CN113980972A - 一种密码子优化的犬λ1干扰素编码基因及其在制备犬λ1干扰素中的应用 - Google Patents
一种密码子优化的犬λ1干扰素编码基因及其在制备犬λ1干扰素中的应用 Download PDFInfo
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Abstract
本发明公开了一种密码子优化的犬λ1干扰素编码基因及其在制备犬λ1干扰素中的应用。具体地公开了密码子优化的犬λ1干扰素基因CaIFNλ1‑MD及利用该基因制备重组犬λ1干扰素的方法。该优化的基因CaIFNλ1‑MD更适合大肠杆菌表达***，在宿主菌的表达能力远高于未优化的基因序列CaIFNλ1‑WT。将CaIFNλ1‑MD基因***到载体pET30a(+)的多克隆位点得到的重组质粒导入大肠杆菌得到了重组菌。利用该重组菌通过诱导培养就可以制备出大量高活性的重组犬λ1干扰素蛋白。本发明提供的基因、重组质粒和重组菌对于重组犬λ1干扰素的生产极具经济价值，对于宠物犬行业也具有重大应用价值和广泛前景。
Description
技术领域
本发明属于基因工程技术领域，涉及一种密码子优化的犬λ1干扰素编码基因及其在制备犬λ1干扰素中的应用，具体涉及重组犬λ1干扰素的高效表达及其制备方法。
背景技术
Ⅲ型干扰素即λ-干扰素(IFN-λ)，是2003年由两个独立的研究小组在脊椎动物中同时发现的分类为Ⅲ型干扰素的细胞因子，因其基因结构与IL-10相似，又被归类为IL-10家族成员。Ⅲ型干扰素包括IFN-λ1(IL-29)、IFN-λ2(IL-28α)、IFN-λ3(IL-28β)。从氨基酸的结构和生物学功能上来看，Ⅲ型干扰素更加类似于I型干扰素，可以激活干扰素刺激反应元件(Interferon stimulated regulatory elements，ISRE)，进而诱导抗病毒基因的转录。IFN-λ的受体也属于异二聚体，由IL-10R2和IL-28Rα组成。由于Ⅲ型干扰素受体仅局限表达于上皮细胞和某些树突状细胞亚群，因此Ⅲ型干扰素的作用有组织特异性，毒副作用小，具有应用于临床治疗的潜力。
IFN-λ具有广谱抗病毒活性，不仅可以抑制RNA病毒的复制，还可抑制多种DNA病毒的复制，其抗病毒活性主要表现在以下几个方面：①可通过促使病毒mRNA降解来抑制感染细胞中病毒mRNA的翻译。②可增强NK细胞对病毒的杀伤能力，并可作为一种可溶性细胞因子用以抑制和干扰病毒的繁殖，并刺激淋巴细胞分泌产生多种广谱抗病毒蛋白质。③通过T和B淋巴细胞或K细胞并在IgG的共同作用下，使感染病毒的宿主细胞溶解死亡从而阻断病毒的繁殖。④能提高细胞表面MHCⅠ类分子的表达水平，向T细胞呈递抗原，促进病毒感染细胞发生溶解。
随着经济发展和生活水平的提高，犬、猫等伴侣动物的饲养量日趋升高，且多数价格昂贵。然而，随着饲养量的增加、病毒的进化变异及外界因素导致疫苗免疫失败，犬、猫的病毒性疾病日趋严重，常造成重要经济损失和精神损失。由于农业部已废止金刚烷胺、阿昔洛韦等多种抗病毒西药的使用，使兽医临床目前缺乏安全有效的抗病毒药物，市场迫切需求能够预防和治疗动物病毒性疾病的高效生物制品。干扰素治疗宠物病毒性疾病是有效手段，但目前犬干扰素制品研究相对滞后，市场上尚没有组织特异性高、副作用小的宠物用IFN-λ等蛋白制剂，因此，研发新型高效宠物用干扰素制剂可以有效防治动物病毒性疾病，对于控制宠物病毒感染具有重要意义。
发明内容
本发明所要解决的技术问题是如何通过优化编码犬λ1干扰素基因来提高重组犬λ1干扰素蛋白的表达水平。所要解决的技术问题不限于如所描述的技术主题，本领域技术人员通过以下描述可以清楚地理解本文未提及的其它技术主题。
为解决上述技术问题，本发明首先提供了DNA分子，所述DNA分子的核苷酸序列可为SEQ ID No.1或SEQ ID No.4。
本发明还提供了一种高效制备重组犬λ1干扰素的方法，所述方法包括将含优化后DNA分子的重组质粒导入到大肠杆菌中，得到重组大肠杆菌；培养所述重组大肠杆菌，诱导表达得到重组犬λ1干扰素，所述DNA分子的核苷酸序列可为SEQ ID No.1或SEQ ID No.4。
所述DNA分子可为密码子优化的犬λ1干扰素(CaIFNλ1-MD)基因；所述CaIFNλ1-MD基因的核苷酸序列为SEQ ID No.1；
所述DNA分子可为优化的犬λ1干扰素(CaIFNλ1-MD)基因与标签蛋白的融合基因；
进一步地，所述标签蛋白可为His标签蛋白；
所述融合基因可为融合基因His-CaIFNλ1-MD，所述融合基因His-CaIFNλ1-MD的核苷酸序列为SEQ ID No.4。
本发明还提供了所述优化的犬λ1干扰素基因(CaIFNλ1-MD)编码的蛋白质，所述蛋白质的氨基酸序列为SEQ ID No.2。
本发明还提供了所述融合基因His-CaIFNλ1-MD编码的蛋白质，所述蛋白质的氨基酸序列为SEQ ID No.5。
本发明还提供了生物材料，所述生物材料可为下述任一种：
A1)含有所述DNA分子CaIFNλ1-MD或His-CaIFNλ1-MD的表达盒；
A2)含有所述DNA分子CaIFNλ1-MD或His-CaIFNλ1-MD的重组载体、或含有A1)所述表达盒的重组载体；
A3)含有所述DNA分子CaIFNλ1-MD或His-CaIFNλ1-MD的重组微生物、或含有A1)所述表达盒的重组微生物、或含有A2)所述重组载体的重组微生物；
A4)含有所述DNA分子CaIFNλ1-MD或His-CaIFNλ1-MD的转基因细胞系、或含有A1)所述表达盒的转基因细胞系、或含有A2)所述重组载体的转基因细胞系。
所述重组载体可为将SEQ ID No.1所示的DNA分子CaIFNλ1-MD***到载体pET30a(+)的多克隆位点得到的重组载体。
进一步地，所述重组载体(即重组质粒pET30a-CaIFNλ1-MD)可为用SEQ ID No.1所示的DNA分子CaIFNλ1-MD替换pET30a(+)的XhoI识别位点和EcoRV识别位点间的片段(Xho I识别位点和EcoR V识别位点间的小片段)，保持pET30a(+)的其它序列不变得到的重组表达载体。
所述重组菌可为将SEQ ID No.1所示的DNA分子CaIFNλ1-MD导入到大肠杆菌BL21(DE3)得到的重组菌；
进一步地，所述重组菌可为将所述重组载体(即重组质粒pET30a-CaIFNλ1-MD)导入到大肠杆菌BL21(DE3)得到的重组菌，其名称为BL21(DE3)pET30a-CaIFNλ1-MD。
本发明还提供了一种制备重组犬λ1干扰素的方法，所述方法包括利用所述重组微生物或转基因细胞系制备重组犬λ1干扰素。
进一步地，本发明提供了一种高效制备重组犬λ1干扰素的方法，所述方法包括将含优化后DNA分子的重组质粒导入到大肠杆菌中，得到重组大肠杆菌；培养所述重组大肠杆菌，诱导表达得到重组犬λ1干扰素。
上述方法中，所述重组微生物为将所述DNA分子CaIFNλ1-MD或His-CaIFNλ1-MD导入到大肠杆菌中，得到的重组大肠杆菌；所述方法包括如下步骤：培养所述重组大肠杆菌，诱导表达得到重组犬λ1干扰素。
进一步地，所述大肠杆菌可为大肠杆菌BL21(DE3)；
进一步地，所述诱导表达可为用0.1mmol/L的IPTG液体，16～37℃诱导3～16小时，如用0.1mmol/L的IPTG液体，37℃诱导3小时。
上述方法中，所述方法具体可包括如下步骤：
(1)将所述DNA分子CaIFNλ1-MD(SEQ ID No.1)导入到大肠杆菌中，得到重组大肠杆菌(BL21(DE3)pET30a-CaIFNλ1-MD)；
(2)将所述重组大肠杆菌加入0.1mmol/L IPTG(异丙基-β-D-硫代半乳糖苷)，16～37℃诱导3～16小时，然后超声破碎菌体，收集包涵体、洗涤包涵体、溶解包涵体并纯化后将其复性，得到含有重组犬λ1干扰素的溶液。
上述方法中，所述方法还包括纯化所述重组犬λ1干扰素的步骤。
本文中任一所述方法制备的重组犬λ1干扰素也在本发明的保护范围内。
本发明还提供了所述DNA分子CaIFNλ1-MD或His-CaIFNλ1-MD，和/或，所述生物材料在制备重组犬λ1干扰素中的应用。
本发明还提供了所述DNA分子CaIFNλ1-MD或His-CaIFNλ1-MD，和/或，所述生物材料在制备用于治疗和/或预防犬病毒性疾病的药物或制剂中的应用。
上述应用中，所述制剂可为病毒抑制剂或提高幼犬存活率的制剂。
本发明提供了一种优化的CaIFNλ1-MD基因，所述基因更适合大肠杆菌表达***，将该基因***pET30a(+)的多克隆位点得到了重组质粒，将重组质粒导入大肠杆菌BL21(DE3)，得到了重组菌。该重组菌通过简单的诱导培养就可以制备出大量重组犬λ1干扰素蛋白。本发明提供的基因、重组质粒和重组菌对于重组犬λ1干扰素的生产极具经济价值，对于宠物行业也具有重大应用价值。
附图说明
图1为CaIFNλ1-MD基因(SEQ ID No.1所示DNA)和CaIFNλ1-WT基因(SEQ ID No.3所示DNA)的序列比对。
图2为SDS-PAGE检测犬干扰素基因CaIFNλ1-MD和CaIFNλ1-WT在大肠杆菌中表达产物结果；UI代表未诱导，I代表诱导，M为低分子量蛋白Marker。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中所有引物与基因合成及测序工作由生工生物工程(上海)股份有限公司与苏州金唯智生物科技有限公司完成。
下述实施例中的。载体pET30a(+)购自Addgene公司，货号#85761。
实施例1、优化的犬λ1干扰素基因(CaIFNλ1-MD基因)的人工合成
一、优化的犬λ1干扰素基因(CaIFNλ1-MD基因)的人工合成
根据密码子的兼并性和大肠杆菌对氨基酸密码子的偏好性，参考Genebank中已经发表的犬λ1干扰素基因序列(Gene ID：612539)，设计了含大肠杆菌偏好密码子的犬λ1干扰素的编码基因，优化后编码犬λ1干扰素(CaIFNλ1-MD基因)的核苷酸序列为SEQ ID No.1，共含573个核苷酸。
SEQ ID No.1所示优化的犬λ1干扰基因编码的蛋白质的氨基酸序列为SEQ IDNo.2。
二、对照基因(CaIFNλ1-WT基因)的克隆
对照基因即Genebank中犬λ1干扰素基因，参考Genebank中的犬λ1干扰素基因序列Gene ID：612539，设计引物扩增对照基因。对照基因(CaIFNλ1-WT)的核苷酸序列如SEQ IDNo.3所示，共含573个核苷酸。
三、CaIFNλ1-MD基因和CaIFNλ1-WT基因的序列比对
CaIFNλ1-MD基因和CaIFNλ1-WT基因的序列比对见图1，CaIFNλ1-MD基因和CaIFNλ1-WT基因均编码SEQ ID No.2所示的重组犬λ1干扰素。
实施例2、重组质粒和重组菌的构建以及基因的表达
一、含有CaIFNλ1-MD基因的重组质粒和工程菌的构建
用核苷酸序列是SEQ ID No.1的DNA分子(CaIFNλ1-MD基因)替换pET30a(+)的XhoI识别位和EcoRV识别位点间的片段(Xho I识别位点和EcoR V识别位点间的小片段)，保持pET30a(+)的其它序列不变得到重组表达载体pET30a-CaIFNλ1-MD。由生工生物工程(上海)股份有限公司测序，经SnapGene软件分析被测序列与优化后的序列一致。pET30a-CaIFNλ1-MD含有核苷酸序列是SEQ ID No.4的His-CaIFNλ1-MD融合基因，所述融合基因是融合了His标签及载体一段序列，His-CaIFNλ1-MD基因的编码序列是SEQ ID No.4，His-CaIFNλ1-MD基因编码氨基酸序列是SEQ ID No.5的蛋白质His-CaIFNλ1-MD。SEQ ID No.5的第48-237位是蛋白质CaIFNλ1的氨基酸序列(即SEQ ID No.2)。SEQ ID No.4的第142-714位是CaIFNλ1基因的核苷酸序列(即SEQ ID No.1)。
将重组质粒pET30a-CaIFNλ1-MD转化进入大肠杆菌BL21(DE3)，得到重组菌BL21(DE3)pET30a-CaIFNλ1-MD。
二、CaIFNλ1-MD基因的表达
将过夜培养的BL21(DE3)pET30a-CaIFNλ1-MD按1:100的比例转接到400ml含有卡那霉素的LB培养基中(卡那霉素浓度为0.5ug/ml)，37℃，220r/min振荡培养3小时(至菌液OD600nm＝1)，加入0.1mmol/L IPTG后37℃，220r/min振荡诱导3小时。分别取诱导前后的培养液1ml于离心管，12000r/min，4℃离心10分钟，弃上清收集沉淀。加入6×Loading Buffer100℃煮10分钟后，进行SDS-PAGE电泳检测。电泳检测结果如图2所示(M为低分子量蛋白Marker。)，诱导后收集的菌体在26kD左右处出现一条蛋白条带，与预期大小相符。
三、重组犬λ1干扰素蛋白变性、纯化和复性
1、变性
经IPTG诱导表达的菌体超声(Φ6探头，超声4秒间隔5秒，30分钟)破碎后，收集包涵体沉淀，工程菌收集菌体重量为0.78g。按照每克菌重加入20ml溶液比例，使用溶液A洗涤包涵体沉淀2～3遍，使用溶液B在4℃条件下搅拌过夜，充分溶解包涵体蛋白。所述溶液A的pH值为8.0，由尿素、Tris-HCl、EDTA、NaCl、β-巯基乙醇、Triton-100和水组成，尿素浓度为2mol/L，Tris-HCl浓度为50mmol/L、EDTA浓度为0.3mmol/L、NaCl浓度为50mmol/L、β-巯基乙醇浓度为2mmol/L、Triton-100浓度为1％；所述溶液B的pH值为8.0，由尿素、Tris-HCl、β-巯基乙醇组成和水组成，尿素浓度为8mol/L，Tris-HCl浓度为50mmol/L、β-巯基乙醇浓度为2mmol/L。
2、纯化
变性后溶液4℃条件下12000r/min离心10min收集上清，使用0.45μm滤膜过滤后，经Ni-NTA亲和层析柱纯化。将含有犬λ1干扰素的溶液，使用GE AKTA Pure蛋白质分离纯化***进行亲和亲和层析柱纯化，使用结合缓冲液以0.5ml/min流速结合目的蛋白，然后用洗杂缓冲液冲洗10个柱体积，最后使用洗脱缓冲液冲洗5-10个柱体积，收集280nm光照下紫外吸收值大于200的洗脱液，得到犬λ1干扰素溶液；所述结合缓冲液pH为8.0，由Tris-HCl、咪唑、尿素和水组成，Tris-HCl浓度为50mmol/L，咪唑为10mmol/L，尿素为8M；所述洗杂缓冲液pH为8.0，由Tris-HCl、咪唑、尿素和水组成，Tris-HCl浓度为50mmol/L，咪唑为80mmol/L，尿素为8M；所述洗脱缓冲液pH为8.0，由Tris-HCl、咪唑、尿素和水组成，Tris-HCl浓度为50mmol/L，咪唑为500mmol/L，尿素为8M。
3、复性
纯化后蛋白在尿素浓度梯度递减的复性缓冲液中透析，每隔3h换一次复性缓冲液，最后在PBS缓冲液中透析12～24h。将透析袋放入大平皿中用PEG8000浓缩，将浓缩好的蛋白过滤除菌并测定浓度，于-80℃保存备用。尿素复性缓冲液pH值为8.0，由尿素、Tris-HCl、NaCl、L-精氨酸、氧化型谷胱甘肽、还原型谷胱甘肽和水组成，尿素浓度为6mol/L、4mol/L、2mol/L、0mol/L梯度递减，Tris-HCl浓度为50mmol/L，NaCl浓度为0.15mol/L，L-精氨酸浓度为0.5mol/L，氧化型谷胱甘肽浓度为0.4mmol/L，还原型谷胱甘肽浓度为2mmol/L。经过步骤1、2、3得到含有重组犬λ1干扰素的溶液(CaIFNλ1-MD溶液和CaIFNλ1-WT溶液)。
四、优化后基因序列CaIFNλ1-MD在宿主菌中的蛋白量测定
按50:1的体积比取BCA蛋白定量试剂盒(购自上海索莱宝生物科技有限公司，货号:PC0020)中的溶液A和溶液B混合成工作液，并将BSA标准品梯度稀释至500mg/ml，400mg/ml，300mg/ml，200mg/ml，150mg/ml，100mg/ml，50mg/ml，取各个浓度的标准品或待测溶液20μl，加200μl的工作液混合，用保鲜膜封好37℃孵育30min，恢复至室温后用酶标仪读562nm处吸光值。根据标准品浓度和吸光值绘制标准曲线，再根据标准曲线计算待测蛋白样品中的蛋白浓度。
CaIFNλ1-MD溶液的蛋白浓度为0.5mg/ml，收获1.5mg蛋白。CaIFNλ1-WT溶液的蛋白浓度为0.2mg/ml,收获0.6mg蛋白。
结果表明从400mL的BL21(DE3)pET30a-CaIFNλ1-MD发酵液(诱导后的培养液，OD600nm＝1.3)，按照上述步骤纯化得到3mL CaIFNλ1-MD溶液的蛋白浓度为0.5mg/ml，共收获1.5mg蛋白；从400mL的BL21(DE3)pET30a-CaIFNλ1-WT的发酵液(诱导后的培养液OD600nm＝1.3))，按照上述步骤纯化得到3mL CaIFNλ1-WT溶液的蛋白浓度为0.2mg/ml，共收获0.6mg蛋白。结果表明，本发明提供的优化后基因序列CaIFNλ1-MD在宿主菌中的表达能力远远高于未优化的CaIFNλ1-WT。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
SEQUENCE LISTING
<110> 山东农业大学
<120> 一种密码子优化的犬λ1干扰素编码基因及其在制备犬λ1干扰素中的应用
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 573
<212> DNA
<213> 人工序列（Artificial sequence）
<400> 1
atggccaccg cgcgcgttct ggttctggtt acggttgtgc tgggtctgac ccgtgcgggt 60
ccagttccaa cgagcaaacc aaccaccacc cgccgcggtt gccatatgga tcgcttccag 120
agtctgagtc cacgcgaact ggaggccttc aaaaaggcca aagacgcgct ggaagagagt 180
ctaagctgga aaaactggag ctgcagtagc cgtctgtttc cgcgtagccg cgatctgcgt 240
ctgctgcaag cgtgggaacg tccagttgcg ctggaagccg aactggatct gacgctgaag 300
gtgctggaga acatgaccga tagcagtctg ggcgttacgc tggatcagcc actgcgcacg 360
ctacaccata tccatagcga actgcaagcg tgcgttccag cgcagccaac cgccgatcca 420
cgaccacatg gccgtctgca tcattggctg caccgtctgc agaaggcccc gaaagaaagc 480
caaggctgtc tggaagcgag cattacgttc aatctgttcc gtctgctgac ccgcgatctg 540
aaatgcgttg cgagccgcga tctctgcgtt taa 573
<210> 2
<211> 190
<212> PRT
<213> 人工序列（Artificial sequence）
<400> 2
Met Ala Thr Ala Arg Val Leu Val Leu Val Thr Val Val Leu Gly Leu
1 5 10 15
Thr Arg Ala Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Arg Arg
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Gly Cys His Met Asp Arg Phe Gln Ser Leu Ser Pro Arg Glu Leu Glu
35 40 45
Ala Phe Lys Lys Ala Lys Asp Ala Leu Glu Glu Ser Leu Ser Trp Lys
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Asn Trp Ser Cys Ser Ser Arg Leu Phe Pro Arg Ser Arg Asp Leu Arg
65 70 75 80
Leu Leu Gln Ala Trp Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Asp
85 90 95
Leu Thr Leu Lys Val Leu Glu Asn Met Thr Asp Ser Ser Leu Gly Val
100 105 110
Thr Leu Asp Gln Pro Leu Arg Thr Leu His His Ile His Ser Glu Leu
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Gln Ala Cys Val Pro Ala Gln Pro Thr Ala Asp Pro Arg Pro His Gly
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Arg Leu His His Trp Leu His Arg Leu Gln Lys Ala Pro Lys Glu Ser
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Gln Gly Cys Leu Glu Ala Ser Ile Thr Phe Asn Leu Phe Arg Leu Leu
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Thr Arg Asp Leu Lys Cys Val Ala Ser Arg Asp Leu Cys Val
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<210> 3
<211> 573
<212> DNA
<213> 犬（Canidae）
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atggctacag ctcgggtcct ggtgctggtg accgtggtgc tgggcttgac cagagccggc 60
cctgtcccta cttccaaacc caccacaacc aggaggggct gccacatgga caggttccag 120
tctctgtcac ccagggagct agaagccttc aagaaggcca aggacgcctt ggaagagtcg 180
ctctcctgga agaactggag ctgcagctct cgcctcttcc ctagatccag ggacctgaga 240
ctcctgcagg cctgggagcg tcctgtggcc ttggaggctg agctagactt gacactgaag 300
gtcctggaga acatgactga ctcatcgctg ggggtgaccc tggaccagcc cctccgcacg 360
ctgcaccaca tccactcgga gctccaggct tgtgtcccgg ctcagcccac agcagacccc 420
aggccccacg gccgcctcca ccactggctg caccggctcc agaaggcccc caaggagtcc 480
cagggctgcc tcgaggcctc catcacgttc aacctcttcc gcctcctcac acgggacctg 540
aaatgtgttg ccagtagaga cctgtgcgtc tga 573
<210> 4
<211> 714
<212> DNA
<213> 人工序列（Artificial sequence）
<400> 4
atgcaccatc atcatcatca ttcttctggt ctggtgccac gcggttctgg tatgaaagaa 60
accgctgctg ctaaattcga acgccagcac atggacagcc cagatctggg taccgacgac 120
gacgacaagg ccatggctga tatggccacc gcgcgcgttc tggttctggt tacggttgtg 180
ctgggtctga cccgtgcggg tccagttcca acgagcaaac caaccaccac ccgccgcggt 240
tgccatatgg atcgcttcca gagtctgagt ccacgcgaac tggaggcctt caaaaaggcc 300
aaagacgcgc tggaagagag tctaagctgg aaaaactgga gctgcagtag ccgtctgttt 360
ccgcgtagcc gcgatctgcg tctgctgcaa gcgtgggaac gtccagttgc gctggaagcc 420
gaactggatc tgacgctgaa ggtgctggag aacatgaccg atagcagtct gggcgttacg 480
ctggatcagc cactgcgcac gctacaccat atccatagcg aactgcaagc gtgcgttcca 540
gcgcagccaa ccgccgatcc acgaccacat ggccgtctgc atcattggct gcaccgtctg 600
cagaaggccc cgaaagaaag ccaaggctgt ctggaagcga gcattacgtt caatctgttc 660
cgtctgctga cccgcgatct gaaatgcgtt gcgagccgcg atctctgcgt ttaa 714
<210> 5
<211> 237
<212> PRT
<213> 人工序列（Artificial sequence）
<400> 5
Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser
1 5 10 15
Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp
20 25 30
Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Met
35 40 45
Ala Thr Ala Arg Val Leu Val Leu Val Thr Val Val Leu Gly Leu Thr
50 55 60
Arg Ala Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Arg Arg Gly
65 70 75 80
Cys His Met Asp Arg Phe Gln Ser Leu Ser Pro Arg Glu Leu Glu Ala
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Phe Lys Lys Ala Lys Asp Ala Leu Glu Glu Ser Leu Ser Trp Lys Asn
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Trp Ser Cys Ser Ser Arg Leu Phe Pro Arg Ser Arg Asp Leu Arg Leu
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Leu Gln Ala Trp Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Asp Leu
130 135 140
Thr Leu Lys Val Leu Glu Asn Met Thr Asp Ser Ser Leu Gly Val Thr
145 150 155 160
Leu Asp Gln Pro Leu Arg Thr Leu His His Ile His Ser Glu Leu Gln
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Ala Cys Val Pro Ala Gln Pro Thr Ala Asp Pro Arg Pro His Gly Arg
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Leu His His Trp Leu His Arg Leu Gln Lys Ala Pro Lys Glu Ser Gln
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Gly Cys Leu Glu Ala Ser Ile Thr Phe Asn Leu Phe Arg Leu Leu Thr
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Arg Asp Leu Lys Cys Val Ala Ser Arg Asp Leu Cys Val
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Claims (10)
1.DNA分子，其特征在于，所述DNA分子的核苷酸序列为SEQ ID No.1或SEQ ID No.4。
2.生物材料，其特征在于，所述生物材料为下述任一种：
A1)含有权利要求1所述DNA分子的表达盒；
A2)含有权利要求1所述DNA分子的重组载体、或含有A1)所述表达盒的重组载体；
A3)含有权利要求1所述DNA分子的重组微生物、或含有A1)所述表达盒的重组微生物、或含有A2)所述重组载体的重组微生物；
A4)含有权利要求1所述DNA分子的转基因细胞系、或含有A1)所述表达盒的转基因细胞系、或含有A2)所述重组载体的转基因细胞系。
3.一种制备重组犬λ1干扰素的方法，其特征在于，所述方法包括利用权利要求2中所述重组微生物或转基因细胞系制备重组犬λ1干扰素。
4.根据权利要求3所述的方法，其特征在于，所述重组微生物为将权利要求1所述的DNA分子导入到大肠杆菌中，得到的重组大肠杆菌；所述方法包括如下步骤：培养所述重组大肠杆菌，诱导表达得到重组犬λ1干扰素。
5.根据权利要求3或4所述的方法，其特征在于，所述方法还包括纯化所述重组犬λ1干扰素的步骤。
6.权利要求3-5中任一所述方法制备的重组犬λ1干扰素。
7.权利要求1所述的DNA分子，和/或，权利要求2所述的生物材料在制备重组犬λ1干扰素中的应用。
8.权利要求1所述的DNA分子，和/或，权利要求2所述的生物材料在制备用于治疗和/或预防犬病毒性疾病的药物或制剂中的应用。
9.根据权利要求8所述的应用，其特征在于，所述制剂为病毒抑制剂或提高幼犬存活率的制剂。
10.根据权利要求8或9所述的应用，其特征在于，所述犬病毒性疾病为犬瘟热、犬细小病毒性肠炎、犬传染性肝炎、犬冠状病毒性肠炎、犬传染性喉气管炎或狂犬病。
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| CN108359004A (zh) * | 2018-01-12 | 2018-08-03 | 中国农业科学院北京畜牧兽医研究所 | 犬重组干扰素-λ1及其制备方法与应用 |
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