CN115003155A - 抗j亚群禽白血病病毒转基因家禽的制备方法 - Google Patents
抗j亚群禽白血病病毒转基因家禽的制备方法 Download PDFInfo
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Abstract
本发明涉及制备抗J亚群禽白血病病毒转基因家禽的方法，其特征在于该家禽携带通过CRISPR/Cas9介导的同源重组引入chNHEI基因的W38缺失，或任选地，携带使用CRISPR/Cas9引入的所述W38缺失和一种特定的sgRNA，以生产对J亚群禽白血病病毒(ALV)具有抗性的家鸡或家养火鸡。
Description
技术领域
本发明涉及家禽对一种严重家禽疾病——由J亚群禽白血病病毒(ALV-J)诱发的禽白血病的经基因工程的、永久性的和基因决定的抗性。
背景技术
禽白血病是由禽白血病病毒(ALV)引起的家禽造血系统的肿瘤性疾病。
禽类白血病的病因与逆转录病毒科(最常见的是A和B亚群的，近年是J亚群的，最近期是K亚群的)的禽类白血病病毒(ALV)有关。家禽群体经常遭受外源ALV的感染(Jurajda,V.,2002)，但一般来说，临床疾病的发生率明显低于例如马克氏病的情况(1-5％的动物受感染)。在白血病的形式中，相对最常见的是淋巴性白血病(LL)；ALV的其他病理表现，包括骨软化症、肉瘤和相关的肿瘤，在本领域是零星的，甚至很少发现。一个例外是由J亚群ALV诱发的骨髓细胞瘤病，其1990年开始在包括捷克共和国在内的全球许多国家的肉用品种和肉鸡中发生。J亚群ALV的经济影响不仅体现在直接死亡损失上，而是更多地体现在对育种表现的负面影响上(降低的增重量和产蛋率)。此外，淋巴样白血病潜伏感染的免疫抑制作用会加剧其他感染的进程。
当感染家禽时，ALV-J病毒通过chNHE1受体进入细胞。然后该逆转录病毒通过逆转录进行转录，并作为原病毒整合到宿主细胞的核DNA中。原病毒DNA转录为病毒RNA，后者在细胞质中翻译并包裹成新形成的粒子。除非家禽具有抗性或受到限制性因素的保护，否则受感染细胞释放的粒子会导致感染扩散到个体体内的允许细胞。
在逆转录病毒生命周期中，对病毒的敏感性或抗性由靶细胞表面是否存在适当的受体分子来定义。chNHE1受体是一种具有离子交换功能的细胞糖蛋白，以同源三聚体的形式出现。其突出的第一个细胞外环包含对与ALV-J相互作用至关重要的氨基酸残基。缺乏这种糖蛋白的鸡细胞对ALV-J感染没有允许性。类似地，氨基酸W38的缺失将导致对ALV-J产生抗性，但不会对所有其他ALV亚组产生抗性。在雉鸡和大多数鸡形目鸟类的NHE1中发现了W38的缺失或替代；因此，敏感物种仅包括家鸡、四种野生家禽、火鸡和几种来自Calipepla属、齿鹑属(Colinus)和山翎鹑属(Oreortyx)的新世界鹌鹑(
J,
M,
D,
F,Stepanets V,Hron T,TrejbalováK,Elleder D,Hejnar J.:Identification of New World quails susceptible to infection with avianleukosis virus subgroup J.Journal of Virology 91(3):e02002-16,2017)。根据我们的研究，家养母鸡品种和原始家养鸡群都不含W38缺失的等位基因。因此，没有遗传变异的天然来源可用于选择性地繁育具抗性动物((
M,
J,
D,
F,Vinkler M,Hejnar J.:Genetic Diversity of NHE1,Receptor for Subgroup J AvianLeukosis Virus,in Domestic Chicken and Wild Anseriform Species.PloS One 11(3):e0150589)。
在家禽业发达的国家，为了消除这种疾病，对患有这种疾病的动物进行了系统性和长期性的筛选；例如，2007年美国消灭了ALV-J感染的最后一个病灶((Malhotra S,Justice J 4th,Lee N,Li Y,Zavala G,Ruano M,Morgan R,Beemon K:Complete genomesequence of an American avian leukosis virus subgroup J isolate that causeshemangiomas and myeloid leukosis.Genome Announcements 3(2):pii:e01586-14,2015)。然而，这需要在繁殖过程中对动物进行严格且昂贵的筛查，这只能在对传染病完全掌控的工厂化养殖中应用。在中国——实际上在整个亚洲和非洲——家禽通常在市场上出售，而且在没有任何管控的小型家庭农场生产，因此尽管近年来已经启动了根除计划，但依然难以摆脱这种疾病。
发明内容
本发明的制备抗J亚群禽白血病病毒的转基因家禽的方法解决了上述问题，该方法基于一个事实，即该家禽携带了利用CRISPR/Cas9介导的同源重组被引入chNHE1基因的W38缺失，或可选地，携带了使用CRISPR/Cas9和特定sgRNA引入的W38缺失，以产生抗(ALV-J)的家鸡或家养火鸡。
发明人创造了转基因的家禽个体，其中通过使用原始生殖细胞(PGC)的基因组编辑，chNHE1离子交换基因中位置38(W38)的色氨酸密码子已被删除。由于chNHE1充当ALV-J受体，而W38是对其受体而非离子交换功能至关重要的氨基酸，这将诱导对ALV-J的完全抗性。本发明基于两项已经获得授权的捷克专利第307102号和第307285号。
根据捷克专利第307102号，我们意识到可以使用家禽胚胎细胞——“原始生殖细胞”(PGC)构建转基因个体，该细胞可在体外培养，基因操控，然后移植到公鸡的辐照灭菌睾丸中。引入的细胞恢复精子形成，这可能使得后代携带与移植的PGC相同的基因修饰。根据捷克专利第307285号，我们知道在鸡成纤维细胞的体外模型中，chHNE1受体中氨基酸W38的缺失诱导对禽白血病的完全抵抗。
本发明的方法实际上代表了上述两项捷克专利成果的结合，创造了携带W38缺失的家鸡的转基因品系。通过防止白血病病毒进入这些个体的细胞，永久性的抵抗力得以诱导。这种保护是由这些鸡的基因决定的，因此可以传递给后代。将这些抗性改良个体纳入育种计划可以将所述特性转移给所有个体，从而显着提高育种群的质量，最终使蛋或家禽肉的生产具有显著经济性。这种新方法可能对家禽健康产生全球性的影响，尤其与家鸡和家养火鸡(统称为家禽)息息相关。
本发明涉及J亚群禽白血病的方法是指：发明人建立的对ALV-J感染具有永久抵抗力的转基因个体模型。因此，它代表了一条根除这种疾病的途径——不仅仅适用于中国和东南亚，也可用于世界其他地区，因为存在这样一种风险，即新的、更难根除的中国ALV-J毒株可能再度被引入到目前没有发现该疾病的地区(包括欧洲和美国)。
如上文中所提到的，捷克专利第307285号专门提到了家鸡对ALV-J感染的敏感性和抵抗力。其构成了如下权利要求：一种编码突变蛋白chNHE1或其片段的分离的DNA分子，其中38位的色氨酸已被删除；此外，也构成了如下权利要求：一种编码突变的chNHE1或其片段的分离的DNA分子，其中38位的色氨酸已被甘氨酸或谷氨酸取代。该专利描述了基于实验性细胞的验证，即含有突变体W38的家鸡的NHE1序列赋予对ALV-J感染的完全抵抗力。在该专利中，发明人发现当chHNE1受体中的色氨酸被甘氨酸取代时，这种取代在鸡成纤维细胞的体外模型中引起对禽白血病的完全抵抗。这一事实只能通过以下方式来肯定/否定：创建转基因家禽，其中使用CRISPR/Cas9基因组编辑将这种缺失或替代引入基因组。为此，发明人使用了根据捷克专利第307102号所新创建的转基因动物制备模型，该模型与移植后在绝育受体公鸡睾丸中产生具有受精能力的鸟类精子的方法直接相关，促成了可完美施行及实践的转基因个体创建，达到约50％的高效。
附图描述
在附图中，图1展示了导致鸡NHE1基因中W38缺失的同源重组图。顶部是chNHE1基因的内含子-外显子结构。外显子1包含gRNA目标序列(中间)，带有W38的标示出的TGG三联体(黄色)作为Cas9切割(剪刀)的限制位点。红色表示核苷酸，其同义突变创建了BsaI核酸内切酶的识别位点，其之后用于检测修饰的等位基因。底部，单链寡核苷酸(ssODN)序列用作鸡和火鸡同源重组的模板。
图2展示了野生型chNHE1等位基因序列(A)与W38密码子缺失后的序列(B)的比较。仅展示了W38的紧邻区域，用箭头表示。该序列表示为具有转录的核苷酸和氨基酸序列的色谱图。
图3展示了来自基因型W38-/-(左上)、W38+/-(右上)和W38+/+(左下)的胚胎的成纤维细胞感染后，通过流式细胞术测量的GFP阳性。x轴，GFP荧光强度，y轴，细胞计数。GFP阳性细胞的百分比显示在直方图的右下角。右下角，图表总结了所有检查胚胎中GFP阳性细胞的百分比(每个基因型四个)。
图4展示了基因型W38-/-、+/-和+/+(在x轴上标记)的鸡在感染报告载体RCASBP(J)GFP后的病毒血症定量分析。在定量RT PCR后，以阴性对照组(由非特异性RCAS-A病毒感染)的相对单位(倍数)评估病毒血症(见y轴上)。
本发明的方法已被the Institute of Molecular Genetics of the CzechAcademy of Sciences,v.v.i.,Prague,CZ,和BIOPHARM a.s.,
CZ的发明者们在实验室中充分验证。
以下本发明的实施例仅用于说明，不以任何方式限制本发明。
实施例
实施例1
具有修饰受体基因chNHE1的家鸡系的制备
家禽转基因品系的制备基于从24至96小时龄的鸡胚胎中衍生原始生殖细胞。这些细胞是从收集的胚胎血样本或胚胎的头部培养出来的，并在体外扩增。使用CRISPR/Cas9将W38缺失引入PGC基因组中，其中gRNA特异于chNHE1基因中的W38区域和包含W38区域的同源重组模板(图1)。通过Amaxa系统中的电穿孔，将具有适当gRNA的编码CRISPR/Cas9的构建体和同源重组模板引入PGC。电穿孔后，通过在流式细胞仪中对单个细胞进行分选来选择与CRISPR/Cas9相关的、对GFP荧光呈阳性的细胞。然后，根据CZ专利第307102号，将生长良好的、经分子学上确认具有W38缺失的扩增克隆体原位移植到已灭菌的受体公鸡。五个月后观察到恢复的精子形成，并且受体公鸡的射精用于母鸡的人工授精，首先在输卵管膨大部内，然后在阴道内。后代完全由杂合了W38缺失的个体组成。达到性成熟后，杂合动物杂交，然后在G2中，所有基因型(即W38+/+、W38+/-和W38-/-)按预期比例分离。与预期比率的一致表明W38缺失并不代表其携带者将背负重大负担，降低其在该品种中的效用。使用分子遗传学方法(即DNA分离、通过聚合酶链式反应(PCR)扩增chNHE1的特定片段和对PCR产物进行测序)，在所有层面(PGC克隆、受体公鸡的射精、G1和G2个体的血液或羽毛髓)验证W38缺失。示例性的DNA序列如图2所示。
实施例2
W38缺失携带者对ALV-J抗性的演示
W38-/-个体的耐药性通过三种独立的方法演示，这些方法是病毒学研究领域的常规方法，并且是专门为ALV-J设立的。
A.胚胎成纤维细胞的体外感染
将G2个体的胚胎培养至发育的第10天，然后制备胚胎成纤维细胞的培养物。W38基因型通过DNA分离、PCR和测序验证。胚胎成纤维细胞的培养物被具有ALV-J受体特异性的病毒感染(特别是被报告载体RCASBP(J)GFP感染)，其中为了便于病毒检测，其转导了绿色荧光蛋白(GFP)的报告基因。使用流式细胞术对GFP进行定量评估。具有W38+/+和W38+/-基因型的胚胎成纤维细胞在大约90％的细胞中显示出相同的GFP阳性，这表明病毒几乎达到完全传播。相比之下，具有W38-/-基因型的胚胎成纤维细胞在不到0.05％的细胞中显示出GFP阳性，这再现了自发荧光给到的天然背景(图3)。可以得出结论，W38-/-基因型在胚胎成纤维细胞水平上对ALV-J感染具有完美的抵抗力。
B.鸡的体内感染
W38+/+、W38+/-和W38-/-基因型的鸡在几天到两个月大时被报告载体RCASBP(J)GFP感染，其不仅保持ALV-J受体特异性，同时比ALV-J的原型菌株，例如HPRS103，更具侵略性。ALV-J感染的正常病程表现为短暂的病毒血症，根据感染者的年龄，它会在几天后发展，通常在10天内达到极点，并在达到极点后保持一到两周。这次病毒在体内的存在既在分子学上验证，即通过使用逆转录酶定量聚合酶链反应(RT-qPCR)对病毒基因组RNA定量检测，也在生物学上验证，即通过一种互补测定法进行验证，在该方法中一种具有复制能力的引入病毒将补充有缺陷的病毒，之后根据形成病灶的转化细胞的数量对该有缺陷的病毒进行二次量化。第二种检测方法验证生物活性病毒的存在，而非仅仅验证RNA的存在。在这两种情况下，测试的材料由在两个时间点，即感染后一周和两周收集的受感染动物的血清表示。使用RT-qPCR时，所有W38-/-基因型的鸡(共5只鸡)在两个时间点均检测为阴性。W38+/+和W38+/-基因型(总共10只鸡)在较晚的时间点呈阳性，但有一个例外；第一个采集时间点，3只鸡为阴性，这意味着第一周病毒血症发病缓慢，第二周病毒血症增强。唯一的阴性情况可能意味着感染不成功(例如，由于接种有缺陷)。这些结果总结在图4中。在互补测定中，在第一个时间采集点，仅在W38+/+和W38+/-基因型中发现了零星的阳性病例。在第二个时间点，具有这些基因型的动物样本呈阳性，只有一个例外(同为此前实验中的那个个体)，而所有W38-/-基因型仍为阴性(表1)。总之，我们可以得出结论，当将病毒接种到雏鸡个体的循环血液中时，W38缺失会诱导对ALV-J的完全抵抗。这种类型的感染最能模拟家养繁殖条件下的感染。
表1展示了W38-/-、+/-和+/+基因型(对照野生型基因型)鸡只在感染报告载体RCASBP(J)GFP后病毒血症的验证。病毒血症确定为感染后6天和13天两个血清样品中补充缺陷病毒16Q的滴度和终末稀释度。
C.ALV-J-假型病毒诱导肿瘤
为了模拟ALV-J感染后实体瘤的形成，我们通过具有ALV-J受体特异性的病毒(即RCASBP(J)GFP载体)对存在于鹌鹑细胞系16Q中并含有v-src癌基因的转化病毒进行了假型分析。这导致病毒急剧转化，癌基因v-src被ALV-J病毒糖蛋白包裹，能够在接种部位(在我们的案例中是翼蹼)诱导快速生长的肉瘤。我们在10日龄至2月龄的鸡中接种，并在1个月内监测肉瘤的发病率和生长情况。所有W38+/+和W38+/-基因型的鸡都发展出逐渐生长的肉瘤，而W38-/-基因型的鸡在任何一种情况下都没有发展出任何肿瘤。W38+/+基因型动物的肿瘤比W38+/-基因型鸡的肿瘤生长得更快，这显示出了携带W38缺失的等位基因的轻度负显性效应。这可能与chNHEI三聚化有关。可以得出结论，该实验已证明在纯合W38缺失的情况下对ALV-J具有(完美)完全抗性。
上述实施例(近日在KoslováA,Trefil P,MucksováJ,
M,
J,Kalina J,
D,Geryk J,KrchlíkováV,
B,Hejnar J:Precise CRISPR/Cas9 Editing of the NHE1 Gene Renders Chickens Resistant to the J Subgroup ofAvian Leukosis Virus.Proc Natl Acad Sci U S A.117::2108-2112,2020中无删节地发表)最终证明了使用本发明的方法所创建的、在chNHEI基因中携带纯合缺失W38的家鸡新品系，对病原体具有完美的抗性，同时能保证品系的正常生长、繁殖和利用特性，没有任何明显的副作用，从而将带来巨大的经济效应。
工业实用性
生产对J亚群禽白血病病毒具有抗性的转基因家禽的新颖方法为根除J型禽白血病ALV提供了一种新颖的、原创的解决方案，并提供了一种新创建的对ALV-J感染具有永久抵抗力的转基因个体模型。因此，它代表了一种彻底根除这种疾病的革命性方法，该方法不仅适用于中国和东南亚等大幅度家禽养殖国家，也同时适用于在世界其他地区，因为这种疾病有被再度引入那些病原体已被根除的地区的潜在风险。
参考文献：
JURAJDA,Vladimír.Nemoci
a ptactva-virovéinfection.1.vyd.Brno:
VFU.Brno,2002.184s.ISBN 80-7305-436-1.
Malhotra S,Justice J 4th,Lee N,Li Y,Zavala G,Ruano M,Morgan R,BeemonK:Complete genome sequence of an american avian leukosis virus subgroup jisolate that causes hemangiomas and myeloid leukosis.Genome Announcements 3(2):pii:e01586-14,2015.
KoslováA,Trefil P,MucksováJ,
M,
J,Kalina J,
D,Geryk J,
V,
B,Hejnar J:Precise CRISPR/Cas9 Editing ofthe NHE1 Gene Renders Chickens Resistant to the J Subgroup of Avian LeukosisVirus.Proc Natl Acad Sci U S A.117::2108-2112,2020.
CZ patent No.307102.
CZ patent No.307285.
Claims (1)
1.一种抗J亚群禽白血病病毒转基因家禽的制备方法，其特征在于，所述家禽携带通过CRISPR/Cas9介导的同源重组引入chNHEI基因的W38缺失，或任选地，携带使用CRISPR/Cas9引入的所述W38缺失和一种特定的sgRNA，以生产对J亚群禽白血病病毒(ALV)具有抗性的家鸡或家养火鸡。
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| ANNA KOSLOVÁ ET AL: "Genetic Resistance to Avian Leukosis Viruses Induced by CRISPR/Cas9 Editing of Specific Receptor Genes in Chicken Cells", VIRUSES, pages 1 - 12 * |
| HONG JO LEE ET AL: "Precise gene editing of chicken Na+/H+ exchange type 1 (chNHEl) confers resistance to avian leukosis virus subgroup J (ALV-J)", pages 5 * |
| 徐望红: "《肿瘤流行病学》", 复旦大学出版社, pages: 48 * |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2020297483A1 (en) | 2021-12-23 |
| WO2020253894A1 (en) | 2020-12-24 |
| EP4075964A1 (en) | 2022-10-26 |
| KR20220018016A (ko) | 2022-02-14 |
| CZ308509B6 (cs) | 2020-10-07 |
| CZ2019392A3 (cs) | 2020-10-07 |
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