个六边形壳粒组成，主要为p72蛋白，占病毒粒子总量的33％，衣壳蛋白p72、pE120R介导成熟病毒粒子从病毒工厂到细胞膜转运、出芽排出，p72具有促进病毒蛋白折叠/降解的伴侣分子样作用，可募集Hsp70促进病毒蛋白的折叠。P54和P30蛋白参与病毒附着的两个不同步骤，介导ASFV病毒与细胞受体之间的特异性相互作用，并且都有助于抗体介导的保护性免疫应答[6]，其中P30蛋白在病毒感染细胞的内化过程中起作用[7]。由于ASFV生物学特性的复杂性，迄今未能研制出安全有效的疫苗[8]。P30、P72蛋白在病毒感染过程中的重要作用，使其成为疫苗开发、抗体治疗和基于抗原的诊断检测的重要靶点。
噬菌体展示技术经过20年的发展完善，已被广泛应用于抗原抗体库的建立、药物设计、疫苗研究、病原检测、基因治疗、抗原表位研究及细胞信号转导研究等[9]。由于噬菌体展示技术实现了基因型和表型的有效转换，因此人们可以在基因分子克隆基础上实现蛋白质构象体外控制，从而为获取具有良好生物学活性的表达产物提供了强有力手段。T7噬菌体展示技术具有稳定性高、特异性强、表达量高、操作简单、易于富集等优势[10]。利用T7噬菌体展示技术将外源蛋白展示于噬菌体表面，可制备重组VLP颗粒。通过VLP制备的新型基因工程疫苗，具有免疫保护效果突出、安全性好、抗原纯度高等特点。同时，还能为机体提供多重保护，有效刺激动物机体产生细胞免疫、体液免疫、粘膜免疫，为机体打造全方位免疫保护，具有良好的应用前景。
发明内容
为了解决现有技术中的问题，本发明提供一种基于非洲猪瘟病毒P30、P72蛋白的病毒样颗粒的制备与应用，解决现有技术中ASFV迄今未能研制出安全有效的疫苗的问题。
本发明目的是通过如下技术方案实现的：
表达ASFV病毒P30-Spycatcher融合蛋白的pET-28a原核表达载体pET-28a-P30-SC，序列特征为SEQ ID No.4。
表达ASFV病毒P72-Spycatcher融合蛋白的pET-28a原核表达载体pET-28a-P72-SC，序列特征为SEQ ID No.5。
表达蛋白-Spycatcher融合蛋白的基因，包含ASFV P30、P72蛋白和Spycatcher标签蛋白，蛋白与标签之间以一段柔性肽连接，P30-SC氨基酸序列特征为SEQ ID No.6，P72-SC氨基酸序列特征为SEQ ID No.7。
一种基于非洲猪瘟病毒P30、P72蛋白的病毒样颗粒的制备方法，构建表达载体pET-28a-P30-SC和pET-28a-P72-SC并表达纯化融合蛋白，将大肠杆菌原核表达系统纯化的P30-SC、P72-SC融合蛋白在缓冲液中，分别与表面展示ST标签蛋白的重组T7噬菌体颗粒T7-ST共同孵育，验证结合情况，通过板式化学发光实验对制备的病毒样颗粒颗粒的免疫原性进行评估。
所述构建表达载体pET-28a-P30-SC和pET-28a-P72-SC并表达纯化融合蛋白，具体包括以下步骤：
(1)ASFV P30、P72基因的PCR扩增：以实验室现存基因所在质粒为模板，设计无缝克隆引物扩增P30、P72蛋白编码基因用于pET-28a-Spycatcher原核载体连接；
(2)Spycatcher基因的合成：将Spycatcher基因序列合成于pET-28a原核表达载体之上，在序列N段加上一段柔性肽序列，得到pET-28a-Spycatcher表达载体，核苷酸序列为SEQ ID No.8；
(3)表达P30-SC、P72-SC原核表达载体的构建及蛋白纯化：通过无缝克隆，将P30、P72基因序列分别克隆至pET-28a-Spycatcher原核表达载体上，得到pET-28a-P30-SC重组质粒，序列特征为SEQ No.4和pET-28a-P72-SC重组质粒，序列特征为SEQ No.5；重组质粒转化大肠杆菌BL21表达菌株，经过蛋白纯化获得P30-SC、P72-SC融合蛋白。
所述表面展示ST标签蛋白的重组T7噬菌体的构建方法为：通过T7噬菌体展示技术，将外源SpyTag蛋白基因片段插入到T7噬菌体基因组中，通过体外包装，获得表面展示ST蛋白的T7噬菌体。具体包括以下步骤：
(1)首先通过基因合成获得ST基因，基因N段加入一段柔性肽，合成序列两端加入EcoR I和Hind III限制性酶切位点序列及相应的保护性碱基，序列特征为SEQ ID No.1；
(2)重组噬菌体基因的构建：通过EcoR I和Hind III两种限制性内切酶双酶切合成的ST基因，之后通过T4连接酶将酶切产物与T7载体臂相连接，从而获得重组噬菌体基因组；
(3)体外包装：将步骤(2)中连接产物与T7包装提取物混合孵育，获得完整的含有重组基因组的T7噬菌体颗粒；
(4)噬菌斑分析：通过双层琼脂培养法，对包装产物进行噬菌斑计数，从而计算噬菌体滴度；
(5)文库鉴定：蛋白酶K法提取T7 DNA，然后用T7通用引物进行PCR鉴定，最终得到所需要的噬菌体；
(6)文库扩增：使用液体裂解液法对噬菌斑进行扩增，待菌液变澄清后，离心将细胞碎片去除；通过PEG/NaCl沉淀法，对上清中的噬菌体进行浓缩，之后通过噬菌斑计数测定浓缩后的噬菌体滴度。
利用上述方法制备的病毒样颗粒在制备用于抗非洲猪瘟病毒药物中的应用。
本发明有益效果：本发明基于在非洲猪瘟病毒感染细胞及内化过程中起重要作用的P30蛋白以及ASFV主要衣壳蛋白P72，利用Spycatcher与SpyTag之间异肽键的特异性相互作用，制成了表面展示病毒致病相关P30、P72蛋白的VLP颗粒。与新型的腺病毒疫苗、mRNA疫苗相比，通过VLP制备的新型基因工程疫苗，具有免疫保护效果突出、安全性好、抗原纯度高等特点。同时，还能为机体提供多重保护，有效刺激动物机体产生细胞免疫、体液免疫、粘膜免疫，为机体打造全方位免疫保护，具有良好的应用前景，为非洲猪瘟病毒的免疫防控提供选择。表面展示ST的工程化T7噬菌体展示文库的构建具有一定的创新性，在为ASFV的防控提供新思路与制剂的同时，也为其他疾病免疫防控制剂的制备奠定了基础。
附图说明
图1：包装产物噬菌斑分析结果；
图2：噬菌体重组率鉴定；
图3：文库扩增后噬菌斑分析测定噬菌体滴度；
图4：P30、P72基因无缝克隆PCR扩增结果；
图5：pET-28a-P30-SC和pET-28a-P72-SC重组质粒转化大肠杆菌后的转化子检测，1-3泳道分别代表P30-SC大肠杆菌转化子菌落PCR检测结果；4-6泳道分别代表P72-SC大肠杆菌转化子菌落PCR检测结果；
图6：P30-SC融合蛋白预表达结果；
图7：P30-SC融合蛋白大规模表达纯化结果；
图8：P72-SC融合蛋白预表达结果；
图9：P72-SC融合蛋白大规模表达纯化结果；
图10：P30-SC展示在T7-ST表面示意图；
图11：SDS-PAGE验证P30-SC、P72-SC与T7-ST结合情况；
图12：Western blot验证P30-SC、P72-SC与T7-ST结合情况；
图13：化学发光实验验证VLP免疫原性结果。
具体实施方式
以下结合实例进一步阐述本发明，其中的内容不应理解为限定该发明的范围。实施例中所述试剂除特别说明，均为商业化试剂。
实施例1.表面展示SpyTag的工程化噬菌体的构建
(1)SpyTag片段的获得
将带有柔性肽和EcoR I/Hind III酶切位点序列(带保护性碱基)的SpyTag片段，设计单链互补引物，退火合成双链基因片段，序列特征为SEQ No.1。
连接反应需要的目的基因片段为0.06pmol，合成的的基因总量为5nmol/管，因此加入50μL的ddH2O溶解后，浓度为0.1nmol，即100pmol，故需将片段再100倍稀释至1pmol后，取1μL进行双酶切。将上述合成基因片段经EcoR I/Hind III双酶切，酶切体系如下：
37℃，酶切2h后，65℃，10min，使酶灭活，这样就得到终浓度为0.1pmol的插入基因片段。
(2)SpyTag片段与T7载体臂的连接
为了获得最大的克隆效率，优化每种插入物制备的插入物：载体比率是有必要的。最佳比例将根据插入片段的性质和质量而变化，但是通常在1：1至3：1的摩尔比(插入片段：载体)下可获得最高的克隆效率。插入制剂应不含干扰物质(盐，引物，核苷酸等)。通过使纯化的寡核苷酸退火而制备的插入物无需进一步处理即可使用。
连接体系如下：
将连接体系置于金属浴上，25℃连接3h。
(3)体外包装
1、让T7 Select包装提取物在冰上解冻(提取物的体积为25μL，可包装高达1μg的载体DNA而不会降低效率)；
2、每25μL提取物加入5μL连接反应。用吸管顶端轻轻搅拌，不要旋转；
3、在室温(22℃)下孵化反应2小时；
4、加入270μL无菌LB培养基停止反应。(如果包装产物在扩增前储存超过24小时，加入20μL氯仿并通过倒置轻轻混合。包装反应可在4℃保存长达一周，滴度没有明显损失。为了长期储存，包装后的噬菌体必须用平板或液体培养的方法进行扩增。)
5、进行如下所述的空斑分析以确定产生的重组子的数量。
(4)噬菌斑分析
1、在平板划线的宿主菌株中，挑取单菌落接种在无抗无菌LB培养基中，在37℃摇动培养至OD600＝1.0。不要使用储存时间超过48小时的宿主细胞。
2、融化上层琼脂培养基，分装至试管中，每管4mL，将其转移到45℃水浴中。
3、使用无菌LB培养基作为稀释剂，准备一系列样品稀释液。通常，重组噬菌体的适当稀释度为103-106。
4、吸取500μL对数期菌液至EP管中，每个EP管中加入5μL的包装产物(T7噬菌体)，吸附5-10min。
6、将上述EP管中的混合物加入到预热的含上层琼脂培养基的试管中，混合均匀后倒入预热(37℃)的无抗无菌LB琼脂平板上。立即(轻轻地)旋转平板，使琼脂均匀分布。
7、让平板静置几分钟，直到顶部琼脂变硬，然后倒置并在37℃孵育3-4小时，或在室温下过夜。
8、计数噬菌斑并计算噬菌体滴度.
菌斑测定结果如图1所示，最终平板上噬菌斑数为2，经计算，噬菌体滴度为200pfu。
(5)噬菌体文库重组率鉴定
1、蛋白酶K法提取病毒DNA。挑取噬菌斑至5mL对数期BL21菌液中，
37℃培养至菌液澄清；取500μL裂解后菌液，5000rpm离心20min后，取250μL上清，加入100μL TE缓冲液，75μL 10％SDS和25μL蛋白酶K，58℃裂解2h；加入500μL氯仿，13000rpm，4℃，离心5min；取上层水相，加入无水乙醇至终浓度为70％，于4℃中过夜醇沉；13000rpm，4℃，离心15min，弃上清，室温下晾干；加入200μL ddH2O溶解。
2、使用T7通用检测引物T7-F(SEQ ID NO.9：
GGAGCTGTCGTATTCCAGTC)和T7-R(SEQ ID NO.10：
AACCCCTCAAGACCCGTTTA)进行噬菌体基因组PCR验证，结果如图2所示，PCR产物大小为176bp，两个噬菌斑所提取的基因组均验证正确，说明噬菌体文库重组率为100％。
(6)文库扩增
1、液体裂解液法扩增噬菌体文库。宿主菌大肠杆菌37℃培养OD600＝0.8左右时，加入噬菌体继续培养；
2、待培养物开始变澄清时，终止培养，8000rpm离心10min，收集上清即为扩增后的噬菌体文库；
3、浓缩纯化噬菌体。慢慢搅动并加入NaCl至终浓度为0.5mol/L，这有利于病毒的沉淀，再等量加入10％PEG；
4、4℃过夜或放置一段时间；
5、8000rpm离心30分钟，收集病毒沉淀；
6、将病毒沉淀加入适量的1×PBS中，4℃过夜；
7、10000r/min离心1小时,沉淀即为浓缩的病毒。还可以用梯度离心或其他方法进一步纯化。
8、对扩增后文库进行噬菌斑测定。
结果如图3所示，108倍稀释的平板上，噬菌斑数量为325，经计算，扩增后噬菌体文库滴度为3.25×1012PFU。
实施例2.P30-SC融合蛋白的原核表达纯化
(1)P30、P72基因的扩增
P30基因序列参考ASFV病毒P30基因序列(GenBank号：JQ764967.1)，以实验室现存质粒为模板，设计无缝克隆引物P30-F(SEQ ID NO.11：cagcaaatgggtcgcggatccATGAAAATGGAGGTCATCTTCAAAA)和P30-R(SEQ ID NO.12：accggaattcgagtcggatccTTTTTTTTTTAAAAGTTTAATAACCATGAG)扩增ASFV P30蛋白编码基因P30用于原核载体连接。
P72基因序列参考ASFV病毒P72基因序列(GenBank号：AY578708.1)，以实验室现存质粒为模板，设计无缝克隆引物P72-F(SEQ ID NO.13：cagcaaatgggtcgcggatccATGGCATCAGGAGGAGCTTTTTGTC)和P72-R(SEQ ID NO.14：accggaattcgagtcggatccGGTATGGCTACACGTTCGCTGCGTA)扩增ASFV P72蛋白编码基因P72用于原核载体连接。
PCR体系为：
使用以下PCR程序进行扩增：
P30 PCR扩增产物大小为603bp，P72 PCR扩增产物大小为854bp，结果图如图4所示。
(2)pET-28a-P30-SC、pET-28a-P72-SC表达载体的构建
将P30、P72基因连接到pET-28a-Spycatcher表达载体上(公司合成，序列特征为SEQ No.8)。pET-28a-Spycatcher载体经BamH I酶切线性化后与无缝引物PCR扩增后的P30、P72基因连接，利用无缝克隆试剂盒(C112-01,Vazyme)获得连接产物，并转化E.coli DH5α。大肠杆菌转化子经卡那霉素抗性平板筛选，并用P30无缝克隆引物(P30-F:
cagcaaatgggtcgcggatccATGAAAATGGAGGTCATCTTCAAAA(SEQ ID NO.11)和P30-R:accggaattcgagtcggatccTTTTTTTTTTAAAAGTTTAATAACCATGAG(SEQ ID NO.12))和P72无缝克隆引物(P72-F:cagcaaatgggtcgcggatccATGGCATCAGGAGGAGCTTTTTGTC(SEQ ID NO.13)和P72-R:accggaattcgagtcggatcc GGTATGGCTACACGTTCGCTGCGTA(SEQ ID NO.14))进行PCR验证及测序。
结果如图5所示，P30菌落PCR扩增产物大小在603bp，阳性转化子为No.1号，P72菌落PCR扩增产物大小在854bp，阳性转化子为No.5号，测序结果表明，与预期结果一致，表明pET-28a-P30-SC与pET-28a-P72-SC表达载体构建成功。
(3)P30-SC、P72-SC融合蛋白的表达纯化
1、P30-SC、P72-SC融合蛋白的试表达。
转化重组质粒：
1.从-80℃冰箱中取出BL21感受态(100μL/tube)置于冰上化冻约5min；
2.取1μL质粒加至感受态细胞中，轻弹EP管壁数下后冰上放置30min；
3.热击：将细胞置于42℃水中热击45sec后即刻插在冰上2min；
4.在EP管中加入600μL无抗LB，在37℃摇床上培育45min；
5.铺细胞：吸取100μL细胞至平板中用涂布棒涂匀；
6.将铺好细胞的平板静置2min后，37℃倒置培养14小时。
诱导试表达：
1.挑取1个典型单菌落至5ml卡那霉素抗性LB培养基中，分别编号，将试管至于37℃摇床中震荡培养5h。
2.取出试管，此时菌体OD600≈1.2
①保种：分别取800μL菌液至保种管中，再加入800μL 50％甘油，混匀，并分别标明蛋白名称、载体名称、菌株名称、抗性、日期、编号，存放于-80℃冰箱；
②对照取样：分别取800μL菌液至Ep管中，分别记录编号；
③诱导：在剩下的菌液中分别加入1.5μL 1M IPTG，将试管至于16℃摇床继续诱导培养5h；
3.取出试管，分别取800μL菌液至Ep管中，剩下的菌液超声破碎菌体后12000rpm，4℃离心10min，将上清和沉淀分开，分别记录编号；
4.制样：分别将对照样品、诱导后样品和沉淀样品离心，弃上清，用100μL无菌水重悬菌液后加入20μL 6×SDS loading buffer，在100μL上清样品中同样加入20μL 6×SDSloading buffer，涡旋震荡混匀后置于100℃金属煮样器中煮10分钟；
5.跑电泳：将煮好的样品离心，点样时尽量吸取上层样品5μL，电压：150V，时常：50min。
6.染色：将胶从胶板中取出用清水清洗后，置于染色盒内加入清水，微波炉高火煮5min，弃水，再加入染色液，微波炉高火煮1min，取出后静置5min，回收染色液，最后用水再煮5min，此时可见条带。
7.鉴定：根据marker大小，对比未诱导组鉴定是否有表达，如果有表达，则可准备下一步大规模生产。
蛋白试表达结果如图6、图8所示，在诱导后的条带中可见目的蛋白成功表达，分子量符合预测大小，为可溶性蛋白。
大规模表达纯化：
提前一天挑取保存的P30 BL21甘油菌至5mL卡那霉素抗性的LB培养基中，活化菌株；第二天将5mL菌液平均转移至两个900mL的卡那霉素抗性LB培养基中，37℃，220rpm培养5h至OD600＝1.2左右；将摇床温度设置为16℃降温1h，然后在培养基中2加入360μL IPTG，16℃，220rpm过夜培养16-17h；收菌：将诱导后的菌液倒至离心瓶中(倒三分之二，不要倒满)，秤好并对称放至离心机中，4000g离心20分钟；上清倒掉，用勺将细菌抠下来，转移至烧杯中，悬菌buffer重悬菌液；菌液中按照1：100加入1M的PMSF，然后使用高压破菌仪破碎菌体；将破碎后菌液转移至超速离心管中，18000rpm，4℃离心30min；将上清进行镍柱亲和层析，20mM咪唑洗杂，300mM咪唑洗脱蛋白，洗脱是分8次，每次1mL；制样：取每管洗脱蛋白20μL，加入4μL 6×SDS Loading Buffer，沸水煮10min后上样。
将所制样品进行SDS-PAGE电泳，结果如图7、图9所示，成功得到P30-SC和P72-SC融合蛋白，P30-SC分子量在37kDa左右，P72-SC分子量在45kDa左右。
实施例3.表面展示P30-SC、P72-SC的T7噬菌体VLP颗粒的制备
使用实施例2中纯化方法得到的蛋白溶于高浓度的咪唑中，因此需要通过透析，将蛋白缓冲液置换为1×PBS。将透析后的蛋白通过浓缩管浓缩后，取20μL蛋白与先前纯化的等体积的T7-ST噬菌体共同孵育，4℃过夜即得到所制备的VLP颗粒(P30-SC::T7-ST，简称P30::T7；P72-SC::T7-ST，简称P72::T7)。结合后得到的VLP结构如图10所示，每个T7-ST噬菌体表面原则上可展示高达415个拷贝的融合蛋白。
实施例4.P30::T7与P72::T7 VLP颗粒的表征
1、SDS-PAGE验证。分别取T7-ST、P30-SC、P30::T7和P72::T7各20μL，加入5μL 5×SDS loading buffer，沸水浴煮样10min，12000rpm离心10min，吸取8μL上清用于12％SDS-PAGE电泳。
图11结果表明，P30-SC蛋白结合T7-ST噬菌体后，分子量为77kDa，大小正确，表明P30-SC成功结合并展示在T7-ST噬菌体表面；P72-SC蛋白结合T7-ST噬菌体后，分子量为85kDa，大小正确，表明P72-SC成功结合并展示在T7-ST噬菌体表面。
2、Western Blot验证。由于T7-ST本身不带有his标签，所以分别取T7-ST、P30-SC、P30::T7、P72::T7样品各20μL，加入5μL 5×SDS loading buffer，沸水浴煮样10min，12000rpm离心10min，吸取5μL上清用于12％SDS-PAGE电泳。
通过湿转法将蛋白分离胶转移至与其同等大小的PVDF膜上，在300mA 100min条件下转膜；室温下用5％脱脂牛奶封闭1小时；将膜完全浸没在用5％BSA 1:2000稀释的鼠抗His单克隆抗体(HT501,Transgene)中，4℃置于摇床上孵育过夜；回收一抗，用1×TBST缓冲液漂洗PVDF膜3次，每次10min；用5％BSA 1:5000稀释的羊抗鼠HRP标记二抗(LK2003,Sungene Biotech)室温孵育1h；用1×TBST缓冲液漂洗PVDF膜3次，每次10min；避光向PVDF膜正面滴加化学发光显色底物(34075,ThermoFisher)，通过Bio-rad化学发光成像仪曝光，观察蛋白展示情况。
结果如图12所示，结合后的P30::T7分子量在77kDa，P72::T7分子量在85kDa，大小正确。Western Blot结果表明P30-SC与P72-SC成功结合并展示在T7-ST噬菌体表面。
3、化学发光实验。
通过酶联抗体竞争吸附板式化学发光实验，来对制备的P30::T7、P72::T7VLP颗粒的免疫原性进行初步的评估。具体步骤如下：
1、包被(优化包被液与包被浓度)
(1)将待包被蛋白稀释2μg/mL浓度，准备发光板，按照100μL每孔包被，4℃包被过夜；
(2)包被过夜后，洗涤液洗涤两次，并拍干；
2、封闭
(1)上述拍干后的发光板，每孔加入350μL封闭液，置于37℃，封闭1小时；
(2)封闭结束后，甩出封闭液，拍干，完成封闭；
3、反应步骤
(1)加入合适稀释倍数及体积的酶标抗原(100μL)和合适稀释液倍数与体积的样本(50μL)；
(2)将其置于37℃孵育箱内，孵育45min；
(3)孵育结束后，利用洗涤液洗涤5次，并拍干；
4、发光步骤
(1)将发光底物A与底物B混合均匀待用；
(2)向上述拍干的发光板中，每孔加入100μL发光底物混合液；
5、结果检测与分析
将加完发光液的发光板立即放入发光检测仪进行检测，并将检测结果复制到Eecel进行结果分析。
结果如图13所示，在与所包被抗原的竞争结合中，P30::T7和P72::T7两组VLP表现出良好的竞争结合抑制，表明我们制备的VLP颗粒具有良好的免疫原性。
尽管本发明内容是结合上述实施例进行说明，但本发明的实施方式并不受上述实施例的限制,本发明的范围由所附权利要求书限定，其他的任何在限定范围内所作的改变或修饰，均应为等效的置换方式，均包含在本发明的保护范围之内。
References:
[1]Sanchez-Vizcaino,J.M.,L.Mur,and B.Martinez-Lopez,African SwineFever:An Epidemiological Update.Transboundary and Emerging Diseases,2012.59:p.27-35.
[2]Vergne,T.,et al.,Pig empire under infectious threat:risk ofAfrican swine fever introduction into the People's Republic ofChina.Veterinary Record,2017.181(5).
[3]Wang,Nan et al.“Architecture of African swine fever virus andimplications for viral assembly.”Science(New York,N.Y.)vol.366,6465(2019):640-644.
[4]Jia N,Ou Y,Pejsak Z,et al.Roles of African swine fever virusstructural proteins in viral infection[J].Journal of Veterinary Research,61(2):135-143.
[5]Revilla,Y.,D.Perez-Nunez,and J.A.Richt,African Swine Fever VirusBiology and Vaccine Approaches,in Advances in Virus Research,Vol 100,M.Kielian,T.C.Mettenleiter,and M.J.Roossinck,Editors.2018.p.41-74.
[6]Oviedo J M,Rodr Guez F,G Mezpuertas P,et al.High level expressionof the major antigenic African swine fever virus proteins p54 and p30 inbaculovirus and their potential use as diagnostic reagents[J].1997,64(1):27-35.
[7]Parisotto G,Souza J S D,Ferr O M F,et al.The African swine fevervirus proteins p54 and p30 are involved in two distinct steps of virusattachment and both contribute to the antibody-mediated protective immuneresponse[J].1998,243(2):461-71.
[8]Rock,D.L.,Challenges for African swine fever vaccinedevelopment-"...perhaps the end of the beginning".Veterinary Microbiology,2017.206:p.52-58.
[9]Sheehan,Jared,and Wayne A Marasco.“Phage and Yeast Display.”Microbiology spectrum vol.3,1(2015):AID-0028-2014.
[10]Piggott,Andrew M,and Peter Karuso.“Identifying the cellulartargets of natural products using T7 phage display.”Natural product reportsvol.33,5(2016):626-36.
序列表
<110> 天津大学
<120> 基于非洲猪瘟病毒P30、P72蛋白的病毒样颗粒（VLP）的制备与评估
<130> 20210714
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 76
<212> DNA
<213> 人工序列()
<400> 1
ccggaattcc ggtagcggcg gtagcggcgc ccacatcgtg atggttgatg cgtataaacc 60
gaccaaaaag cttggg 76
<210> 2
<211> 561
<212> DNA
<213> African Swine fever virus
<400> 2
atgaaaatgg aggtcatctt caaaacggat ttaagatcat cttcacaagt tgtgtttcat 60
gcgggtagcc tgtataattg gttttctgtt gagattatca atagcggtag aattgttacg 120
accgctataa aaacattgct tagtactgtt aagtatgata ttgtgaaatc tgctcgtata 180
tatgcagggc aagggtatac tgaacatcag gctcaagaag aatggaatat gattctgcat 240
gtgctgtttg aagaggagac ggaatcctca gcatcttcgg agaacattca tgaaaaaaat 300
gataatgaaa ccaatgaatg cacatcctcc tttgaaacgt tgtttgagca agagccctca 360
tcgaaggtac ctaaagactc caagctgtat atgcttgcac aaaagactgt gcaacatatt 420
gaacaatatg gaaaggcacc tgattttaac aaggttatta gagcacataa ttttattcaa 480
accatttatg gaacccctct aaaggaagaa gaaaaagagg tggtaagact catggttatt 540
aaacttttaa aaaaaaaata a 561
<210> 3
<211> 813
<212> DNA
<213> African Swine fever virus
<400> 3
atggcatcag gaggagcttt ttgtcttatt gctaacgatg ggaaggccga caagattata 60
ttggcccaag acttgctgaa tagcaggatc tctaacatta aaaatgtgaa caaaagttat 120
gggaaacccg atcccgaacc cactttgagt caaatcgaag aaacacattt ggtgcatttt 180
aatgcgcatt ttaagcctta tgttccagta gggtttgaat acaataaagt acgcccgcat 240
acgggtaccc ccaccttggg aaacaagctt acctttggta ttccccagta cggagacttt 300
ttccatgata tggtgggcca tcatatattg ggtgcatgtc attcatcctg gcaggatgct 360
ccgattcagg gcacgtccca gatgggggcc catgggcagc ttcaaacgtt tcctcgcaac 420
ggatatgact gggacaacca aacaccctta gagggcgccg tttacacgct tgtagatcct 480
tttggaagac ccattgtacc cggcacaaag aatgcgtacc gaaacttggt ttactactgc 540
gaataccccg gagaacgact ttatgaaaac gtaagattcg atgtaaatgg aaattcccta 600
gacgaatata gttcggatgt cacaacgctt gtgcgcaaat tttgcatccc aggggataaa 660
atgactggat ataagcactt ggttggccag gaggtatcgg tggagggaac cagtggccct 720
ctcctatgca acattcatga tttgcacaag ccgcaccaaa gcaaacctat tcttaccgat 780
gaaaatgata cgcagcgaac gtgtagccat acc 813
<210> 4
<211> 6199
<212> DNA
<213> African Swine fever virus
<400> 4
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600
tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660
actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720
gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780
aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840
agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900
cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960
aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020
tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080
tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140
taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200
ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260
tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320
tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380
cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280
tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340
caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400
ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520
gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580
gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640
aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700
ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760
acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820
ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880
tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940
tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000
cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060
gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120
ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180
catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240
ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300
gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360
gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420
ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480
atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600
tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660
ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720
aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780
atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840
cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900
gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960
tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020
agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080
gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140
ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200
catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260
tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320
tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380
gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440
ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500
tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560
catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620
cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680
tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740
ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800
ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860
cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920
gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980
aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040
ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100
agcagcggcc tggtgccgcg cggcagccat atggctagca tgactggtgg acagcaaatg 5160
ggtcgcggat ccatgaaaat ggaggtcatc ttcaaaacgg atttaagatc atcttcacaa 5220
gttgtgtttc atgcgggtag cctgtataat tggttttctg ttgagattat caatagcggt 5280
agaattgtta cgaccgctat aaaaacattg cttagtactg ttaagtatga tattgtgaaa 5340
tctgctcgta tatatgcagg gcaagggtat actgaacatc aggctcaaga agaatggaat 5400
atgattctgc atgtgctgtt tgaagaggag acggaatcct cagcatcttc ggagaacatt 5460
catgaaaaaa atgataatga aaccaatgaa tgcacatcct cctttgaaac gttgtttgag 5520
caagagccct catcgaaggt acctaaagac tccaagctgt atatgcttgc acaaaagact 5580
gtgcaacata ttgaacaata tggaaaggca cctgatttta acaaggttat tagagcacat 5640
aattttattc aaaccattta tggaacccct ctaaaggaag aagaaaaaga ggtggtaaga 5700
ctcatggtta ttaaactttt aaaaaaaaaa taaggatccg actcgaattc cggttccggc 5760
ggcagcggcg gcgatagcgc gacccacatc aaattcagca aacgcgacga ggatggtaaa 5820
gaactggcgg gcgcgaccat ggaactgcgt gatagcagcg gcaaaaccat ctctacctgg 5880
atcagcgacg gccaggttaa agatttctat ctgtatccgg gcaaatacac cttcgttgaa 5940
accgccgcgc cggacggcta cgaagttgcg accgccatta ccttcaccgt taacgaacag 6000
ggtcaggtca ccgttaacgg cggtggctaa gcggccctcg agcaccacca ccaccaccac 6060
tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag 6120
caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa 6180
ggaggaacta tatccggat 6199
<210> 5
<211> 6451
<212> DNA
<213> African Swine fever virus
<400> 5
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600
tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660
actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720
gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780
aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840
agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900
cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960
aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020
tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080
tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140
taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200
ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260
tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320
tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380
cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280
tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340
caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400
ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520
gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580
gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640
aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700
ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760
acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820
ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880
tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940
tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000
cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060
gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120
ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180
catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240
ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300
gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360
gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420
ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480
atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600
tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660
ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720
aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780
atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840
cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900
gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960
tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020
agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080
gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140
ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200
catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260
tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320
tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380
gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440
ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500
tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560
catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620
cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680
tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740
ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800
ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860
cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920
gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980
aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040
ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100
agcagcggcc tggtgccgcg cggcagccat atggctagca tgactggtgg acagcaaatg 5160
ggtcgcggat ccatggcatc aggaggagct ttttgtctta ttgctaacga tgggaaggcc 5220
gacaagatta tattggccca agacttgctg aatagcagga tctctaacat taaaaatgtg 5280
aacaaaagtt atgggaaacc cgatcccgaa cccactttga gtcaaatcga agaaacacat 5340
ttggtgcatt ttaatgcgca ttttaagcct tatgttccag tagggtttga atacaataaa 5400
gtacgcccgc atacgggtac ccccaccttg ggaaacaagc ttacctttgg tattccccag 5460
tacggagact ttttccatga tatggtgggc catcatatat tgggtgcatg tcattcatcc 5520
tggcaggatg ctccgattca gggcacgtcc cagatggggg cccatgggca gcttcaaacg 5580
tttcctcgca acggatatga ctgggacaac caaacaccct tagagggcgc cgtttacacg 5640
cttgtagatc cttttggaag acccattgta cccggcacaa agaatgcgta ccgaaacttg 5700
gtttactact gcgaataccc cggagaacga ctttatgaaa acgtaagatt cgatgtaaat 5760
ggaaattccc tagacgaata tagttcggat gtcacaacgc ttgtgcgcaa attttgcatc 5820
ccaggggata aaatgactgg atataagcac ttggttggcc aggaggtatc ggtggaggga 5880
accagtggcc ctctcctatg caacattcat gatttgcaca agccgcacca aagcaaacct 5940
attcttaccg atgaaaatga tacgcagcga acgtgtagcc ataccggatc cgactcgaat 6000
tccggttccg gcggcagcgg cggcgatagc gcgacccaca tcaaattcag caaacgcgac 6060
gaggatggta aagaactggc gggcgcgacc atggaactgc gtgatagcag cggcaaaacc 6120
atctctacct ggatcagcga cggccaggtt aaagatttct atctgtatcc gggcaaatac 6180
accttcgttg aaaccgccgc gccggacggc tacgaagttg cgaccgccat taccttcacc 6240
gttaacgaac agggtcaggt caccgttaac ggcggtggct aagcggccct cgagcaccac 6300
caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct 6360
gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt 6420
tttttgctga aaggaggaac tatatccgga t 6451
<210> 6
<211> 282
<212> PRT
<213> African Swine fever virus
<400> 6
Met Lys Met Glu Val Ile Phe Lys Thr Asp Leu Arg Ser Ser Ser Gln
1 5 10 15
Val Val Phe His Ala Gly Ser Leu Tyr Asn Trp Phe Ser Val Glu Ile
20 25 30
Ile Asn Ser Gly Arg Ile Val Thr Thr Ala Ile Lys Thr Leu Leu Ser
35 40 45
Thr Val Lys Tyr Asp Ile Val Lys Ser Ala Arg Ile Tyr Ala Gly Gln
50 55 60
Gly Tyr Thr Glu His Gln Ala Gln Glu Glu Trp Asn Met Ile Leu His
65 70 75 80
Val Leu Phe Glu Glu Glu Thr Glu Ser Ser Ala Ser Ser Glu Asn Ile
85 90 95
His Glu Lys Asn Asp Asn Glu Thr Asn Glu Cys Thr Ser Ser Phe Glu
100 105 110
Thr Leu Phe Glu Gln Glu Pro Ser Ser Lys Val Pro Lys Asp Ser Lys
115 120 125
Leu Tyr Met Leu Ala Gln Lys Thr Val Gln His Ile Glu Gln Tyr Gly
130 135 140
Lys Ala Pro Asp Phe Asn Lys Val Ile Arg Ala His Asn Phe Ile Gln
145 150 155 160
Thr Ile Tyr Gly Thr Pro Leu Lys Glu Glu Glu Lys Glu Val Val Arg
165 170 175
Leu Met Val Ile Lys Leu Leu Lys Lys Lys Asp Ser Asn Ser Gly Ser
180 185 190
Gly Gly Ser Gly Gly Asp Ser Ala Thr His Ile Lys Phe Ser Lys Arg
195 200 205
Asp Glu Asp Gly Lys Glu Leu Ala Gly Ala Thr Met Glu Leu Arg Asp
210 215 220
Ser Ser Gly Lys Thr Ile Ser Thr Trp Ile Ser Asp Gly Gln Val Lys
225 230 235 240
Asp Phe Tyr Leu Tyr Pro Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala
245 250 255
Pro Asp Gly Tyr Glu Val Ala Thr Ala Ile Thr Phe Thr Val Asn Glu
260 265 270
Gln Gly Gln Val Thr Val Asn Gly Gly Gly
275 280
<210> 7
<211> 367
<212> PRT
<213> African Swine fever virus
<400> 7
Met Ala Ser Gly Gly Ala Phe Cys Leu Ile Ala Asn Asp Gly Lys Ala
1 5 10 15
Asp Lys Ile Ile Leu Ala Gln Asp Leu Leu Asn Ser Arg Ile Ser Asn
20 25 30
Ile Lys Asn Val Asn Lys Ser Tyr Gly Lys Pro Asp Pro Glu Pro Thr
35 40 45
Leu Ser Gln Ile Glu Glu Thr His Leu Val His Phe Asn Ala His Phe
50 55 60
Lys Pro Tyr Val Pro Val Gly Phe Glu Tyr Asn Lys Val Arg Pro His
65 70 75 80
Thr Gly Thr Pro Thr Leu Gly Asn Lys Leu Thr Phe Gly Ile Pro Gln
85 90 95
Tyr Gly Asp Phe Phe His Asp Met Val Gly His His Ile Leu Gly Ala
100 105 110
Cys His Ser Ser Trp Gln Asp Ala Pro Ile Gln Gly Thr Ser Gln Met
115 120 125
Gly Ala His Gly Gln Leu Gln Thr Phe Pro Arg Asn Gly Tyr Asp Trp
130 135 140
Asp Asn Gln Thr Pro Leu Glu Gly Ala Val Tyr Thr Leu Val Asp Pro
145 150 155 160
Phe Gly Arg Pro Ile Val Pro Gly Thr Lys Asn Ala Tyr Arg Asn Leu
165 170 175
Val Tyr Tyr Cys Glu Tyr Pro Gly Glu Arg Leu Tyr Glu Asn Val Arg
180 185 190
Phe Asp Val Asn Gly Asn Ser Leu Asp Glu Tyr Ser Ser Asp Val Thr
195 200 205
Thr Leu Val Arg Lys Phe Cys Ile Pro Gly Asp Lys Met Thr Gly Tyr
210 215 220
Lys His Leu Val Gly Gln Glu Val Ser Val Glu Gly Thr Ser Gly Pro
225 230 235 240
Leu Leu Cys Asn Ile His Asp Leu His Lys Pro His Gln Ser Lys Pro
245 250 255
Ile Leu Thr Asp Glu Asn Asp Thr Gln Arg Thr Cys Ser His Thr Asp
260 265 270
Ser Asn Ser Gly Ser Gly Gly Ser Gly Gly Asp Ser Ala Thr His Ile
275 280 285
Lys Phe Ser Lys Arg Asp Glu Asp Gly Lys Glu Leu Ala Gly Ala Thr
290 295 300
Met Glu Leu Arg Asp Ser Ser Gly Lys Thr Ile Ser Thr Trp Ile Ser
305 310 315 320
Asp Gly Gln Val Lys Asp Phe Tyr Leu Tyr Pro Gly Lys Tyr Thr Phe
325 330 335
Val Glu Thr Ala Ala Pro Asp Gly Tyr Glu Val Ala Thr Ala Ile Thr
340 345 350
Phe Thr Val Asn Glu Gln Gly Gln Val Thr Val Asn Gly Gly Gly
355 360 365
<210> 8
<211> 5632
<212> DNA
<213> 人工序列()
<400> 8
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600
tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660
actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720
gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780
aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840
agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900
cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960
aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020
tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080
tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140
taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200
ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260
tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320
tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380
cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280
tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340
caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400
ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520
gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580
gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640
aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700
ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760
acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820
ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880
tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940
tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000
cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060
gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120
ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180
catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240
ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300
gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360
gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420
ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480
atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600
tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660
ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720
aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780
atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840
cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900
gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960
tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020
agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080
gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140
ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200
catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260
tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320
tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380
gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440
ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500
tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560
catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620
cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680
tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740
ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800
ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860
cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920
gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980
aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040
ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100
agcagcggcc tggtgccgcg cggcagccat atggctagca tgactggtgg acagcaaatg 5160
ggtcgcggat ccgactcgaa ttccggttcc ggcggcagcg gcggcgatag cgcgacccac 5220
atcaaattca gcaaacgcga cgaggatggt aaagaactgg cgggcgcgac catggaactg 5280
cgtgatagca gcggcaaaac catctctacc tggatcagcg acggccaggt taaagatttc 5340
tatctgtatc cgggcaaata caccttcgtt gaaaccgccg cgccggacgg ctacgaagtt 5400
gcgaccgcca ttaccttcac cgttaacgaa cagggtcagg tcaccgttaa cggcggtggc 5460
taagcggccc tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga 5520
aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc 5580
tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg at 5632
<210> 9
<211> 20
<212> DNA
<213> 人工序列()
<400> 9
ggagctgtcg tattccagtc 20
<210> 10
<211> 20
<212> DNA
<213> 人工序列()
<400> 10
aacccctcaa gacccgttta 20
<210> 11
<211> 46
<212> DNA
<213> 人工序列()
<400> 11
cagcaaatgg gtcgcggatc catgaaaatg gaggtcatct tcaaaa 46
<210> 12
<211> 51
<212> DNA
<213> 人工序列()
<400> 12
accggaattc gagtcggatc cttttttttt taaaagttta ataaccatga g 51
<210> 13
<211> 46
<212> DNA
<213> 人工序列()
<400> 13
cagcaaatgg gtcgcggatc catggcatca ggaggagctt tttgtc 46
<210> 14
<211> 46
<212> DNA
<213> 人工序列()
<400> 14
accggaattc gagtcggatc cggtatggct acacgttcgc tgcgta 46
Claims (8)
1.表达ASFV病毒P30-Spycatcher融合蛋白的pET-28a原核表达载体pET-28a-P30-SC，序列特征为SEQ ID No.4。
2.表达ASFV病毒P72-Spycatcher融合蛋白的pET-28a原核表达载体pET-28a-P72-SC，序列特征为SEQ ID No.5。
3.表达蛋白-Spycatcher融合蛋白的基因，包含ASFV P30、P72蛋白和Spycatcher标签蛋白，蛋白与标签之间以一段柔性肽连接，P30-SC氨基酸序列特征为SEQ ID No.6，P72-SC氨基酸序列特征为SEQ ID No.7。
4.一种基于非洲猪瘟病毒P30、P72蛋白的病毒样颗粒的制备方法，其特征在于，构建表达载体pET-28a-P30-SC和pET-28a-P72-SC并表达纯化融合蛋白，将大肠杆菌原核表达系统纯化的P30-SC、P72-SC融合蛋白在缓冲液中，分别与表面展示ST标签蛋白的重组T7噬菌体颗粒T7-ST共同孵育，验证结合情况，通过板式化学发光实验对制备的病毒样颗粒颗粒的免疫原性进行评估。
5.根据权利要求4所述制备方法，其特征在于，所述构建表达载体pET-28a-P30-SC和pET-28a-P72-SC并表达纯化融合蛋白，具体包括以下步骤：
(1)ASFV P30、P72基因的PCR扩增：以实验室现存基因所在质粒为模板，设计无缝克隆引物扩增P30、P72蛋白编码基因用于pET-28a-Spycatcher原核载体连接；
(2)Spycatcher基因的合成：将Spycatcher基因序列合成于pET-28a原核表达载体之上，在序列N段加上一段柔性肽序列，得到pET-28a-Spycatcher表达载体，核苷酸序列为SEQID No.8；
(3)表达P30-SC、P72-SC原核表达载体的构建及蛋白纯化：通过无缝克隆，将P30、P72基因序列分别克隆至pET-28a-Spycatcher原核表达载体上，得到pET-28a-P30-SC重组质粒，序列特征为SEQ No.4和pET-28a-P72-SC重组质粒，序列特征为SEQ No.5；重组质粒转化大肠杆菌BL21表达菌株，经过蛋白纯化获得P30-SC、P72-SC融合蛋白。
6.根据权利要求4所述制备方法，其特征在于，所述表面展示ST标签蛋白的重组T7噬菌体的构建方法为：通过T7噬菌体展示技术，将外源SpyTag蛋白基因片段插入到T7噬菌体基因组中，通过体外包装，获得表面展示ST蛋白的T7噬菌体。
7.根据权利要求6所述制备方法，其特征在于，具体包括以下步骤：
(1)首先通过基因合成获得ST基因，基因N段加入一段柔性肽，合成序列两端加入EcoRI和Hind III限制性酶切位点序列及相应的保护性碱基，序列特征为SEQ ID No.1；
(2)重组噬菌体基因的构建：通过EcoR I和Hind III两种限制性内切酶双酶切合成的ST基因，之后通过T4连接酶将酶切产物与T7载体臂相连接，从而获得重组噬菌体基因组；
(3)体外包装：将步骤(2)中连接产物与T7包装提取物混合孵育，获得完整的含有重组基因组的T7噬菌体颗粒；
(4)噬菌斑分析：通过双层琼脂培养法，对包装产物进行噬菌斑计数，从而计算噬菌体滴度；
(5)文库鉴定：蛋白酶K法提取T7 DNA，然后用T7通用引物进行PCR鉴定，最终得到所需要的噬菌体；
(6)文库扩增：使用液体裂解液法对噬菌斑进行扩增，待菌液变澄清后，离心将细胞碎片去除；通过PEG/NaCl沉淀法，对上清中的噬菌体进行浓缩，之后通过噬菌斑计数测定浓缩后的噬菌体滴度。
8.利用权利要求4所述方法制备的病毒样颗粒在制备非洲猪瘟病毒疫苗中的应用。
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