CN116855459A - 一种基于CRISPR-Cas9技术的表达新城疫病毒La Sota毒株HN蛋白的重组血清4型禽腺病毒及其制备方法 - Google Patents
一种基于CRISPR-Cas9技术的表达新城疫病毒La Sota毒株HN蛋白的重组血清4型禽腺病毒及其制备方法 Download PDFInfo
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Abstract
本发明涉及一种基于CRISPR‑Cas9技术的表达新城疫病毒La Sota毒株HN蛋白的重组血清4型禽腺病毒及其制备方法，所述新城疫病毒La Sota毒株HN蛋白，序列如SEQ ID NO.1所示。所述重组血清4型禽腺病毒，其中使用新城疫病毒La Sota毒株HN基因替换FAdV‑4的Fiber‑2基因，替换的是FAdV‑4的Fiber‑2基因的253位核苷酸到1440位核苷酸，FAdV‑4的Fiber‑2基因序列如SEQ ID NO.2所示。通过本发明，利用当下流行的血清4型禽腺病毒作为载体插入新城疫病毒HN基因，成功获得新城疫病毒HN蛋白的重组血清4型禽腺病毒，为同时免疫防控血清4型禽腺病毒和新城疫病毒提供技术支撑和弱毒二联疫苗候选。
Description
技术领域
本发明涉及一种基于CRISPR-Cas9技术的表达新城疫病毒La Sota毒株HN蛋白的重组血清4型禽腺病毒及其制备方法，属于生物技术领域。
背景技术
禽腺病毒(Fowl adenovirus，FAdV)属于腺病毒科禽腺病毒属。基于限制性酶切图谱分析和六邻体序列，禽腺病毒分为A-E共5个基因型。基于血清交叉中和试验，5个基因型又分为12个血清型(1-7，8a，8b，9-11)。从2015年6月至今，爆发了由高致病性血清4型禽腺病毒(FAdV-4)引起的肝炎-心包积液综合征(hepatitis-hydropericardium syndrome，HHS)给家禽养殖业造成巨大的经济损失，极大的威胁着养禽业的健康持续发展。与此同时，鸡新城疫(Newcastle Disease，ND)是由新城疫病毒(Newcastle Disease Virus,NDV)引起的一种急性、烈性传染病，发病率和死亡率可高达90％。因此，临床上FAdV-4与NDV的联防联控迫在眉睫。虽然国内在2021年获批了2款新城疫-禽流感-禽腺病毒(I群4型)病三联灭活疫苗，但远不能满足国内市场对FAdV-4及其他疫病的联防联控需求。值得注意的是，与灭活疫苗比较，基因工程多价或多联的弱毒疫苗不仅能同时诱导有效的体液免疫以及细胞免疫，而且具有一次扩增同时制备两种抗原、成本低等优点。HN蛋白是NDV表面主要的中和保护性抗原，其在病毒感染复制过程中介导病毒的吸附、入侵和释放,是疫苗研制的主要靶标。本发明以FAdV-4为病毒载体，通过CRISPR/Cas9技术和Cre-LoxP重组系统，靶向Fiber-2，利用双荧光体系构建了表达新城疫病毒La Sota毒株HN蛋白的重组禽腺病毒FAdV4-HN,为同时免疫防控血清4型禽腺病毒病和新城疫提供了技术支撑和弱毒二联疫苗候选。
发明内容
本发明的目的是基于CRISPR-Cas9技术构建表达新城疫病毒HN蛋白的重组血清4型禽腺病毒，提供一种基于CRISPR-Cas9技术的表达新城疫病毒La Sota毒株HN蛋白的重组血清4型禽腺病毒及其制备方法，本发明的关键技术是在FAdV-4中寻找到合适的位置插入外源基因；同时利用CRISPR-Cas9技术在血清4型禽腺病毒中插入新城疫病毒HN基因以及带有LoxP位点的RFP表达盒，由于利用RFP蛋白进行病毒的纯化，最后获得可以稳定表达新城疫病毒HN蛋白的重组血清4型禽腺病毒。
本发明的目的是通过以下技术方案实现的，一种基于CRISPR-Cas9技术的表达新城疫病毒La Sota毒株HN蛋白的重组血清4型禽腺病毒，其特征是，所述新城疫病毒La Sota毒株HN蛋白，序列如SEQ ID NO.1所示。
所述重组血清4型禽腺病毒，其中使用新城疫病毒La Sota毒株HN基因替换FAdV-4的Fiber-2基因，替换的是FAdV-4的Fiber-2基因的253位核苷酸到1440位核苷酸，FAdV-4的Fiber-2基因序列如SEQ ID NO.2所示。
重组血清4型禽腺病毒的制备方法，该制备方法包括以下步骤：
(1)利用sgRNA在线设计网站设计针对血清4型禽腺病毒Fiber-2基因的sgRNA，其序列如表3所示；
表3针对Fiber-2基因的sgRNA序列
(2)通过PCR和同源重组的方法构建携带新城疫病毒La Sota毒株的HN基因和RFP表达盒的供体质粒，其中的RFP表达盒两端分别带有LoxP位点，PCR使用的引物序列如表4所示；
表4构建供体质粒所用的PCR引物
(3)将生长良好的LMH细胞经胰酶消化后接种六孔板培养，第二天转染sgRNA和供体质粒各3μg，转染6h后换成细胞生长液；转染12h后感染表达EGFP的重组病毒FA4-EGFP，感染2h后换成细胞维持液；
(4)感染血清4型禽腺病毒后观察RFP红色荧光，利用空斑试验和有限稀释的方法进行红色荧光重组病毒的纯化，获得带有RFP表达盒的表达新城疫病毒HN蛋白的重组血清4型禽腺病毒；
(5)获得的红色荧光重组病毒接种LMH细胞扩增后转使用PCR、IFA和Western Blot的方法对表达新城疫病毒La Sota毒株HN蛋白的重组血清4型禽腺病毒进行鉴定。
所述表达EGFP的重组病毒FA4-EGFP，其特点是能够表达绿色荧光EGFP，区别于重组病毒的红色荧光蛋白RFP，便于快速纯化重组病毒。
通过本发明，提供的一种基于CRISPR-Cas9技术的表达新城疫病毒La Sota毒株HN蛋白的重组血清4型禽腺病毒及其制备方法，利用当下流行的血清4型禽腺病毒作为载体插入新城疫病毒HN基因，成功获得新城疫病毒HN蛋白的重组血清4型禽腺病毒，为同时免疫防控血清4型禽腺病毒和新城疫病毒提供技术支撑和弱毒二联疫苗候选。与传统的重组禽腺病毒构建方法不同，本发明中靶向毒力基因fiber-2，使用了双荧光体系，且整合了CRISPR/Cas9技术和Cre-loxP重组系统，不需要对禽腺病毒全基因组进行克隆，使得重组禽腺病毒的构建、筛选及其纯化非常快捷及高效。与灭活疫苗比较，基因工程多价或多联的弱毒疫苗不仅能同时诱导有效的体液免疫以及细胞免疫，而且具有一次扩增同时制备两种抗原、成本低等优点。
本发明涉及借助于CRISPR-Cas9技术构建的表达新城疫病毒HN蛋白的重组血清4型禽腺病毒，构建的表达新城疫病毒HN蛋白的重组血清4型禽腺病毒，为制备血清4型禽腺病毒和新城疫病毒二联弱毒疫苗奠定了基础。因此，本发明具有良好的市场应用价值。
附图说明
图1为供体质粒的构建；其中：
a：线性化的HR1-Fiber-2-RFP-HR2 in pMD 19载体；b：带有部分载体片段的HN基因。
图2为表达新城疫病毒HN蛋白的重组病毒的构建策略示意图。
图3为初代重组病毒FAdV4-HN(NDV)-RFP的荧光图；其中：
a：重组病毒FAdV4-HN(NDV)-RFP；b：母本病毒FA4-EGFP；c：阴性对照(LMH细胞)。
图4为PCR鉴定重组病毒FAdV4-HN(NDV)-RFP的纯度；其中：
a.FAdV4-HN(NDV)-RFP；b：母本病毒FA4-EGFP；
c:1.NDV HN基因M：DNA marker；2：未纯化的重组病毒；3：纯化后的重组病毒；4：母本病毒FA4-EGFP；5：野生型FAdV-4。
图5为重组病毒FAdV4-HN(NDV)-RFP中HN蛋白的表达鉴定；其中：
a：利用针对NDV的多抗鉴定重组病毒；c：阴性LMH细胞对照；d：WB鉴定重组病毒中HN蛋白的表达。
具体实施方式
实施例：
1.病毒基因组的制备与反转录：使用天根公司的基因组提取试剂盒严格按照说明书进行提取，在提取完新城疫病毒RNA后用Takara公司的PrimeScript RT reagent Kit反转录试剂盒制备cDNA，具体操作按照说明书进行，反应体系反转录体系1所示。混匀后置于42℃孵育2min，再立即冰浴2min。
表1反转录体系1
| 成分 | 剂量 |
| RNA(2μg) | 7μL |
| gDNA Eraser | 1μL |
| 5xgDNA Eraser | 2μL |
在冰浴期间，按表反转录体系2所述反应体系混合4种反应成分，而后再将反应体系1中的冰浴混合物加入反应体系2中进行恒温孵育，反应条件为：37℃15min，85℃5s。NDVcDNA制备完成后分装保存于-80℃。
表2反转录体系2
| 成分 | 剂量 |
| PrimeScript RT Enzyme Mix | 1μL |
| 5xPrimeScript Bμffer 2 | 4μL |
| RT Primer Mix | 1μL |
| RNase Free H2O | 4μL |
2.供体质粒的构建：供体质粒的构建：由南京擎科生物科技有限公司合成带有两个连续LoxP序列的pΜC-57载体，利用同源重组的方法在上述载体中插入RFP表达盒，构建的pΜC-57-LoxP-RFP载体由本实验室保存。以pΜC-57-LoxP-RFP载体为模板扩增带有LoxP序列的RFP表达盒(如图1a)；以pMD19-T载体为模板将pMD19-T载体线性化；以血清4型禽腺病毒基因组为模板扩增Fiber-2基因及其左端同源臂HR1和右端同源臂HR2，并克隆至pMD19-T载体中；琼脂糖凝胶电泳后进行胶回收，通过同源重组的方法按照HR1-Fiber-2-RFP-HR2的顺序组装在pMD19-T上，最终获得的供体质粒送南京擎科生物科技有限公司测序并由实验室保存。构建供体质粒所用的引物序列见表3。
以HR1-Fiber-2-RFP-HR2 in pMD 19质粒载体为模板，利用线性化引物将载体线性化(如图1a)，利用带有质粒部分序列的引物以新城疫病毒的cDNA为模板扩增新城疫病毒的HN基因(如图1b)，琼脂糖凝胶电泳后进行胶回收，回收得到的线性化的载体和新城疫的HN基因在同源重组酶的作用下将HN基因连接到HR1-Fiber-2-RFP-HR2 in pMD 19质粒上，最终获得携带新城疫病毒La Sota毒株的带有HN基因和RFP表达盒的供体质粒。最终获得的供体质粒送南京擎科生物科技有限公司测序并由实验室保存。构建供体质粒所用的引物序列见表4。
表4构建供体质粒所用的PCR引物
3.sgRNA表达载体的构建：根据FAdV-4的Fiber-2基因序列利用sgRNA在线设计网站(http://crispor.tefor.net/)进行sgRNA的设计。将设计好的sgRNA克隆到lentiCRISPRv2质粒，通过测序进行sgRNA表达载体的验证。具体的sgRNA序列见表3，由南京擎科生物科技有限公司合成。
表3针对Fiber-2基因的sgRNA序列
4.红色荧光重组病毒的拯救：构建策略如图2，在6孔板中铺LMH细胞，次日，将3μgsgRNA、3μg的供体质粒以及6μL的转染试剂Mirus加入到200μL的Opti-MEM中，室温孵育45min。随后加入到LMH细胞中，转染6h后换成细胞生长液。转染12h后，弃去细胞生长液，用0.1MOI的FA4-EGFP感染LMH细胞，感染2h后换成细胞维持液。感染病毒3天后，去上清12000rpm离心10分钟后，盲传至新的96孔板的LMH细胞中，每天通过荧光显微镜观察红色荧光簇。红色荧光簇的出现表明成功构建了重组病毒(如图3)，并命名为FAdV4-HN(NDV)-RFP。
5.红色荧光重组病毒的纯化及鉴定：将拯救的红色荧光重组病毒利用空斑试验和有限稀释法纯化，获得纯化的红色荧光重组病毒，通过PCR鉴定纯度，结果如图4。结果表明成功获得纯的重组病毒FAdV4-HN(NDV)-RFP。PCR所用的引物见表5。
表5 PCR鉴定重组病毒的引物
6.重组病毒中HN蛋白的表达鉴定：将纯化后的FAdV4-HN(NDV)-RFP重组病毒接种LMH细胞，3天后将LMH细胞进行固定，利用针对新城疫病毒的鸡多抗血清进行WB和IFA鉴定。结果如图5，视野中可检测到特异性绿色荧光，且与阴性对照LHM细胞上清相比较，在73kDa处有一明显条带，表明HN成功插入FAdV-4中，并获得良好表达。
SEQ ID NO.1
新城疫病毒HN基因序列：
ATGGACCGCGCGGTTAACAGGGTCGTGCTGGAGAATGAGGAAAGAGAAGCAAAGAACACATGGCGCCTAGTTTTCCGGATCGCAGTCTTACTTTTAATGGTAATGACTCTAGCTATCTCCGCGGCTGCCCTGGCACACAGCATGGGGGCCAGTACGCCGCACGACCTCGCAGGCATATCGACTGTGATCTCCAAGACAGAAGACAAGGTTACGTCTTTACTCAGTTCAAGTCAAGATGTGATAGATAGGATATACAAGCAGGTAGCTCTTGAATCCCCGCTGGCACTACTAAACACCGAATCTATAATTATGAATGCAATAGCCTCTCTTTCTTATCAAATTAACGGGGCTGAGAACAATAGCGGATGTGGTGCGCCTGTTCATGACCCAGATTATATCGGGGGGATAGGCAAAGAACTCATAGTGGACGACATCAGTGATGTCACATCATTTTATCCTTCTGCATATCAAGAACACTTGAATTTCATCCCGGCGCCTACTACAGGATCCGGTTGCACTCGGATACCCTCATTTGACATGAGCACCACCCATTATTGTTATACTCACAATGTGATACTATCTGGTTGCAGAGACCACTCACACTCACATCAATACTTAGCACCTGGTGTGCTTCGGACATCTGCAACAGGGAGGGTATTCTTTTCTACTCTGCGCTCCATCAATTTAGATGACACCCAAAATCGGAAGTCCTGCAGTGTGAGTGCAACCCCTTTAGGTTGTGATATGCTGTGCTCTAAGGTCACAGGGACTGAAGAGGAGGATTACAAGTCAGTTGCCCCCACATCAATGGTGCACGGAAGGCTAGGGTTTGACGGTCAATACCATGAGAAGGACTTAGACACCACGGTCTTATTTAAGGATTGGGTGGCAAATTACCCGGGAGTGGGAGGAGGGTCTTTTATTGACGGCCGTGTATGGTTCCCAGTTTACGGAGGGCTCAAACCCAATTCACCCAGTGACGCTGCACAAGAAGGGAAATATGTAATATACAAGCGTCATAACAACACATGCCCCGATGAACAAGATTACCAAATTCGGATGGCTAAGTCCTCATATAAACCCGGGCGATTTGGTGGAAAGCGCGTACAGCAAGCCATCTTATCCATCAAAGTGTCAACATCCCTGGGTAAGGACCCGGTGCTGACTATTCCACCTAATACAATCACACTCATGGGAGCTGAAGGCAGAATCCTCACAGTAGGGACATCTCACTTCTTGTACCAACGAGGGTCTTCATATTTCTCCCCTGCCTTATTGTATCCCATGACAGTAAATAACAAAACGGCTACACTCCATAGTCCTTACATGTTTAATGCTTTCACTCGGCCAGGTAGTGTCCCTTGCCAGGCATCAGCAAGATGCCCCAACTCATGCATTACTGGGGTCTATACCGATCCATATCCCTTAATCTTCCATAGGAATCATACTCTACGAGGGGTCTTCGGGACGATGCTTGATGATGAACAAGCGAGGCTTAACCCCGTATCTGCAGTATTTGACAACATATCTCGCAGTCGTGTCACCCGGGTGAGTTCAAGCAGCACCAAGGCAGCATACACGACATCGACATGTTTTAAAGTTGTCAA GACCAATAAAGCTTATTGTCTTAGTATCGCAGAAATATCCAATACCCTATTCGGGGAATTTAGGATCGTTCCCTTACTAGTTGAGATCCTCAAGGATGATAGAGTTTAA
SEQ ID NO.2
血清4型禽腺病毒Fiber-2基因序列：
ATGCTCCGGGCCCCTAAAAGAAGACATTCCGAAAACGGGAAGCCCGAGACCGAAGCGGGACCTTCCCCGGCTCCAATCAAGCGCGCCAAACGCATGGTGAGAGCATCCCAGCTTGACCTGGTTTATCCTTTCGATTACGTGGCCGACCCCGTCGGAGGGCTCAACCCGCCTTTTTTGGGAGGCTCAGGACCCCTAGTGGACCAGGGCGGACAGCTTACGCTCAACGTCACCGATCCCATCATCATCAAGAACAGATCGGTGGACTTGGCCCACGACCCCAGTCTCGATGTCAACGCCCAAGGTCAACTGGCGGTGGCCGTTGACCCCGAAGGGGCCCTGGACATCACCCCCGATGGACTGGACGTCAAGGTCGACGGAGTGACCGTAATGGTCAACGATGACTGGGAACTGGCCGTAAAAGTCGACCCGTCCGGCGGATTGGATtCCACCGCGGGTGGACTGGGGGTCAGCGTGGACGACACCTTGCTCGTGGATCAGGGAGAACTGGGCGTACACCTCAACCAACAAGGACCCATCACTGCCGATAGCAGTGGTATCGACCTCGAGATCAATCCTAACATGTTCACGGTCAACACCTCGACCGGAAGCGGAGTGCTGGAACTCAACCTAAAAGCGCAGGGAGGCATCCAAGCCGACAGTTCGGGAGTGGGCGTTTCCGTGGATGAAAGCCTACAGATTGTCAACAACACTCTGGAAGTGAAACCGGATCCCAGCGGACCGCTTACGGTCTCCGCCAATGGCCTAGGGCTGAAGTACGACACTAATACCCTAGCGGTGACCGCGGGCGCTTTAACCGTGGTCGGAGGGGGGAGCGTCTCCACACCCATCGCTACTTTTGTCTCGGGAAGTCCCAGCCTCAACACCTACAATGCCACGACCGTCAATTCCAGCGCGAACGCCTTCTCTTGCGCCTACTACCTTCAACAGTGGAACATACAGGGGCTCCTTGTTACCTCCCTCTACTTGAAATTGGACAGCGCCACCATGGGGAATCGCCCTGGGGACCTCAACTCCGCCAATGCCAAATGGTTCACCTTTTGGGTGTCCGCCTATCTCCAGCAATGCAACCCCTCCGGGATTCAAGCGGGAACGGTCAGCCCCTCCACCGCCACCCTCACGGACTTTGAACCCATGGCCAATAGGAGCGTGACCAGCCCATGGACGTACTCGGCCAATGGATACTATGAACCATCCATCGGGGAATTCCAAGTGTTCAGCCCGGTGGTAACAGGTGCCTGGAACCCGGGAAACATAGGGATCCGCGTCCTCCCCGTGCCGGTTTCGGCCTCCGGAGAGCGATACACCCTTCTATGCTATAGTCTGCAGTGCACGAACGCGAGCATTTTTAATCCAAACAACAGCGGAACCATGATCGTGGGACCCGTGCTCTACAGCTGTCCAGCGGCCTCCCTCCCGTAA
Claims (4)
1.一种基于CRISPR-Cas9技术的表达新城疫病毒La Sota毒株HN蛋白的重组血清4型禽腺病毒，其特征是，所述新城疫病毒La Sota毒株HN蛋白，序列如SEQ ID NO.1所示。
2.根据权利要求1所述的一种基于CRISPR-Cas9技术的表达新城疫病毒La Sota毒株HN蛋白的重组血清4型禽腺病毒，其特征是，所述重组血清4型禽腺病毒，其中使用新城疫病毒La Sota毒株HN基因替换FAdV-4的Fiber-2基因，替换的是FAdV-4的Fiber-2基因的253位核苷酸到1440位核苷酸，FAdV-4的Fiber-2基因序列如SEQ ID NO.2所示。
3.权利要求1所述的重组血清4型禽腺病毒的制备方法，其特征是，该制备方法包括以下步骤：
(1)利用sgRNA在线设计网站设计针对血清4型禽腺病毒Fiber-2基因的sgRNA，其序列如表3所示；
表3针对Fiber-2基因的sgRNA序列
(2)通过PCR和同源重组的方法构建携带新城疫病毒La Sota毒株的HN基因和RFP表达盒的供体质粒，其中的RFP表达盒两端分别带有LoxP位点，PCR使用的引物序列如表4所示；
表4构建供体质粒所用的PCR引物
(3)将生长良好的LMH细胞经胰酶消化后接种六孔板培养，第二天转染sgRNA和供体质粒各3μg，转染6h后换成细胞生长液；转染12h后感染表达EGFP的重组病毒FA4-EGFP，感染2h后换成细胞维持液；
(4)感染血清4型禽腺病毒后观察RFP红色荧光，利用空斑试验和有限稀释的方法进行红色荧光重组病毒的纯化，获得带有RFP表达盒的表达新城疫病毒HN蛋白的重组血清4型禽腺病毒；
(5)获得的红色荧光重组病毒接种LMH细胞扩增后转使用PCR、IFA和Western Blot的方法对表达新城疫病毒La Sota毒株HN蛋白的重组血清4型禽腺病毒进行鉴定。
4.根据权利要求3所述的重组血清4型禽腺病毒的制备方法，其特征是，所述的表达EGFP的重组病毒FA4-EGFP，其特点是能够表达绿色荧光EGFP，区别于重组病毒的红色荧光蛋白RFP，便于快速纯化重组病毒。
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