CN114716570A - 一种融合蛋白及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种融合蛋白以及密码子优化后的融合蛋白的核苷酸序列,所述序列如SEQ ID NO.1所示。本发明还公开了一种使用所述融合蛋白制备的疫苗。本发明公开的融合蛋白具有良好的免疫原性,能够比常规的狂犬病毒G蛋白产生更高的抗体滴度,为狂犬病毒亚单位疫苗的研发提供了重要的参考。另外,本发明筛选到的狂犬病毒G蛋白的6条抗原表位多肽,具有较好的抗原性,为狂犬病毒G蛋白的研究提供了重要的参考,也为后续开发狂犬病毒G蛋白的单克隆抗体提供了重要的抗原线索。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种融合蛋白及其制备方法和应用。
背景技术
狂犬病毒(Rabies virus,RV)属于弹状病毒科(Rhabdoviridae)狂犬病毒属(Lyssavirus)。外形呈弹状,核衣壳呈螺旋对称,表面具有包膜,内含有单链RNA,是引起狂犬病的病原体。狂犬病毒具有两种主要抗原:一种是病毒外膜上的糖蛋白抗原,能与乙酰胆碱受体结合使病毒具有神经毒性,并使体内产生中和抗体及血凝抑制抗体,中和抗体具有保护作用;另一种为内层的核蛋白抗原,可使体内产生补体结合抗体和沉淀素,无保护作用。狂犬病是由狂犬病毒(Rabies Virus)引起的人畜共患的传染病。
狂犬病病毒为核糖核酸型弹状病毒,一端圆凸;一端平凹,形如子弹,直径65-80nm,长约130~240nm。病毒易为日光、紫外线、甲醛、升汞季胺类化合物(如新洁尔灭)、脂溶剂、50%~70%酒精等灭活,其悬液经56℃30-60分钟或100℃2分钟即灭活。病毒于-70℃或冻干后置0~4℃中可保持活力数年。被感染的组织可保存于50%甘油内送验。狂犬病毒含5种蛋白,即糖蛋白(G)、核蛋白(N)、双聚酶(L)、磷蛋白(NS)及基质(M)等。后二者为小分子蛋白。G蛋白可导致体内形成中和抗体,可对抗病毒攻击。N蛋白导致的抗体但不具中和力,可用检测浆内包涵体。
狂犬病毒的G蛋白是一种三聚体,大约67kD,是诱导生成病毒中和抗体的主要抗原,并且可以诱导机体产生对狂犬病毒致命感染的免疫。G蛋白同时含有毒力决定簇。G基因是第一种被克隆和测序的狂犬病毒基因。从核苷酸序列中可以推导出,它编码含524个氨基酸的多肽,其中包括由19个氨基酸构成的信号序列。在位点333的精氨酸对病毒毒力具有重要作用,它与神经侵袭力和跨突触传播能力相关,能使病毒在神经系统中扩散的速度更快。
发明内容
为了弥补现有技术的不足,本发明的目的在于提供一种融合蛋白及其制备方法。
因此,一方面,本发明提供了一种融合蛋白,所述融合蛋白的氨基酸序列为:KLTNKHSPEAAYAAYCTGVVTEAETYTNFVGYVTTTFKRKAAYAAYMAGDPRYEESLHNPYPDYHWLRTVKAAYAAYSKGSKTCGFVDERGLYKSLKGACKLAAYAAYIHDFRSDEIEHLVVEELVKKREECLAAYAAYVSFRRLSHLRKLVPGFGKAYTIFNKAAYAAYADPSTVFKDGDEAEDFVEVHLPDVHAAYAAYQARESKDNKIGKTENPHHHHHH。
优选地,本发明所述的融合蛋白的密码子优化后的核苷酸序列如SEQ ID NO.1所示。
优选地,本发明所述的融合蛋白包括6条狂犬病毒G蛋白的抗原表位多肽,所述的6条狂犬病毒G蛋白的抗原表位多肽的序列分别为:
抗原表位多肽4:CTGVVTEAETYTNFVGYVTTTFKRK;
抗原表位多肽5:MAGDPRYEESLHNPYPDYHWLRTVK;
抗原表位多肽12:SKGSKTCGFVDERGLYKSLKGACKL;
抗原表位多肽16:IHDFRSDEIEHLVVEELVKKREECL;
抗原表位多肽19:VSFRRLSHLRKLVPGFGKAYTIFNK;
抗原表位多肽27:ADPSTVFKDGDEAEDFVEVHLPDVH。
优选地,本发明所述的融合蛋白N端含有一段可诱导IFN-γ的产生的多肽,所述多肽序列为KLTNKHSPE。
优选地,本发明所述的融合蛋白C端含有B细胞抗原表位,所述B细胞抗原表位序列为QARESKDNKIGKTENP。
再一方面,本发明还提供了一种疫苗,所述疫苗包含所述的融合蛋白和药学上可接受的佐剂。
优选地,本发明所述疫苗中的融合蛋白的含量为25μg/ml。
优选地,本发明所述药学上可接受的佐剂为ISA 201VG佐剂。
另一方面,本发明还提供了一种所述的融合蛋白在狂犬病毒疫苗制备中的应用。
本发明公开的融合蛋白具有良好的免疫原性,能够比常规的狂犬病毒G蛋白产生更高的抗体滴度,为狂犬病毒亚单位疫苗的研发提供了重要的参考。另外,本发明筛选到的狂犬病毒G蛋白的6条抗原表位多肽,具有较好的抗原性,为狂犬病毒G蛋白的研究提供了重要的参考,也为后续开发狂犬病毒G蛋白的单克隆抗体提供了重要的抗原线索。
附图说明
图1去除信号肽的G蛋白跨膜区预测。Inside表示胞内区,inside数值越大,表示该氨基酸位于胞内区的可能性越大;Outside表示胞外区,outside数值越大,表示该氨基酸位于胞外区的可能性越大。Transmembrane表示跨膜区,Transmembrane数值越大,表示该氨基酸在跨膜区的可能性越大。
图2融合蛋白的SDS-PAGE图。其中1为纯化后的融合蛋白。
图3融合蛋白制备疫苗免疫小鼠后抗体效价结果。
图4融合蛋白制备疫苗免疫小鼠后IFN-γ的含量检测结果。
具体实施方式
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。除非特别说明,以下实施例所用试剂和材料均为市购。
实施例1:狂犬病毒G蛋白抗原表位多肽设计和筛选
选择GenBank:MW055078.1的G蛋白,通过对该蛋白序列的分析和研究,去除N端的19氨基酸(信号肽序列)和C端的55个氨基酸(跨膜区和胞内区,见图2所示),设计合成了30条抗原表位多肽(表1)。将30条抗原表位多肽分别免疫小鼠,采集小鼠血清进行ELISA抗体效价检测,筛选抗体效价最高的抗原表位多肽。经过检测,共有6条抗原表位多肽的抗体效价最高(表1),因此,选择这6条抗原表位多肽(第4、第5、第12、第16、第19和第27)作为后续研究的重点。
使用常规的ELISA方法检测抗体效价,简述为:使用常规真核表达外源蛋白的方法制备G蛋白(参见CN 113252893 A实施例1所述)包被酶标板,100ng/孔,0.1ml/孔,4℃包被过夜;洗涤3次后,使用含2%的BSA的PBST封闭,37℃孵育2h;用PBS将小鼠血清做10倍系列稀释(10~107),再加到包被的酶标板中,0.1ml/孔,37℃孵育1h;洗涤3次后,加入1:5000倍稀释的HRP标记的羊抗鼠IgG二抗,0.1ml/孔,37℃孵育30min;洗涤3次后,加入0.1ml TMB单组分显色液(购自北京索莱宝生物科技有限公司),37℃孵育10min;加入终止液(2M硫酸),测定OD450nm值;当OD450nm值大于0.1时判定为阳性。
表1 G蛋白抗原表位多肽
实施例2:狂犬病毒G蛋白融合蛋白的设计和制备
合并实施例1筛选到的6条抗原表位多肽,两两之间通过“AAYAAY”进行连接,并在N端添加一段氨基酸序列(KLTNKHSPE,可诱导产生IFN-γ的产生),在C端添加一段氨基酸序列(QARESKDNKIGKTENP,为B细胞抗原表位之一),从而形成一条完整的融合蛋白,该融合蛋白的序列为:
KLTNKHSPEAAYAAYCTGVVTEAETYTNFVGYVTTTFKRKAAYAAYMAGDPRYEESLHNPYPDYHWLRTVKAAYAAYSKGSKTCGFVDERGLYKSLKGACKLAAYAAYIHDFRSDEIEHLVVEELVKKREECLAAYAAYVSFRRLSHLRKLVPGFGKAYTIFNKAAYAAYADPSTVFKDGDEAEDFVEVHLPDVHAAYAAYQARESKDNKIGKTENPHHHHHH。
将融合蛋白序列交付南京金斯瑞生物科技有限公司进行大肠杆菌密码子优化以及全基因合成;合成的基因连接到pET22b载体的BamHI和EcoRI的酶切位点之间,合成的的基因序列如SEQ ID NO.1所示。将重组质粒转化到E.coli BL21(DE3)宿主中,经过抗性筛选及重组质粒鉴定后做菌种保藏,将其作为表达重组蛋白的工程菌。
将上述工程菌以1%比例接种含20μg/ml卡纳霉素LB液体培养基,在37℃条件下以200r/min振荡培养2~4小时,至菌液OD600nm为0.6~0.8时,加入终浓度1.0mM IPTG继续诱导4小时。诱导好的重组菌5000g离心10分钟弃上清,沉淀部分使用PBS溶解10分钟后,用超声细胞破碎仪破碎。破碎后4℃离心(12000r/min)30分钟弃上清,沉淀包涵体部分用裂解液(50mM Tris,8M Urea,0.5M NaCl)裂解10分钟,12000r/min离心30分钟取上清过镍柱纯化。使用10倍柱体积裂解液清洗柱体;将上清加入镍柱中,蛋白液缓慢上样;10倍柱体积裂解液洗涤,去除杂蛋白;5倍柱体积洗脱液(50mM Tris,8M Urea,0.5M NaCl,300mM咪唑)洗脱收集纯化的融合蛋白于-70℃以下保存。融合蛋白使用SDS-PAGE蛋白凝胶进行电泳检验,在26KD左右有一条清晰条带,纯度大于90%(图2);使用BCA检测蛋白浓度,融合蛋白浓度为1.58mg/ml。
实施例3:狂犬病毒融合蛋白疫苗的制备和应用
疫苗制备:将实施例2制备的融合蛋白稀释至50μg/ml,按照质量比1:1的比例与ISA 201VG佐剂(购自法国赛彼克公司)乳化制备疫苗,疫苗中含融合蛋白浓度约为25μg/ml。按照现行《中国兽药典》附录进行检验,均无细菌、支原体和外源病毒污染;粘度为25.8cP,<200cP;吸取疫苗10ml加入离心管中,以3000r/min离心15分钟,管底均为析出,稳定性好。制备及检验合格的疫苗置于2~8℃保存备用。参照此疫苗制备方法,将实施例中用于ELISA检测的真核系统表达的G蛋白制备疫苗,以用于比对使用。
小鼠免疫:将6周龄的雌性Balb/c小鼠随机分组,每组5只,按照表2进行分组和免疫,于首次免疫前、首次免疫后14日(二免前)、首次免疫后28日(二免14日)采集小鼠血清,对小鼠血清效价和IFN-γ进行检测。
表2小鼠免疫分组
组别 | 免疫剂量和方式 | 抗原含量 | 免疫次数 |
组1(融合蛋白) | 0.2ml/只,腿部肌肉注射 | 5μg/只/次 | 免疫一次 |
组2(融合蛋白) | 0.2ml/只,腿部肌肉注射 | 5μg/只/次 | 间隔2周加强免疫一次 |
组3(G蛋白) | 0.2ml/只,腿部肌肉注射 | 5μg/只/次 | 免疫一次 |
组4(G蛋白) | 0.2ml/只,腿部肌肉注射 | 5μg/只/次 | 间隔2周加强免疫一次 |
对照组 | / | / | / |
使用常规的ELISA方法检测小鼠血清抗体效价,简述为:使用常规真核表达外源蛋白的方法制备G蛋白(参见CN 113252893 A实施例1所述)包被酶标板,100ng/孔,0.1ml/孔,4℃包被过夜;洗涤3次后,使用含2%的BSA的PBST封闭,37℃孵育2h;用PBS将小鼠血清做2倍系列稀释(100、200、400、800、1600......),再加到包被的酶标板中,0.1ml/孔,37℃孵育1h;洗涤3次后,加入1:5000倍稀释的HRP标记的羊抗鼠IgG二抗,0.1ml/孔,37℃孵育30min;洗涤3次后,加入0.1ml TMB单组分显色液(购自北京索莱宝生物科技有限公司),37℃孵育10min;加入终止液(2M硫酸),测定OD450nm值;当P/N值(样品OD450nm值/空白对照孔OD450nm值)大于2.1且样品ODOD450nm值>0.1时判定为阳性。
抗体检测结果显示(图3):两组单次免疫组相比,组1(融合蛋白)较组3(G蛋白)的抗体效价要高,且有显著性差异;两组二次免疫组相比,组2(融合蛋白)较组4(G蛋白)的抗体效价要高,且有显著性差异;单次免疫组和二次免疫组相比,二次免疫组的抗体效价要高。综合来看,本发明设计制备的融合多肽制备疫苗后具有更好的免疫效果。
参照试剂盒的使用说明(购自北京索莱宝生物科技有限公司的Mouse IFN-γELISA Kit)对血清中细胞因子IFN-γ的含量进行检测。结果显示(图4),两组单次免疫组相比,组1(融合蛋白)较组3(G蛋白)的要高,且有显著性差异;两组二次免疫组相比,组2(融合蛋白)较组4(G蛋白)的要高,且有显著性差异;单次免疫组和二次免疫组相比,二次免疫组的要高。综合来看,本发明设计制备的融合多肽制备疫苗后具有更好的免疫效果,能够诱导产生更高滴度的IFN-γ。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序 列 表
<110> 魏慢慢
<120> 一种融合蛋白及其制备方法和应用
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 669
<212> DNA
<213> 密码子优化后的融合蛋白的核苷酸序列
<400> 1
aaactgacca acaaacactc cccggaagcc gcgtatgctg cgtattgcac cggtgttgtt 60
accgaagcag aaacctatac caatttcgtg ggttacgtga ccacgacctt caagcgcaaa 120
gctgcatacg cggcttacat ggctggtgat ccgcgctatg aggaatccct gcacaacccg 180
tatcctgatt atcactggct gcgtactgtg aaagcggctt atgctgccta ctccaaaggc 240
tctaaaacct gcggttttgt tgatgaacgt ggcctgtaca aatctctgaa aggtgcttgc 300
aaactggctg cctacgctgc ttacatccat gatttccgtt ccgatgaaat tgaacacctg 360
gtggtcgaag aactggtgaa aaaacgtgaa gaatgcctgg ctgcatatgc tgcctacgtt 420
tccttccgtc gcctgagcca cctgcgcaaa ctggtgcctg gctttggtaa agcttatacc 480
atcttcaaca aagcggctta cgcagcgtac gcggacccat ctacggtttt caaagatggc 540
gacgaagcag aagacttcgt tgaagttcac ctgccagacg tgcacgctgc ttacgccgca 600
taccaggcac gcgaaagcaa agacaataaa atcggcaaaa ccgaaaaccc gcaccatcat 660
caccatcat 669
Claims (9)
1.一种融合蛋白,其特征在于,所述的融合蛋白的氨基酸序列为:
KLTNKHSPEAAYAAYCTGVVTEAETYTNFVGYVTTTFKRKAAYAAYMAGDPRYEESLHNPYPDYHWLRTVKAAYAAYSKGSKTCGFVDERGLYKSLKGACKLAAYAAYIHDFRSDEIEHLVVEELVKKREECLAAYAAYVSFRRLSHLRKLVPGFGKAYTIFNKAAYAAYADPSTVFKDGDEAEDFVEVHLPDVHAAYAAYQARESKDNKIGKTENPHHHHHH。
2.根据权利要求1所述的融合蛋白,其特征在于,所述的融合蛋白的密码子优化后的核苷酸序列如SEQ ID NO.1所示。
3.根据权利要求1所述的融合蛋白,其特征在于,所述的融合蛋白包括6条狂犬病毒G蛋白的抗原表位多肽,所述的6条狂犬病毒G蛋白的抗原表位多肽的序列分别为:
抗原表位多肽4:CTGVVTEAETYTNFVGYVTTTFKRK;
抗原表位多肽5:MAGDPRYEESLHNPYPDYHWLRTVK;
抗原表位多肽12:SKGSKTCGFVDERGLYKSLKGACKL;
抗原表位多肽16:IHDFRSDEIEHLVVEELVKKREECL;
抗原表位多肽19:VSFRRLSHLRKLVPGFGKAYTIFNK;
抗原表位多肽27:ADPSTVFKDGDEAEDFVEVHLPDVH。
4.根据权利要求1所述的融合蛋白,其特征在于,所述的融合蛋白N端含有一段可诱导IFN-γ的产生的多肽,所述多肽序列为KLTNKHSPE。
5.根据权利要求1所述的融合蛋白,其特征在于,所述的融合蛋白C端含有B细胞抗原表位,所述B细胞抗原表位序列为QARESKDNKIGKTENP。
6.一种疫苗,其特征在于,所述疫苗包含权利要求1所述的融合蛋白和药学上可接受的佐剂。
7.根据权利要求6所述的疫苗,其特征在于,所述疫苗中的融合蛋白的含量为25μg/ml。
8.根据权利要求6所述的疫苗,其特征在于,所述药学上可接受的佐剂为ISA 201VG佐剂。
9.一种如权利要求1~5任一权利要求所述的融合蛋白在狂犬病毒疫苗制备中的应用。
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CN116789858A (zh) * | 2023-06-26 | 2023-09-22 | 广东兴亚生物科技有限公司 | 一种伪狂犬病毒重组融合蛋白及其制备方法和应用 |
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CN116789858A (zh) * | 2023-06-26 | 2023-09-22 | 广东兴亚生物科技有限公司 | 一种伪狂犬病毒重组融合蛋白及其制备方法和应用 |
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