CN104877029A - 一种Vimentin-Fc融合蛋白及其应用 - Google Patents
一种Vimentin-Fc融合蛋白及其应用 Download PDFInfo
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Abstract
本发明公开了一种Vimentin-Fc融合蛋白及其应用,本发明利用分子生物学和细胞生物学技术,构建波形蛋白Vimentin与人IgG的Fc片段的融合蛋白,所述的融合蛋白包括中间丝的波形蛋白和人免疫球蛋白的Fc片段,波形蛋白与Fc片段之间直接融合或通过连接序列融合;本发明提供的融合蛋白可用于乙型脑炎病毒感染的预防或治疗或作为生物制剂用于Vimentin和Fc蛋白功能的相关科学研究。
Description
技术领域
本发明属于生物和医药技术领域,具体涉及一种新的重组Vimentin-Fc融合蛋白及其用途。
背景技术
日本脑炎病毒(Japanese encephalitis virus,JEV),我国又称乙脑病毒,属于黄病毒科黄病毒属。JEV引起的流行性乙型脑炎(乙脑),是一种严重的急性中枢神经系统传染病,主要流行于东亚、东南亚和南亚,临床以高热、惊厥、意识障碍及脑膜刺激征为主。JEV每年引起大约3~5万人感染,1~1.5万人死亡,幸存者中高达50%伴有严重的神经系统后遗症。乙脑是2004年我国卫生部公布的病死率最高的十大传染病之一,2006年山西等地再次出现疫情。近年来在一些乙脑非流行地区,如塞班岛、巴基斯坦和澳洲北部也有病例报道,表明乙脑已经扩大流行,并且有继续扩大流行的趋势。JEV更可作为生物战剂使用,其重要性不应忽视。
目前对JEV的病原学研究已较深入,病毒基因组和蛋白的结构与功能已基本清楚。但JEV宿主细胞上受体分子尚未完全明确,JEV的致病机制尚未阐明,正在使用的疫苗尚不理想,灭活疫苗免疫力不持久、伴随一些副作用,减毒活疫苗有潜在的毒力变异危险性。JEV的发病机理和抗病毒研究成为当前研究的重点和难点。
Vimentin是目前发现的JEV的重要潜在受体之一,研究证实,波形蛋白是一个能够与JEV病毒粒子相互作用的细胞表面分子,并且体外原核表达出的重组波形蛋白竞争性抑制JEV病毒的感染。因此对于JEV的感染起着关键作用。
“病毒受体陷阱(virus receptor trap)疗法”是指利用人工合成的可溶性病毒受体来结合病毒,进而阻断病毒与靶细胞膜上的受体结合及随后的病毒内吞。病毒受体陷阱已被成功应用于实验性小鼠CVB3心肌炎的治疗,且表现出良好的抗病毒效果。目前国内外基于病毒受体融合蛋白的“病毒受体陷阱”研究在其它病毒的研究中则非常有限。
该融合蛋白将JEV受体基因的cDNA融合入人IgGl的Fc区即构建成重组二聚体蛋白(Vimentin-Fc),Vimentin-Fc的Vimentin段相当于IgG的Fab区,因此Vimentin-Fc有可能作为JEV感染神经细胞的抑制剂。此融合蛋白的成功研制将为新型抗病毒药物开发提供思路,并对提高乙型脑炎的防控和治疗效果具有重要意义和实用价值。
发明内容
为了解决上述的技术问题,本发明的目的在于提供一种Vimentin-Fc融合蛋白,融合蛋白的氨基端是中间丝的波形蛋白,羧基端是人免疫球蛋白的Fc片段,波形蛋白与Fc片段之间直接融合或通过连接序列融合;所述Vimentin-Fc融合蛋白为P1-L-P2或P1-P2形式,其中P1为Vimentin蛋白全长,P2为人IgG Fc区段,L为连接肽。
所述波形蛋白氨基酸序列如SEQ ID NO:1所示。
所述人免疫球蛋白的Fc片段的氨基酸序列如SEQ ID NO:2所示。
所述的连接肽序列为GlySerGly4。
本发明另一目的在于提供编码上述Vimentin-Fc融合蛋白的基因序列;
本发明还提供了编码Vimentin全长区段、Linker与Fc融合蛋白的编码基因在融合后的核苷酸序列如SEQ ID NO:3所示。
本发明另一目的在于提供上述Vimentin-Fc融合蛋白在制备用于抗乙型脑炎病毒感染药物中的应用。
本发明的技术方案通过下述具体方法和步骤来实现:
a) 根据Vimentin和人IgG Fc基因序列分别设计引物,并分别在人IgG Fc和Vimentin基因的正向和反向引物中引入Linker蛋白对应的基因序列GGATCAGGCGGGGGTGGG,以便在随后的融合PCR中在Vimentin和人IgG Fc的融合基因间引入Linker序列;
b) 将步骤a)中的设计并合成的引物分别对成神经瘤细胞和人淋巴细胞的cDNA进行扩增,分别获得Vimentin和人IgG Fc基因片段后,再以两个基因片段为模板,以Vimentin正向引物和人IgG Fc基因反向引物为上下游引物,进行融合PCR扩增,获得Vimentin-Fc融合基因;
c) 利用融合基因两端引入的EcoRI和Not I酶切位点,将融合基因片段引入表达载体中,获得Vimentin-Fc融合蛋白的重组表达载体;
d) 将步骤c)中的重组表达载体转入宿主细胞,筛选获得表达Vimentin-Fc融合蛋白的工程菌;
e) 培养步骤d)获得的工程菌;
f) 从步骤e)中的培养产物中以亲和纯化的方式分离Vimentin-Fc融合蛋白,以PBS溶液进行蛋白复性。
所述步骤c)中的表达载体可以选用各种已知的载体,优选为载体pET32a或载体pPIC9K。
所述步骤d)中的宿主细胞优先为与载体pET32a对应的Rosetta菌或与载体pPIC9K对应的毕赤酵母菌。
本发明一个优选的实施例中,上述Vimentin-Fc融合蛋白的制备方法为:将筛选得到并测序正确的工程菌在LB培养基中培养条件为37℃,0.5mM IPTG条件下诱导表达4小时,9000rpm离心后,收集菌体,超声破碎,用8M尿素裂解过夜,再用Ni-NTA分离Vimentin-Fc融合蛋白。
本发明通过基因克隆和基因重组技术合成一种Vimentin-Fc融合蛋白,该融合蛋白中的Vimentin部分是Vimentin蛋白全长,通过其乙型脑炎病毒受体的功能来结合并达到捕获病毒的目的;而Fc部分则起效应功能,如抗体依赖的细胞毒作用和补体依赖的细胞毒作用,从而达到使捕获的病毒易于机体免疫系统识别和清除的作用。通过将Vimentin-Fc融合蛋白的基因克隆到一个表达载体中,转化受体细胞得到工程菌,扩大培养制备Vimentin-Fc融合蛋白。通过蛋白复性,使得Vimentin-Fc融合蛋白具有较高的生物活性。
附图说明
图1是本发明中重组质粒的电泳结果示意图;其中泳道M为DNA相对分子质量标记;泳道1为Vimentin-Fc融合基因菌液PCR结果;
图2是本发明中重组质粒的酶切鉴定结果示意图;其中泳道M为DNA相对分子质量标记;泳道1为双酶切鉴定重组质粒;
图3是本发明中蛋白的SDS-PAGE检测结果示意图;其中泳道M为蛋白相对分子质量标准;泳道1为未诱导菌液样品;泳道2为已诱导菌液样品;
图4是本发明中纯化后的蛋白的SDS-PAGE检测示意图;其中泳道M为蛋白相对分子质量标准;泳道1为未诱导菌液样品;泳道2为已诱导菌液样品;泳道3-7为纯化条带;
图5是本发明不同浓度Vimentin-Fc重组蛋白对JEV复制的影响结果。
具体实施方式
下面用实施例对本发明做进一步阐述,应当理解为,这些实施例仅用于说明本发明而非对本发明有任何限制,本领域技术人员在本说明书的启示下对本发明实施中所做的任何变动都将落在权利要求书的范围内。
实施例1:融合蛋白Vimentin-Fc的载体构建和蛋白表达
Vimentin-Fc融合蛋白的表达过程包括以下步骤:克隆基因;把基因插入表达载体,将表达载体转化进入Rosetta菌;利用表达载体的Kana抗性筛选出正确转入载体的Rosetta菌株,培养该菌株,利用IPTG作为诱导剂,诱导蛋白表达,最后从包涵体中分离纯化Vimentin-Fc蛋白,具体包括:
1、基因克隆
在基因合成的专业公司(上海生工生物工程技术服务有限公司)合成引物,利用引物通过融合PCR技术从人淋巴细胞和成神经瘤细胞中克隆SEQ ID NO:3所示的核苷酸序列。其中1-6是EcoR I的酶切位点;7-1404是编码中间丝的波形蛋白的核苷酸序列,1405-1422是插入的linker序列,1423-2091是编码IgG Fc片段的核苷酸序列;2091-2099是Not I的酶切位点。
1.1 Vimentin基因的获得
1.1.1 病毒培养
将培养瓶内中汇合率长至90%的成神经瘤细胞,以PBS溶液洗1遍,再以1ml Trizol裂解液加入裂解细胞。
1.1.2 RNA提取
将Trizol裂解的细胞静置5min,加200μl氯仿,剧烈震荡5s。室温静置5min,然后4℃,12000r/min离心15min。取上层水相移至另一个Ep管中。加入500μl异丙醇轻柔颠倒混匀,室温静置5min,13000r/min离心5min弃上清。加入1ml 75%乙醇洗涤沉淀,温和震荡离心管,使沉淀悬浮。13000r/min离心15min弃上清,室温晾干15min。加入30μl DEPC水溶解RNA,用spectro photometer检测RNA浓度。
1.1.3 反转录
使用RevertAidFirst Stand cDNA Synthesis Kit,按照使用说明操作,反应体系:
轻摇混匀,离心 65℃ 5min再放置冰上,加入反应体系:
轻弹混匀,42℃ 60min,即反转录为cDNA。
1.1.4 PCR反应体系:dNTP 4μl;rTaq 0.25μl;10×缓冲液 5μl;MgCl2:3μl;水:32.75μl;引物各2μl;DNA片段1μl,共50μl;循环参数:94℃×5min→(94℃×30s→58℃×30s→72℃×90s)×30→72℃×10min→4℃;
引物序列:F1:5- GGAATTCATGTCTACCAGGTCTGTGTC -3
R1:5- CCCACCCCCGCCTGATCCTTCAAGGTCATCGTGATGCT -3
1.2 mFc基因的获得
1.2.1淋巴细胞提取
取正常人的新鲜全血4.5ml,将全血及组织稀释液1:1混匀后小心加于等体积细胞分离液,随即呈现混合液在上、分离液在下的状态,2000r/min离心20min后溶液会分为四层,淋巴细胞层在第二层。收集第二层淋巴细胞放入含细胞洗涤液10ml的试管中,充分混匀后以1800r/min离心20min弃去上清,留沉淀重新悬浮起,重复洗涤2次即可得到所需淋巴细胞。
1.2.2 RNA提取
步骤同1.1.2;
1.2.3 反转录
步骤同1.1.3;
1.2.4 PCR反应体系
dNTP 4μl;rTaq 0.25μl;10×缓冲液 5μl;MgCl2:3μl;水:32.75μl;引物各2μl;DNA片段1μl,共50μl;循环参数:94℃×5min→(94℃×30s→65℃×30s→72℃×45s)×30→72℃×10min→4℃;
引物序列:F2:5- GGATCAGGCGGGGGTGGGGGTTGTAAGCCTTGCATATG -3
R2:5- ATAGTTTAGCGGCCGCTCATTTACCAGGAGAGTGGG -3;
1.3 用含有酶切位点的上下游引物对Vimentin基因与mFc基因进行重叠PCR,获得目的序列。
PCR反应体系:dNTP 4μl;rTaq 0.25μl;10×缓冲液 5μl;MgCl2:3μl;水:32.75μl;引物各2μl;DNA片段1μl,共50μl。循环参数:94℃×5min→(94℃×30s→58℃×30s→72℃×150s)×30→72℃×10min→4℃;
引物序列:F1:5- GGAATTCATGTCTACCAGGTCTGTGTC-3
R2:5- ATAGTTTAGCGGCCGCTCATTTACCAGGAGAGTGGG-3;
2、克隆载体和表达载体的构建
2.1 在用引物合成上述基因后,用EcoRI和Not I双酶切基因,反应体系如下所示:
37℃过夜。
2.2 琼脂糖凝胶电泳,分别回收基因和载体的酶切片段,采用小量琼脂糖凝胶DNA回收试剂盒,按试剂盒说明操作。
2.3 连接目的基因与载体,反应体系:
16℃连接过夜,构建克隆载体pMD-19T/Vimentin-Fc。将连接产物转化大肠杆菌LB平板培养。挑取多个克隆培养,扩增上述表达载体。抽提质粒后,用EcoR I和Not I双酶切表达载体pMD-19T/Vimentin-Fc和载体pET-28a,反应体系同2.1,分别回收基因和载体的酶切片段,连接目的基因与载体,反应体系同2.3,构建表达载体PET-28a/ Vimentin-Fc。图1中融合基因重组质粒基因片段与预期大小一致,图2融合基因的重组质粒双酶切出两条条带,大小与预期相符。
3、Vimentin-Fc融合蛋白的生产
利用LB培养基,在37℃,0.5mM IPTG条件下诱导表达4小时,9000 rpm离心后,收集菌体,超声破碎,用8M尿素裂解过夜,再用蛋白亲和层析分离纯化得到Vimentin-Fc融合蛋白(图3、4)。表达和纯化出的重组蛋白与预期大小一致。
实施例2 :Vimentin-Fc融合蛋白抑制乙型脑炎病毒复制实验
Vimentin-Fc融合蛋白的病毒感染实验包括以下步骤:细胞培养;病毒与纯化后的蛋白混合后感染细胞;培养48小时后裂解细胞并提取RNA进行荧光定量PCR,具体包括:
1、细胞培养
培养成神经瘤细胞至长满细胞瓶,利用胰酶消化细胞,加入培养基用枪头吹打混匀后吸取约104个/孔铺至24孔板中,培养过夜。
2、以设计的荧光定量PCR引物,从JEV的基因组RNA反转录出的cDNA作为模板,克隆基因片段至pMD-19T,抽提质粒测定浓度,作为荧光定量PCR基因模板。
3、病毒感染抑制实验
将纯化并复性的Vimentin-Fc融合蛋白过0.22nm滤膜除后,取200μl至4#灭菌EP管中,吸取100μl至3#EP管,加入等体积无菌DMEM培养基至3#管中,混匀后取100μl至2#EP管中,依次将重组蛋白稀释至3个梯度。再分别取40μl病毒液至每个EP管中,使最终MOI值为1,混匀室温静置作用30 min。将24孔板标号,2-4#加入等量病毒和不同浓度的蛋白,1#只加入等量的病毒,培养48小时后收取上清,提取RNA,反转录为cDNA,进行荧光定量PCR。
4、荧光定量PCR
采用SYBR Green I法进行荧光定量试验,以构建的pMD-19T重组质粒为模板进行绝对定量测定,阴性对照为不含模板基因的双蒸水,以ABI公司的7500荧光定量PCR仪进行,分析不同样品中病毒的拷贝数差异。
如图5所示,融合蛋白Vimentin-Fc融合蛋白具有明显的抑制JEV病毒复制的作用,且呈现出一定的浓度依赖效应,在重组蛋白终浓度为2145 ng/ml时,对JEV的抑制率达到99%以上,具有极显著的差异。
序列表
<110> 昆明理工大学
<120> 一种Vimentin-Fc融合蛋白及其应用
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 460
<212> PRT
<213> 成神经瘤细胞
<400> 1
Met Ser Thr Arg Ser Val Ser Ser Ser Ser Tyr Arg Arg Met Phe Gly
1 5 10 15
Gly Pro Gly Thr Ser Asn Arg Gln Ser Ser Asn Arg Ser Tyr Val Thr
20 25 30
Thr Ser Thr Arg Thr Tyr Ser Leu Gly Ser Ala Leu Arg Pro Ser Thr
35 40 45
Ser Arg Ser Leu Tyr Ser Ser Ser Pro Gly Gly Ala Tyr Val Thr Arg
50 55 60
Ser Ser Ala Val Arg Leu Arg Ser Ser Met Pro Gly Val Arg Leu Leu
65 70 75 80
Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu Phe
85 90 95
Lys Asn Thr Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp
100 105 110
Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn
115 120 125
Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Gly Lys Ser
130 135 140
Arg Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln
145 150 155 160
Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu Val Glu Arg Asp
165 170 175
Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu
180 185 190
Met Leu Gln Arg Glu Glu Ala Glu Ser Thr Leu Gln Ser Phe Arg Gln
195 200 205
Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val
210 215 220
Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys Lys Leu His Asp Glu
225 230 235 240
Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln Glu Gln His Val Gln Ile
245 250 255
Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val
260 265 270
Arg Gln Gln Tyr Glu Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu
275 280 285
Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg
290 295 300
Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln Glu Ser Asn Glu Tyr Arg
305 310 315 320
Arg Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr
325 330 335
Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala
340 345 350
Leu Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Arg Leu Gln Asp Glu
355 360 365
Ile Gln Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln
370 375 380
Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr
385 390 395 400
Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro
405 410 415
Asn Phe Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu Glu Ser Leu Pro
420 425 430
Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu Ile Lys Thr Val Glu
435 440 445
Thr Arg Asp Gly Gln Val Ile Asn Glu Thr Ser Gln
450 455 460
<210> 2
<211> 222
<212> PRT
<213> 人淋巴细胞
<400> 2
Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe
1 5 10 15
Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro
20 25 30
Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val
35 40 45
Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr
50 55 60
Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu
65 70 75 80
Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys
85 90 95
Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser
100 105 110
Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro
115 120 125
Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile
130 135 140
Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly
145 150 155 160
Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp
165 170 175
Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp
180 185 190
Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His
195 200 205
Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
210 215 220
<210> 3
<211> 2099
<212> DNA
<213> 人工合成
<400> 3
gaattcatgt ctaccaggtc tgtgtcctcg tcctcttacc gcaggatgtt cggtggcccc 60
ggcacctcga accggcagag ctccaaccgg agctatgtga ccacgtccac ccgcacctac 120
agcctgggca gcgcactgcg ccccagcacc agtcgcagcc tctattcctc atctcccggt 180
ggcgcctatg tgacccgatc ctctgcggtg cgcctgcgga gcagcatgcc cggcgtgagg 240
ctgctgcagg actcggtgga cttctcgctg gccgacgcca ttaacaccga gttcaagaac 300
acccgcacca acgagaaggt agaactgcag gagctgaatg accgcttcgc caactacatc 360
gacaaggtgc gcttcctgga gcagcagaac aaaatcctgc tagccgagct cgagcaactc 420
aagggtcagg gcaagtcgcg cctgggcgac ctctatgagg aggagatgcg ggagctgcgc 480
cggcaggtgg atcagctcac caacgacaag gcacgcgtcg aggtggagcg tgacaacctg 540
gctgaggaca tcatgcggct gcgagaaaaa ttgcaggagg agatgctcca gagagaggaa 600
gcggagagca ccctgcagtc cttcagacag gatgttgaca atgcctctct ggcacgcctc 660
gaccttgaac gtaaagtgga atccttgcaa gaagagattg cctttttgaa gaaactgcat 720
gatgaagaga tccaggagct acaggcccag attcaggagc aacatgtcca gattgacgtg 780
gatgtttcta agcccgacct cactgctgcc ctgcgcgatg tccgccagca gtatgaaagt 840
gtggctgcca agaacctcca ggaggcggag gaatggtaca agtccaagtt tgccgacctc 900
tctgaagctg ccaaccggaa caatgatgcc ctgcgccagg caaagcagga gtcaaatgag 960
taccggagac aggtgcagtc actcacctgc gaagtggatg cacttaaagg aactaatgag 1020
tctctggaac gccagatgcg tgagatggaa gagaattttg cccttgaagc tgctaactac 1080
caggacacta ttggccgcct gcaggatgag attcagaaca tgaaggaaga gatggctcgt 1140
caccttcgtg aataccaaga cctgctcaat gtcaagatgg ctcttgacat tgagattgcc 1200
acctacagga agctactgga aggcgaggag agcaggattt ctctgcctct tcccaacttt 1260
tcttccctga acctgagaga aactaatctg gagtcactcc ctctggttga cacccactca 1320
aaaagaacac tcctgattaa gacagtggaa accagggatg gacaggtgat caatgaaacc 1380
tctcagcatc acgatgacct tgaaggatca ggcgggggtg ggggttgtaa gccttgcata 1440
tgtacagtcc cagaagtatc atctgtcttc atcttccccc caaagcccaa ggatgtgctc 1500
accattactc tgactcctaa ggtcacgtgt gttgtggtag acatcagcaa ggatgatccc 1560
gaggtccagt tcagctggtt tgtagatgat gtggaggtgc acacagctca gacgcaaccc 1620
cgggaggagc agttcaacag cactttccgc tcagtcagtg aacttcccat catgcaccag 1680
gactggctca atggcaagga gttcaaatgc agggtcaaca gtgcagcttt ccctgccccc 1740
atcgagaaaa ccatctccaa aaccaaaggc agaccgaagg ctccacaggt gtacaccatt 1800
ccacctccca aggagcagat ggccaaggat aaagtcagtc tgacctgcat gataacagac 1860
ttcttccctg aagacattac tgtggagtgg cagtggaatg ggcagccagc ggagaactac 1920
aagaacactc agcccatcat ggacacagat ggctcttact tcgtctacag caagctcaat 1980
gtgcagaaga gcaactggga ggcaggaaat actttcacct gctctgtgtt acatgagggc 2040
ctgcacaacc accatactga gaagagcctc tcccactctc ctggtaaatg agcggccgc 2099
Claims (6)
1.一种Vimentin-Fc融合蛋白,其特征在于:融合蛋白包括中间丝的波形蛋白和人免疫球蛋白的Fc片段,波形蛋白与Fc片段之间直接融合或通过连接序列融合。
2.根据权利要求1所述的Vimentin-Fc融合蛋白,其特征在于:所述波形蛋白氨基酸序列如SEQ ID NO:1所示。
3.根据权利要求1或2所述的Vimentin-Fc融合蛋白,其特征在于:所述人免疫球蛋白的Fc片段的氨基酸序列如SEQ ID NO:2所示。
4.编码权利要求1-3中任一项所述Vimentin-Fc融合蛋白的核酸分子。
5.根据权利要求4所述的核酸分子,其特征在于,其核苷酸序列如SEQ ID NO:3所示。
6.权利要求1所述的Vimentin-Fc融合蛋白在制备抗乙型脑炎病毒感染药物中的应用。
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Cited By (1)
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CN112218884A (zh) * | 2018-05-07 | 2021-01-12 | 免疫分子中心 | 由白介素-2突变蛋白和i型干扰素构成的融合蛋白 |
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CN101260157A (zh) * | 2008-04-30 | 2008-09-10 | 中国人民解放军第三军医大学 | 融合多肽及其用于制备抗肿瘤、抗病毒和胞内寄生菌感染药物的应用 |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN112218884A (zh) * | 2018-05-07 | 2021-01-12 | 免疫分子中心 | 由白介素-2突变蛋白和i型干扰素构成的融合蛋白 |
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