CN116970575A - 一种基于CRISPR-Cas9技术表达H7N9禽流感病毒HA蛋白的重组血清4型禽腺病毒及其的制备方法 - Google Patents
一种基于CRISPR-Cas9技术表达H7N9禽流感病毒HA蛋白的重组血清4型禽腺病毒及其的制备方法 Download PDFInfo
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Abstract
本发明涉及一种基于CRISPR‑Cas9技术表达H7N9禽流感病毒HA蛋白的重组血清4型禽腺病毒及其的制备方法，H7N9禽流感病毒HA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H7N9禽流感病毒HA基因替换血清4型禽腺病毒Fiber‑2基因，替换的是血清4型禽腺病毒Fiber‑2基因的253位核苷酸到1440位核苷酸。本发明的关键技术是利用CRISPR‑Cas9技术在血清4型禽腺病毒中插入H7N9禽流感病毒HA基因以及带有LoxP位点的RFP表达盒，利用RFP蛋白进行病毒的纯化，纯化完成后使用Cre重组酶敲除RFP表达盒，最后获得可以稳定表达H7N9禽流感病毒HA蛋白的重组血清4型禽腺病毒。本发明构建的表达H7N9禽流感病毒HA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H7N9禽流感病毒二联疫苗奠定了基础。
Description
技术领域
本发明涉及一种基于CRISPR-Cas9技术表达H7N9禽流感病毒HA蛋白的重组血清4型禽腺病毒及其的制备方法，属于基因工程技术领域。
背景技术
禽流感病毒感染严重威胁国内外养禽业持续健康发展，同时部分亚型禽流感可跨宿主感染哺乳动物，包括人类，对公共安全卫生造成严重威胁。目前H7亚型禽流感病毒和血清4型禽腺病毒都是我国急需控制的两种疾病，本发明以血清4型禽腺病毒为载体，将H7N9禽流感病毒的HA基因替换血清4型禽腺病毒的Fiber-2基因，成功构建了表达H7N9禽流感病毒HA蛋白的重组血清4型禽腺病毒，为联合防控血清4型禽腺病毒和H7N9禽流感病毒提供了二联候选疫苗毒株。
发明内容
本发明的目的是提供一种基于CRISPR-Cas9技术表达H7N9禽流感病毒HA蛋白的重组血清4型禽腺病毒及其的制备方法。
为了实现上述发明目的，本发明采用的技术方案如下：一种基于CRISPR-Cas9技术表达H7N9禽流感病毒HA蛋白的重组血清4型禽腺病毒，其特征是，H7N9禽流感病毒HA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H7N9禽流感病毒HA基因替换血清4型禽腺病毒Fiber-2基因，替换的是血清4型禽腺病毒Fiber-2基因的253位核苷酸到1440位核苷酸。
所述的重组血清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1所示；
表1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H7N9禽流感病毒HA基因和RFP表达盒的供体质粒，其中RFP表达盒两端都带有LoxP位点，PCR使用的引物序列如表2所示；
表2构建供体质粒所用的PCR引物
(3)6孔板接种LMH细胞培养过夜，共转染sgRNA和供体质粒各3μg，转染6h后去除转染上清并加培养液；转染12h后感染表达EGFP的重组4型腺病毒FA4-EGFP，感染2h后换成细胞维持液；
(4)感染FA4-EGFP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H7N9禽流感病毒HA蛋白的重组血清4型禽腺病毒；
(5)纯化后的红色荧光重组病毒接种转染Cre重组酶的LMH细胞，4d后取感染的细胞上清再次接种转染Cre重组酶的LMH细胞，重复该过程直至红色荧光病毒完全消失；使用PCR、IFA和Western Blot的方法对去除RFP表达盒的表达H7N9禽流感病毒HA蛋白的重组血清4型禽腺病毒进行鉴定。
所述的表达EGFP的重组4型腺病毒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，重组病毒FA4-EGFP的详细构建方法见专利CN112877303A。
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H7N9禽流感病毒HA基因，成功获得了表达H7N9禽流感病毒HA蛋白的重组血清4型禽腺病毒，此重组病毒可作为H7N9以及血清4型腺病毒二联疫苗候选株。
本发明的关键技术是利用CRISPR-Cas9技术在血清4型禽腺病毒中插入H7N9禽流感病毒HA基因以及带有LoxP位点的RFP表达盒，利用RFP蛋白进行病毒的纯化，纯化完成后使用Cre重组酶敲除RFP表达盒，最后获得可以稳定表达H7N9禽流感病毒HA蛋白的重组血清4型禽腺病毒。
综上，本发明涉及借助于CRISPR-Cas9技术构建的表达H7N9禽流感病毒HA蛋白的重组血清4型禽腺病毒；本发明构建的表达H7N9禽流感病毒HA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H7N9禽流感病毒二联疫苗奠定了基础。
附图说明
图1为表达H7N9禽流感病毒HA蛋白的重组病毒的构建策略示意图。
图2为初代重组病毒FAdV4-HA(H7)-RFP的荧光图；
a：母本病毒FA4-EGFP；b：重组病毒FAdV4-HA(H7)-RFP；c：阴性对照。
图3为PCR鉴定重组病毒FAdV4-HA(H7)的纯度；
M：DNA marker；1：未纯化的重组病毒；2：纯化后的重组病毒；3：母本病毒FA4-EGFP；4：野生型FAdV-4。
图4为重组病毒FAdV4-HA(H7)中HA蛋白的表达鉴定；
a：利用针对FAdV-4的单抗鉴定重组病毒；b：3A11单抗鉴定重组病毒中HA蛋白的表达；c：阴性LMH细胞对照；d：WB鉴定重组病毒中HA蛋白的表达。
图5为重组病毒FAdV4-HA(H7)的生长曲线测定。
图6为重组病毒FAdV4-HA(H7)在LMH细胞中的稳定性测试；
a：3A11单抗鉴定重组病毒中HA蛋白的表达；b：利用抗FAdV-4的单抗鉴定重组病毒。
具体实施方式
下面结合附图以及附图说明对本发明做进一步说明。
实施例：
1.血清4型腺病毒基因组的制备：根据天根公司的基因组提取试剂盒说明书进行提取，取血清4型禽腺病毒培养上清200μL置于1.5mL指形管内，加入200μL Proteinase K溶液，充分混匀后加入200μL缓冲液GB，混匀后置于70℃水浴作用10min，瞬时离心后加入200μL无水乙醇，充分振荡混匀15sec。将混匀后的溶液加入吸附柱CB3中(吸附柱放在收集管中)，12000rpm离心30sec，倒掉废液，将吸附柱CB3放回收集管中。向吸附柱CB3中加入500μL缓冲液GD，12000rpm离心30sec，倒掉废液，将吸附柱CB3放回收集管中。向吸附柱CB3中加入600μL漂洗液PW，12000rpm离心30sec，倒掉废液，将吸附柱CB3放回收集管中，重复加入600μL漂洗液PW，12000rpm离心30sec，倒掉废液，将吸附柱CB3放回收集管中。12000rpm离心2min，倒掉废液，将吸附柱CB3置于室温放置数分钟以晾干吸附材料中的漂洗液。将吸附柱CB3转入1.5mL指形管中，向吸附膜的中间部位悬空滴加200μL洗脱缓冲液TE，室温放置2min，12000rpm离心2min，收集到离心管中的溶液即为病毒的基因组。
2.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1，由南京擎科生物科技有限公司合成。
表1针对Fiber-2基因的sgRNA序列
3.供体质粒的构建：由南京擎科生物科技有限公司合成带有LoxP系统的pUC-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pUC-57-LoxP-RFP载体由本实验室保存。以pUC-57-LoxP-RFP载体为模板扩增带有LoxP系统的RFP表达盒；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左端同源臂HR1和右端同源臂HR2；以pDP2002-HA质粒为模板扩增H7N9禽流感病毒的HA基因。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2。
表2构建供体质粒所用的PCR引物
4.红色荧光重组病毒的拯救：6孔板中接种LMH细胞，培养过夜。待细胞密度生长至80％左右进行转染转染，在200μL的Opti-MEM培养基中加入3μgsgRNA、3μg供体质粒以及6μL的转染试剂Mirus，室温孵育45min后进行转染，转染6h后换成细胞生长液。转染12h后，弃去细胞生长液，感染0.1MOI的FA4-EGFP病毒，感染2h后换成细胞维持液。感染72h后，取病毒培养上清并进行盲传，每天观察LMH细胞时候出现红色荧光簇。红色荧光簇的出现表明成功构建了重组病毒。
5.红色荧光重组病毒的纯化及鉴定：将拯救的具有红色荧光重组病毒利用空斑试验和有限稀释法进行纯化，通过PCR鉴定病毒是否已纯化，结果如图3。结果表明成功获得纯的重组病毒FAdV4-HA(H7)-RFP。PCR所用的引物见表3。
表3PCR鉴定重组病毒的引物
6.RFP表达盒的敲除：用0.1MOI的红色荧光重组病毒感染转染Cre重组酶的LMH细胞，感染2h后换成细胞维持液，每天观察红色荧光病毒的数量，4d后取感染的细胞上清再次接种转染Cre重组酶的LMH细胞，重复该过程直至红色荧光病毒完全消失。通过PCR、IFA和Western Blot的方法对去除RFP表达盒的重组病毒进行鉴定。
7.重组病毒中HA蛋白的表达鉴定：用0.01MOI纯化后不带RFP表达盒的重组病毒接种LMH细胞，3天后将LMH细胞进行固定，利用针对H7N9禽流感病毒HA蛋白的单抗和针对FAdV-4的单抗进行IFA鉴定。结果如图4，同一视野中可同时检测到HA蛋白和FAdV-4，表明HA成功插入FAdV-4中，并获得良好表达。
8.删除RFP表达盒的重组病毒的生长曲线测定：在6孔板中铺LMH细胞，次日分别用0.1MOI的野生型FAdV-4和重组病毒接种LMH细胞，2h后换成1％维持液，感染后24h、48h、72h、96h和120h收取细胞上清。之后利用TCID50测定病毒各个时间点的病毒滴度，绘制病毒的生长曲线。结果显示，重组病毒的复制能力与野生型FAdV-4的生长速度和滴度接近(如图5)。
9.删除RFP表达盒的重组病毒的稳定性测试：将重组病毒FAdV4-HA(H7)进行连续传代10代，每隔5代进行提基因组和测序鉴定，并在第10代进行IFA鉴定。结果如图6所示，重组病毒FAdV4-HA(H7)在LMH细胞中进行稳定复制，并且在第10代病毒仍然能够用针对HA蛋白的单抗通过IFA检测到HA蛋白，表明连续传代后的重组病毒可以稳定表达HA蛋白。
SEQ ID NO.1
H7N9禽流感病毒HA基因序列：
ATGAACACTCAAATCCTGGTATTCGCTCTGATTGCGATCATTCCAACAAATGCAGACAAAATCTGCCTCGGACATCATGCCGTGTCAAACGGAACCAAAGTAAACACATTAACTGAAAGAGGAGTGGAAGTCGTCAATGCAACTGAAACAGTAGAACGAACAAACATCCCCAGGATCTGCTCAAAAGGGAAAAAGACAATTGACCTCGGTCAATGTGGACTACTGGGGACAATCACTGGACCACCTCAATGTGACCAATTCCTAGAATTTTCAGCCGATTTAATTATTGAGAGGCGAGAAGGAAGTGATGTCTGTTATCCTGGAAAATTCGTGAATGAAGAAGCTCTGAGGCAAATTCTCAGAGAATCAGGCGGAATTGACAAGGAAGCAATGGGATTCACATACAGTGGAATAAGAACTAATGGAGCAACCAGTGCATGTAGGAGATCAGGATCTTCATTCTATGCAGAAATGAAATGGCTCCTGTCAAACACAGATAATGCTACATTCCCGCAGATGACTAAGTCATATAAAAATACAAGAAAAAGCCCAGCTCTAATAGTATGGGGGATCCATCATTCTGTATCAACTGCAGAGCAAACCAAACTATATGGGAGTGGAAACAAACTGGTGACAGTTGGGAGTTCTAATTATCAACAATCTTTTGTACCGAGTCCAGGAGCGAGACCACAAGTTAATGGTCAATCTGGAAGAATTGACTTTCATTGGCTAATGCTAAATCCCAATGATACAGTCACTTTCAGTTTCAATGGGGCTTTCATAGCTCCAGACCGTGCAAGCTTCCTGAGAGGAAAATCTATGGGAATCCAGAGTGGAGTACAGGTTGATGCCAATTGTGAAGGGGACTGCTATCATAGTGGAGGAACAATAACAAGTAACTTGCCATTTCAGAACATAGATAGCAGGGCAGTTGGAAAATGTCCGAGATATGTTAAGCAAAAGAGTCTGCTGCTAGCAACAGGGATGAAGAATGTTCCTGAGATTCCAAAGGGAAGAGGCCTATTTGGTGCTATAGCGGGTTTCATTGAAAATGGATGGGAAGGCCTAATTGATGGTTGGTATGGTTTCAGACACCAGAATGCACAGGGAGAGGGAACTGCTGCAGATTACAAAAGCACTCAATCGGCAATTGATCAAATAACAGGAAAATTAAACCGGCTTATAGAAAAAACCAACCAACAATTTGAGTTGATAGACAATGAATTCAATGAGGTAGAGAAACAAATCGGTAATGTGATAAATTGGACCAGAGATTCTATAACAGAAGTGTGGTCATACAATGCTGAACTCTTGGTAGCAATGGAGAACCAGCATACAATTGATCTGGCTGATTCAGAAATGGACAAACTGTACGAACGAGTGAAAAGACAGCTGAGAGAGAATGCTGAAGAAGATGGCACTGGTTGCTTTGAAATATTTCACAAGTGTGATGATGACTGTATGGCCAGTATTAGAAATAACACCTATGATCACAGCAAGTACAGGGAAGAGGCAATGCAAAATAGAATACAGATTGACCCAGTCAAACTAAGCAGCGGCTACAAAGATGTGATACTTTGGTTTAGCTTCGGGGCATCATGTTTCATACTTCTAGCCATTGTAATGGGCCTTGTCTTCATATGTGTAAAGAATGGAAACATGCGGTGCACTATTTGTATATAA
SEQ ID NO.2
血清4型禽腺病毒Fiber-2基因序列：
ATGCTCCGGGCCCCTAAAAGAAGACATTCCGAAAACGGGAAGCCCGAGACCGAAGCGGGACCTTCCCCGGCTCCAATCAAGCGCGCCAAACGCATGGTGAGAGCATCCCAGCTTGACCTGGTTTATCCTTTCGATTACGTGGCCGACCCCGTCGGAGGGCTCAACCCGCCTTTTTTGGGAGGCTCAGGACCCCTAGTGGACCAGGGCGGACAGCTTACGCTCAACGTCACCGATCCCATCATCATCAAGAACAGATCGGTGGACTTGGCCCACGACCCCAGTCTCGATGTCAACGCCCAAGGTCAACTGGCGGTGGCCGTTGACCCCGAAGGGGCCCTGGACATCACCCCCGATGGACTGGACGTCAAGGTCGACGGAGTGACCGTAATGGTCAACGATGACTGGGAACTGGCCGTAAAAGTCGACCCGTCCGGCGGATTGGATtCCACCGCGGGTGGACTGGGGGTCAGCGTGGACGACACCTTGCTCGTGGATCAGGGAGAACTGGGCGTACACCTCAACCAACAAGGACCCATCACTGCCGATAGCAGTGGTATCGACCTCGAGATCAATCCTAACATGTTCACGGTCAACACCTCGACCGGAAGCGGAGTGCTGGAACTCAACCTAAAAGCGCAGGGAGGCATCCAAGCCGACAGTTCGGGAGTGGGCGTTTCCGTGGATGAAAGCCTACAGATTGTCAACAACACTCTGGAAGTGAAACCGGATCCCAGCGGACCGCTTACGGTCTCCGCCAATGGCCTAGGGCTGAAGTACGACACTAATACCCTAGCGGTGACCGCGGGCGCTTTAACCGTGGTCGGAGGGGGGAGCGTCTCCACACCCATCGCTACTTTTGTCTCGGGAAGTCCCAGCCTCAACACCTACAATGCCACGACCGTCAATTCCAGCGCGAACGCCTTCTCTTGCGCCTACTACCTTCAACAGTGGAACATACAGGGGCTCCTTGTTACCTCCCTCTACTTGAAATTGGACAGCGCCACCATGGGGAATCGCCCTGGGGACCTCAACTCCGCCAATGCCAAATGGTTCACCTTTTGGGTGTCCGCCTATCTCCAGCAATGCAACCCCTCCGGGATTCAAGCGGGAACGGTCAGCCCCTCCACCGCCACCCTCACGGACTTTGAACCCATGGCCAATAGGAGCGTGACCAGCCCATGGACGTACTCGGCCAATGGATACTATGAACCATCCATCGGGGAATTCCAAGTGTTCAGCCCGGTGGTAACAGGTGCCTGGAACCCGGGAAACATAGGGATCCGCGTCCTCCCCGTGCCGGTTTCGGCCTCCGGAGAGCGATACACCCTTCTATGCTATAGTCTGCAGTGCACGAACGCGAGCATTTTTAATCCAAACAACAGCGGAACCATGATCGTGGGACCCGTGCTCTACAGCTGTCCAGCGGCCTCCCTCCCGTAA
Claims (3)
1.一种基于CRISPR-Cas9技术表达H7N9禽流感病毒禽流感病毒HA蛋白的重组血清4型禽腺病毒，其特征是，通过CRISPR-Cas9技术将血清4型腺病毒的Fiber-2基因替换成H7N9禽流感病毒HA基因，替换Fiber-2基因的具体位置是253位核苷酸到1440位核苷酸。H7N9禽流感病毒HA蛋白序列如SEQ ID NO.1所示；
2.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，母本血清4型腺病毒带有EGFP基因，在H7N9禽流感病毒HA基因添加RPF基因，可发红光便于阳性克隆筛选，同时添加LoxP位点，以便后期对RFP切割。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1所示；
表1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H7N9禽流感病毒HA基因和RFP表达盒的供体质粒，其中RFP表达盒两端都带有LoxP位点，PCR使用的引物序列如表2所示；
表2构建供体质粒所用的PCR引物
(3)6孔板接种LMH细胞培养过夜，共转染sgRNA和供体质粒各3μg，转染6h后去除转染上清并加培养液；转染12h后感染表达EGFP的重组4型腺病毒FA4-EGFP，感染2h后换成细胞维持液；
(4)感染FA4-EGFP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H7N9禽流感病毒HA蛋白的重组血清4型禽腺病毒；
(5)纯化后的红色荧光重组病毒接种转染Cre重组酶的LMH细胞，4d后取感染的细胞上清再次接种转染Cre重组酶的LMH细胞，重复该过程直至红色荧光病毒完全消失；使用PCR、IFA和Western Blot的方法对去除RFP表达盒的表达H7N9禽流感病毒HA蛋白的重组血清4型禽腺病毒进行鉴定。
3.根据权利要求2所述的方法，其特征是通过CRISPR-Cas9技术对血清4型腺病毒做定向改造，双荧光筛选的方法具有快速准确等特点。所述的表达EGFP的重组4型腺病毒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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