CN116463297A - 一种表达鸡传染性贫血病病毒vp1蛋白的重组血清4型禽腺病毒及其制备方法 - Google Patents
一种表达鸡传染性贫血病病毒vp1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
本发明涉及一种表达鸡传染性贫血病病毒VP1蛋白的重组血清4型禽腺病毒及其制备方法,属于基因工程技术领域。本发明的关键技术是在血清4型禽腺病毒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型禽腺病毒中插入鸡传染性贫血病病毒VP1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鸡传染性贫血病病毒VP1蛋白的重组血清4型禽腺病毒。本发明构建的表达鸡传染性贫血病病毒VP1蛋白的重组血清4型禽腺病毒，为血清4型禽腺病毒和鸡传染性贫血病病毒的联防联控提供了二联候选疫苗。因此，本发明具有良好的市场应用价值。
Description
技术领域
本发明涉及一种表达鸡传染性贫血病病毒VP1蛋白的重组血清4型禽腺病毒及其制备方法，具体涉及一种利用CRISPR-Cas9和Cre-LoxP重组系统构建的表达鸡传染性贫血病病毒VP1蛋白的重组血清4型禽腺病毒，属于基因工程技术领域。
背景技术
鸡传染性贫血病病毒(Chicken Infectious Anemia Virus，CIAV)属指环病毒科(Anelloviridae)圆圈病毒属(Gyrovirus)。CIAV感染主要引起以雏鸡障碍性贫血及全身性淋巴组织萎缩为特征的免疫抑制，同时伴随着对其他病原(如病毒、细菌、真菌)易感性的增加。该病在2-4周的鸡群开始发病，1-7日龄雏鸡对该病易感性最高，死亡率一般在10-20％，偶尔可达60％，亚临床感染也可造成重大损失。自1979年Yuasa等首次分离到Gifu-1毒株以来，目前呈世界范围流行。我国于1992年首次在黑龙江省分离到CIAV。近年来，由于缺乏有效的防控策略，CIAV的流行与感染给我国养鸡业带来了严重的经济损失。此外，近几年高致病性血清4型禽腺病毒(FAdV-4)导致的肝炎-心包积液综合征在国内鸡群尤为流行，同样极大的威胁着养禽业的健康持续发展。然而，目前市场上尚无针对CIAV和FAdV-4的二联疫苗。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，利用双荧光体系成功构建了表达CIAV病毒T1P6毒株VP1蛋白的重组FAdV-4病毒FAdV4-VP1(CIAV)，为CIAV和FAdV-4的有效联防联控提供了技术支撑和疫苗候选。
发明内容
本发明的目的在于针对上述现有的不足，提供一种表达鸡传染性贫血病病毒VP1蛋白的重组血清4型禽腺病毒及其制备方法，基于CRISPR-Cas9技术构建表达鸡传染性贫血病病毒VP1蛋白的重组血清4型禽腺病毒。本发明的关键技术是在血清4型禽腺病毒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型禽腺病毒中插入鸡传染性贫血病病毒VP1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鸡传染性贫血病病毒VP1蛋白的重组血清4型禽腺病毒。
本发明的目的是这样实现的：一种基于CRISPR-Cas9技术的表达鸡传染性贫血病病毒T1P6毒株VP1蛋白的重组血清4型禽腺病毒，其特征是：
所述鸡传染性贫血病病毒T1P6毒株VP1蛋白，其序列如SEQ ID NO.1所示。
重组血清4型禽腺病毒，其中使用鸡传染性贫血病病毒T1P6毒株VP1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鸡传染性贫血病病毒T1P6毒株VP1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2所示：
表2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表达盒的表达鸡传染性贫血病病毒VP1蛋白的重组血清4型禽腺病毒。
还包括步骤(5)，获得的红色荧光重组病毒接种转染Cre重组酶的LMH细胞，2-3d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表达盒的表达鸡传染性贫血病病毒T1P6毒株VP1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。
本发明方法先进科学，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鸡传染性贫血病病毒VP1基因，成功获得表达鸡传染性贫血病病毒VP1蛋白的重组血清4型禽腺病毒，为同时免疫防控血清4型禽腺病毒和鸡传染性贫血病病毒提供技术支撑和疫苗候选。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。
本发明构建的表达鸡传染性贫血病病毒VP1蛋白的重组血清4型禽腺病毒，为血清4型禽腺病毒和鸡传染性贫血病病毒的联防联控提供了二联候选疫苗。因此，本发明具有良好的市场应用价值。
附图说明
图1为鸡传染性贫血病病毒VP1基因的扩增。
图2为表达鸡传染性贫血病病毒VP1蛋白的重组病毒的构建策略示意图。
图3为IFA鉴定初代重组血清4型禽腺病毒FAdV4-VP1(CIAV)-RFP；
其中，a：重组病毒FAdV4-VP1(CIAV)-RFP；b：母本病毒FA4-EGFP；c：阴性LMH对照。
图4为PCR鉴定重组血清4型禽腺病毒FAdV4-VP1(CIAV)；
其中，1：纯化的重组病毒FAdV4-VP1(CIAV)-RFP；2：纯化的重组病毒FAdV4-VP1(CIAV)；3：母本病毒FA4-EGFP；4：野生型FAdV-4；5：阴性LMH细胞对照；M：DNA marker。
图5为IFA鉴定重组血清4型禽腺病毒FAdV4-VP1(CIAV)中VP1蛋白的表达；
其中，a：2E3单抗鉴定重组病毒中VP1蛋白的表达；b：利用针对FAdV-4的多抗鉴定重组病毒；c：阴性LMH细胞对照。
图6为WB鉴定重组血清4型禽腺病毒FAdV4-VP1(CIAV)中VP1蛋白的表达；
其中，1：重组病毒FAdV4-VP1(CIAV)；2：母本病毒FA4-EGFP；3：阴性LMH细胞对照。
图7为重组血清4型禽腺病毒FAdV4-VP1(CIAV)的生长曲线测定。
具体实施方式
下面将结合本发明中的附图，对本发明中的技术方案进行清楚、完整地描述。
实施例：
1.病毒基因组的制备：使用天根公司的基因组提取试剂盒进行提取，取禽腺病毒上清200uL于1.5mL指形管内，加入200uL Proteinase K溶液，混匀。加入200uL缓冲液GB，充分颠倒混匀，70℃水浴10min，短暂离心后加入200uL无水乙醇，充分振荡混匀15sec。将上述溶液加入一个吸附柱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500uL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600uL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600uL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200ul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：根据血清4型禽腺病毒的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lentiCRISPR v2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两个连续LoxP序列的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序列的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；以鸡传染性贫血病病毒基因组为模板扩增鸡传染性贫血病病毒VP1基因(如图1)。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：构建策略如图2，在6孔板中铺LMH细胞，次日，将3ugsgRNA、3ug的供体质粒以及6uL的转染试剂Mirus加入到200uL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-VP1(CIAV)-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-VP1(CIAV)-RFP。PCR所用的引物见表3。
表3PCR鉴定重组病毒的引物
6.RFP表达盒的删除：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，观察接种病毒的细胞中无红色荧光，但是存在CPE的LMH细胞，挑取该处细胞进行有限稀释，获得删除RFP表达盒的重组病毒。通过PCR，IFA和WB对去除RFP表达盒的重组病毒进行鉴定，成功获得重组病毒FAdV4-VP1(CIAV)(如图4，图5，图6)。
7.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远低于野生型血清4型禽腺病毒(如图7)。
SEQ ID NO.1
传染性贫血病病毒T1P6毒株VP1基因序列：
ATGGCAAGACGAGCTCGCAGACCGAGAGGCCGATTTTACGCCTTCAGAAGAGGACGGTGGCACCACCTCAAGCGACTTCGACGAAGATATAAATTTCGACATCGGAGGAGACAGCGGTATCGTAGACGAGCTTTTAGGAAGGCCTTTCACAACCCCCGCCCCGGTACGTATAGTGTGAGGCTGCCAAACCCCCAATCTACTATGACTATCCGCTTCCAAGGAGTCATCTTTCTCACGGAAGGACTCATTTTGCCTAAAAACAGCACAGCGGGGGGCTATGCAGACCACATGTACGGGGCGAGAGTCGCCAAGATCTCTGTGAACCTCAAGGAGTTCCTCCTAGCGTCAATGAACCTCACGTACGTGAGCAAACTCGGAGGCCCCATCGCCGGTGAGTTGATTGCGGACGGGTCTAAATCACAAGCCGCGGAGAACTGGCCTAACTGCTGGCTACCGCTAGATAATAACGTGCCCTCCGCGACACCATCGGCATGGTGGAGATGGGCCTTAATGATGATGCAGCCCACGGACTCTTGCCGGTTCTTTAATCACCCTAAGCAAATGACCCTGCAAGACATGGGTCGCATGTTTGGGGGCTGGCACCTGTTCCGACACATTGAAACCCGCTTTCAGCTCCTTGCCACTAAGAATGAGGGATCCTTCAGCCCCGTGGCGAGTCTTCTCTCCCAGGGAGAGTACCTCACGCGTCGGGACGATGTTAAGTACAGCAGCGATCACCAGAACCGGTGGCGAAAAGGCGAACAACCGATGACGGGGGGTATTGCTTATGCGACCGGGAAAATGAGACCGGACGAGCAACAGTACCCTGCTATGCCCCCAGACCCCCCGATAATCACCAGTACTACAGCGCAAGGCACGCAAGTCCGCTGCATGAATAGCACGCAAGCTTGGTGGTCATGGGACACATATATGAGCTTTGCAACACTCACAGCGCTCGGTGCACAATGGTCTTTTCCTCCAGGGCAACGTTCAGTTTCTAGACGGTCCTTCAACCACCACAAGGCGAGAGGAGCTGGGGACCCCAAAGGCCAGAGATGGCACACGCTGGTGCCGCTCGGCACGGAGACCATCACCGACAGCTACATGGGAGCACCCGCATCAGAGATAGACACAAATTTCTTTACGCTTTACGTAGCGCAAGGCACAAATAAGTCGCAGCAGTACAAGTTCGGCACAGCTACATACGCGCTAAAGGAGCCGGTAATGAAGAGCGATTCATGGGCAGTGGTACGCGTCCAGTCGGTGTGGCAACTGGGTAACAGGCAAAGGCCATACCCATGGGACGTCAACTGGGCCAACAGCACCATGTACTGGGGGTCGCAGCCCTGA
SEQ ID NO.2
血清4型禽腺病毒Fiber-2基因序列：
ATGCTCCGGGCCCCTAAAAGAAGACATTCCGAAAACGGGAAGCCCGAGACCGAAGCGGGACCTTCCCCGGCTCCAATCAAGCGCGCCAAACGCATGGTGAGAGCATCCCAGCTTGACCTGGTTTATCCTTTCGATTACGTGGCCGACCCCGTCGGAGGGCTCAACCCGCCTTTTTTGGGAGGCTCAGGACCCCTAGTGGACCAGGGCGGACAGCTTACGCTCAACGTCACCGATCCCATCATCATCAAGAACAGATCGGTGGACTTGGCCCACGACCCCAGTCTCGATGTCAACGCCCAAGGTCAACTGGCGGTGGCCGTTGACCCCGAAGGGGCCCTGGACATCACCCCCGATGGACTGGACGTCAAGGTCGACGGAGTGACCGTAATGGTCAACGATGACTGGGAACTGGCCGTAAAAGTCGACCCGTCCGGCGGATTGGATtCCACCGCGGGTGGACTGGGGGTCAGCGTGGACGACACCTTGCTCGTGGATCAGGGAGAACTGGGCGTACACCTCAACCAACAAGGACCCATCACTGCCGATAGCAGTGGTATCGACCTCGAGATCAATCCTAACATGTTCACGGTCAACACCTCGACCGGAAGCGGAGTGCTGGAACTCAACCTAAAAGCGCAGGGAGGCATCCAAGCCGACAGTTCGGGAGTGGGCGTTTCCGTGGATGAAAGCCTACAGATTGTCAACAACACTCTGGAAGTGAAACCGGATCCCAGCGGACCGCTTACGGTCTCCGCCAATGGCCTAGGGCTGAAGTACGACACTAATACCCTAGCGGTGACCGCGGGCGCTTTAACCGTGGTCGGAGGGGGGAGCGTCTCCACACCCATCGCTACTTTTGTCTCGGGAAGTCCCAGCCTCAACACCTACAATGCCACGACCGTCAATTCCAGCGCGAACGCCTTCTCTTGCGCCTACTACCTTCAACAGTGGAACATACAGGGGCTCCTTGTTACCTCCCTCTACTTGAAATTGGACAGCGCCACCATGGGGAATCGCCCTGGGGACCTCAACTCCGCCAATGCCAAATGGTTCACCTTTTGGGTGTCCGCCTATCTCCAGCAATGCAACCCCTCCGGGATTCAAGCGGGAACGGTCAGCCCCTCCACCGCCACCCTCACGGACTTTGAACCCATGGCCAATAGGAGCGTGACCAGCCCATGGACGTACTCGGCCAATGGATACTATGAACCATCCATCGGGGAATTCCAAGTGTTCAGCCCGGTGGTAACAGGTGCCTGGAACCCGGGAAACATAGGGATCCGCGTCCTCCCCGTGCCGGTTTCGGCCTCCGGAGAGCGATACACCCTTCTATGCTATAGTCTGCAGTGCACGAACGCGAGCATTTTTAATCCAAACAACAGCGGAACCATGATCGTGGGACCCGTGCTCTACAGCTGTCCAGCGGCCTCCCTCCCGTAA
Claims (5)
1.一种基于CRISPR-Cas9技术的表达鸡传染性贫血病病毒T1P6毒株VP1蛋白的重组血清4型禽腺病毒，其特征是：
所述鸡传染性贫血病病毒T1P6毒株VP1蛋白，其序列如SEQ IDNO.1所示。
2.根据权利要求1所述的重组血清4型禽腺病毒，其特征是：使用鸡传染性贫血病病毒T1P6毒株VP1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鸡传染性贫血病病毒T1P6毒株VP1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2所示：
表2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表达盒的表达鸡传染性贫血病病毒VP1蛋白的重组血清4型禽腺病毒。
4.根据权利要求1所述的重组血清4型禽腺病毒的制备方法，其特征是：还包括步骤(5)，获得的红色荧光重组病毒接种转染Cre重组酶的LMH细胞，2-3d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表达盒的表达鸡传染性贫血病病毒T1P6毒株VP1蛋白的重组血清4型禽腺病毒进行鉴定。
5.根据权利要求1所述的重组血清4型禽腺病毒的制备方法，其特征是：表达EGFP的重组病毒FA4-EGFP，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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