CN102210860B - 一种结核分枝杆菌tb10.4-f1融合蛋白疫苗及制备方法 - Google Patents
一种结核分枝杆菌tb10.4-f1融合蛋白疫苗及制备方法 Download PDFInfo
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Abstract
本发明公开了一种制备结核分枝杆菌TB10.4-F1融合蛋白疫苗及其制备方法,该方法将结核分枝杆菌TB10.4蛋白基因与呼吸道合胞病毒蛋白的F1蛋白基因连接在一起,转化到大肠杆菌表达菌株中,经IPTG诱导表达TB10.4-F1融合蛋白,并通过金属离子亲和层析的方法获得了纯度较高的蛋白;该融合蛋白具有较强的免疫原性,并能激发出较为平衡的Th1/Th2免疫反应,能预防结核分枝杆菌及呼吸道合胞病毒的感染。
Description
技术领域
本发明涉及生物技术领域,特别是一种利用大肠杆菌细胞表达结核分枝杆菌融合蛋白TB10.4-F1的制备方法。
背景技术
结核分枝杆菌(Mycobacterium tuberculosis,MTB) 和人类呼吸道合胞病毒(Human Respiratory Syncytial virus, RSV)都是通过呼吸道侵染机体并引起一系列呼吸道疾病的病原体。
MTB可引起人类结核病,以肺结核最为多见,该病每年引致死亡的人数有200万到300万。鉴于结核对公众健康所造成的威胁,研究对抗和控制结核感染的新方法已经成为国际热点研究问题。世界卫生组织已在多个地区推广全程督导短程化疗(DOTS)计划,但仍未能控制住结核病的全球性流行,也未能预防高耐药性结核菌株(MDR)的出现和增长。目前应用最广的结核疫苗是卡介苗,虽然它在儿童上有很好的保护效果,但却不能防止结核的隐性感染和成人肺结核的复发(Fine PE,Lancet,1995,346(8986):1339-1345)。
结核分枝杆菌属于分枝杆菌属,细胞壁脂质含量较高。侵入肺部后,即使被肺泡内的吞噬细胞所吞噬,也可以有效抵抗溶菌酶而继续在细胞内繁殖。尽管机体可以对结核分枝杆菌产生抗体,但抗体只能通过直接接触细菌的方式去杀灭菌体,而对于胞内菌则不能直接作用。因此研究新型结核疫苗的关键在于提高T细胞介导的细胞免疫,加强CD8+细胞毒性T细胞介导的杀伤被感染宿主细胞CTL效应,杀伤靶细胞进而释放病原体,从而使体液免疫有效发挥作用。
MTB分泌蛋白是目前发现的保护性最好的一组抗原蛋白。动物实验证明,将结核杆菌短期培养过滤液 (short-term culture filtrate,ST-CF)作为抗原免疫豚鼠,可使动物获得抗结核的免疫保护作用(Horwitz MA et al,Proc Natl Acad Sci USA, 1995, 92(5): 1530-1534)。ST-CF中主要成分便是MTB生长早期分泌到胞外的一些蛋白混合物,能在感染初期便被患者的淋巴细胞识别,有效提高T细胞介导的细胞免疫,加强CD8+细胞毒性T细胞介导的杀伤被感染宿主细胞CTL效应,杀伤靶细胞进而释放病原体,从而使体液免疫有效发挥作用。这些蛋白便成为研制结核新疫苗的靶抗原。近年来研究较多的有Ag85复合物、ESAT-6家族以及MPT-64等。
RSV是全世界范围内婴幼儿下呼吸道感染最重要的病原体,免疫缺陷病人及老年人也是RSV的易感人群。临床表现以低热、下呼吸道感染病变多见,婴幼儿多呈阵咳、憋喘症状,甚至死亡。只有采用特异抗体进行被动免疫治疗,但给患儿和家长带来极大的痛苦和财产损失。世界卫生组织(WHO)早已将RSV 疫苗列为全球疫苗发展计划中优先发展的疫苗之一。
公知的RSV疫苗有灭活疫苗、减毒活疫苗、亚单位疫苗。60年代使用RSV灭活疫苗免疫儿童时可以诱导中和抗体的产生,但不具保护性,而且疫苗使用后增强了幼儿后继感染野毒株RSV的发病程度,住院治疗和死亡率显著提高。而RSV的减毒活疫苗给予1~2月龄未感染过RSV的婴儿时,会导致上呼吸道的轻微至中度充血,表明还需要进一步减毒。虽然减毒活疫苗的临床试验没有引起疫苗增强疾病,但减毒活疫苗仍存在免疫原性差、毒力令人难以接受和遗传不稳定性,人们对使用该疫苗仍有疑虑。
亚单位疫苗被认为是使用很安全的,不存在减毒活疫苗的毒力返祖和免疫原性差等问题,没有RSV灭活疫苗使用后病情加重的担忧。RSV囊膜上的膜融合(F)和粘附(G)糖蛋白可以诱导RSV中和抗体,成为病毒疫苗研究开发的重要蛋白。相比较而言,G蛋白为体液免疫的保护性抗原,而F蛋白既可诱发体液免疫也可以诱发一定的细胞免疫,且同源性较高。所以在RSV的保护性抗原中,F蛋白比G蛋白更重要。F蛋白是一个典型的 型糖蛋白,其全长包括574个氨基酸,其中含F2区(AA1-109) 、裂解多肽(AA110-136)和F1区(AA137-574),F1和F2通过二硫键相连接。F1蛋白的疏水性N端负责病毒与靶细胞膜和靶细胞膜之间的融合,它包括两个七肽重复区(HR1和HR2)。因为HR序列可以与脂质相结合,因此认为它们可能通过分别与病毒包膜和靶细胞膜的结合形成发夹结构来促进融合(Scott PD et al,J Infect Dis, 2006, 193(1): 59-67)。所以F1对于F蛋白的融合活性具有至关重要的作用。
TB10.4属于结核分枝杆菌早期分泌蛋白中的TB10.4 家族,是免疫主导的蛋白,在分枝杆菌的感染过程中能刺激机体产生抗体,促进IFN-γ的分泌(Skjot RL, et al,Infect Immun, 2002, 70(10): 5446-5453 )。因此TB10.4在机体中有强烈的Th1型反应。而在RSV 感染过程中,机体免疫Th1/Th2 处于失衡状态,Th2 类细胞因子表达和释放增强,Th1 类细胞因子相对受抑制。Th2 极化是RSV 导致哮喘的主要原因,所以在婴儿期要维持Th1/Th2平衡,以避免哮喘的发生。鉴于F1蛋白和TB10.4蛋白在诱导机体免疫失衡方面的不足,设想利用基因工程手段,将结核分枝杆菌早期分泌抗原TB10.4蛋白基因与呼吸道合胞病毒F1蛋白基因融合,构建融合基因,在大肠杆菌中表达TB10.4-F1融合蛋白,发挥各自的免疫优势,达到免疫平衡效果。
重组融合蛋白中,TB10.4位于N端,F1位于C端,中间用linker连接,从而增加融合蛋白的柔韧性,有利于两种蛋白的正确折叠。连接子的氨基酸序列为:GAGSGA。
因此,本发明用TB10.4-F1融合蛋白免疫机体,提供一种能够平衡机体体液免疫和细胞免疫的结核分枝杆菌融合蛋白疫苗。
发明内容
本发明提供一种结核分枝杆菌TB10.4-F1融合蛋白疫苗,旨在平衡亚单位疫苗激发的体液免疫和细胞免疫,用于人体或动物预防接种,抵抗结核分枝杆菌及其呼吸道合胞病毒的感染,TB10.4-F1融合蛋白碱基序列如SEQ ID NO.1所示,氨基酸序列如SEQ ID NO.2所示。
本发明另一目的在于提供一种结核分枝杆菌TB10.4-F1融合蛋白疫苗的制备方法,包括如下步骤:
1)重组亚克隆载体TB10.4(N/B)/pMD18T和F1(B/X)/pMD18T的构建
以结核分枝杆菌标准菌株H37Rv全基因组为模板,PCR扩增获得TB10.4(N/B)基因,并引入NdeⅠ和BamHⅠ酶切位点。以F/pMD18T质粒为模板,PCR扩增获得F1(B/X)基因,并引入BamHⅠ和XhoⅠ酶切位点和6×His标签,扩增产物胶回收纯化后与pMD18T载体连接,获得重组亚克隆载体。
(2)重组表达载体TB10.4-F1/pET30a的构建
TB10.4(N/B)/pMD18T质粒经NdeⅠ和BamHⅠ双酶切后得到带有粘性末端的目的基因TB10.4(N/B),并与同样双酶切过的表达载体pET30a相连接,形成重组表达载体TB10.4(N/B)/pET30a。
F1(B/X)/pMD18T质粒经BamHⅠ和XhoⅠ双酶切后得到带有粘性末端的目的基因F1(B/X),并与同样双酶切过的重组载体TB10.4(N/B)/pET30a相连接,即形成重组表达载体TB10.4-F1/pET30a。而TB10.4(N/B)和F1(B/X)之间则以Linker相连,重组融合蛋白基因碱基序列以SEQ ID NO.1所示。
(3)TB10.4-F1蛋白的诱导表达及其纯化
重组表达载体TB10.4-F1/pET30a转化到大肠表达菌株中,形成重组工程菌。重组工程菌经过发酵培养,IPTG诱导表达TB10.4-F1融合蛋白。采用超声破菌,离心,最后通过金属离子亲合层析纯化,4℃下,采用梯度透析复性的方法对已纯化好变性蛋白复性,即得到疫苗蛋白,氨基酸序列以SEQ ID NO.2所示。
本发明所述的方法中大肠杆菌表达载体为质粒pET30a及其适用于大肠杆菌系统的pET系列表达载体。
本发明所述的方法中大肠杆菌表达菌株为E.coli BL21(DE3)、E.coli BL21(DE3) pLys、Origami或Rosetta。
本发明所述的方法中转化大肠杆菌表达菌株的方法为热休克转化法,电转化法或原生质体转化法。
本发明所述的方法中金属离子亲合层析纯化法为镍离子亲和层析法。
与现有技术相比,本发明具有以下有益效果:
(1)在RSV疫苗的临床试验中,F蛋白亚单位疫苗可激发机体的体液免疫应答,而细胞免疫应答不强,造成Th1/Th2失衡,容易引发哮喘,限制了该疫苗的进一步开发。相反,TB10.4蛋白可激发机体的细胞免疫应答,体液免疫应答不强。所以,本发明将结核分枝杆菌TB10.4蛋白与呼吸道合胞病毒F1蛋白用linker连接,增加两种蛋白的柔韧性,使正确折叠,发挥各自的免疫优势,达到体液免疫和细胞免疫的平衡。
(2)在大肠杆菌中原核表达融合蛋白TB10.4-F1,可以提高蛋白疫苗的表达水平。由于大肠杆菌结构简单、生长快速、易于培养和发酵、生产成本低、目的蛋白产量高,这对于规模化生产及其临床应用具有重要的现实意义。
附图说明
图1是TB10.4(N/X),TB10.4(N/B),F1(B/X)基因PCR扩增产物电泳图。A是PCR扩增获得的TB10.4(N/X)片段(M:DNA Marker DL5000;1:TB10.4(N/X)目的基因片段);B是PCR获得TB10.4(N/B)和F1(B/X)片段(M:DNA Marker DL2000;1和2:TB10.4(N/B)基因片段;3和4:F1(B/X)基因片段)。
图2是重组亚克隆载体TB10.4(N/X)/pMD18T,TB10.4(N/B)/pMD18T和F1(B/X) /pMD18T的双酶切鉴定电泳图。A是TB10.4(N/X)/pMD18T双酶切鉴定图(M:DNA Marker DL2000;1:TB10.4(N/X)/pMD18T原始质粒;2:TB10.4(N/X)/pMD18T质粒NdeⅠ和XhoⅠ双酶切结果;3:TB10.4(N/X)/pMD18T质粒BamHⅠ和XhoⅠ双酶切结果);B是TB10.4(N/B)/pMD18T双酶切鉴定图(M:DNA Marker DL5000;1:TB10.4(N/B)/pMD18T原始质粒;2:TB10.4(N/B)/pMD18T质粒NdeⅠ和BamHⅠ双酶切结果);C是F1(B/X)/pMD18T双酶切鉴定图(M:DNA Marker DL2000;1:F1(B/X)/pMD8T质粒BamHⅠ和XhoⅠ双酶切结果)。
图3是重组表达载体TB10.4/pET28a,TB10.4(N/B)/pET30a,TB10.4-F1/pET30a的双酶切鉴定电泳图。A是TB10.4/pET28a的双酶切鉴定图(M:DNA Marker DL5000;1:TB10.4/pET28a质粒NdeⅠ和XhoⅠ双酶切结果);B是TB10.4(N/B)/pET30a双酶切鉴定图(M:DNA Marker DL5000;1:TB10.4(N/B)/pET30a的原始质粒;2:TB10.4(N/B) )/pET30a质粒NdeⅠ和BamHⅠ双酶切结果);C是TB10.4-F1/pET30a双酶切鉴定图(M:DNA Marker DL5000;1: TB10.4-F1/pET30a质粒XbaⅠ和XhoⅠ双酶切结果)。
图4是TB10.4蛋白和TB10.4-F1蛋白的SDS-PAGE检测和Western blot检测图。A是SDS-PAGE检测TB10.4蛋白(M:蛋白分子量标准;1:未诱导的TB10.4/pET28a/BL21全菌蛋白;2:诱导后的TB10.4/pET28a/BL21全菌蛋白;3:诱导后的TB10.4/pET28a/BL21破菌上清;4:诱导后的TB10.4/pET28a/BL21破菌沉淀);B是SDS-PAGE检测TB10.4-F1蛋(M:蛋白分子量标准;1:诱导后的pET30a/BL21全菌蛋白;2:未诱导的TB10.4-F1/pET30a/BL21全菌蛋白;3:诱导后的TB10.4-F1/pET30a/BL21全菌蛋白;4:诱导后TB10.4-F1/pET30a/BL21
破菌上清;5:诱导后的TB10.4-F1/pET30a/BL21破菌沉淀);C是Western blot检测TB10.4蛋白和TB10.4-F1蛋白(M:蛋白分子量标准;1:重组TB10.4蛋白;2:阴性对照;3:重组TB10.4-F1蛋白;4:阴性对照)。
图5是TB10.4蛋白和TB10.4-F1蛋白的纯化结果的SDS-PAGE检测图。A是TB10.4蛋白的纯化结果(M:蛋白分子量标准;1:未纯化样品;2:流穿峰;3:0.05mol/L 咪唑洗脱峰;4:0.3mol/L咪唑洗脱峰);B是TB10.4-F1蛋白的纯化结果(M:蛋白分子量标准;1:未纯化样品;2:流穿峰;3:0.05mol/L 咪唑洗脱峰;4:0.08mol/L咪唑洗脱峰;5:0.3mol/L咪唑洗脱峰)。
图6是动物免疫指标分析图。A是三次免疫后血清中总IgG的变化;B是免疫结束后检测IgG1/IgG2a比值。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明。这些实施例仅用于举例说明本发明,而不对本发明的范围构成任何限制。
实施例1: TB10.4(N/X),TB10.4(N/B),F1(B/X)基因的获得。
以结核分枝杆菌标准菌株H37Rv的全基因组为模板,用TaqPlus DNA聚合酶(来源于TIANGEN公司,J8422)扩增TB10.4基因,采用的引物如下:THP1: 5’-CATATGTCGCAAATCATGTACAACTACCC -3’,THP2: 5’-GCGCCGGATCCGGCGCCGCCGCCCCATTTGGCGGCTTCGGCCGTGTC-3’,TBP2: 5'-CTCGAGTTAGTGGTGGTGGTGGTGGTGGCCGCCCCATTTGGCGGCTTC-3'。其中,THP1和THP2用于扩增TB10.4(N/B)基因,并引入NdeⅠ(来源于TAKALA公司,CK8852A),BamHⅠ(来源于TAKALA公司,CK8701A)两个酶切位点以及linker序列,用以构建融合基因TB10.4-F1;THP1和TBP2用于扩增TB10.4(N/X)基因,并引入NdeⅠ和XhoⅠ(来源于TAKALA公司,CK1701A)两个酶切位点和6×His标签,用于单独表达TB10.4蛋白;PCR扩增条件为:95℃ 10min;95 ℃ 30s,52℃30s,72℃ 20s (30个循环);72℃ 10min;回收扩增产物,1%琼脂糖凝胶电泳鉴定,扩增得到了大小为310bp的TB10.4(N/X)基因(见附图1 A)和TB10.4(N/B)基因(见附图1 B)。
扩增F(B/X)/18T/DH5α菌株(本实验室保存,含有RSV融合蛋白F的全基因),然后采用博大泰克公司质粒抽提试剂盒提取质粒(以下质粒抽提均按说明书方法操作),以此质粒为模板,用TIANGEN公司的TaqPlus DNA聚合酶扩增F1(B/X)基因,引入了BamHⅠ,NdeⅠ两个酶切位点以及6×His标签,采用的引物序列为:F1P1: 5’- ccGGATCCGGCGCCTTTCTTGGCTTTTTGTTAG-3’,F1P2: 5’-GGCTCGAGTTAGTGGTGGTGGTGGTGGTGGTTACTAAATGCAATATTAT-3’;PCR扩增条件为:95℃ 10min;95 ℃ 30s,52℃ 30s,72℃ 1min30s(30个循环);72℃ 10min;回收扩增产物,1%琼脂糖凝胶电泳鉴定,扩增得到了大小为1340bp的F1(B/X)基因(见附图1 B)。
实施例2:重组亚克隆载体TB10.4(N/X)/pMD18T,TB10.4(N/B)/pMD18T和F1(B/X) /pMD18T的构建。
采用博大泰克公司的胶回收试剂盒纯化TB10.4(N/X),TB10.4(N/B)和F1(B/X)基因(以下胶回收纯化均按说明书方法操作)后,各取50ng分别与50ng的pMD18T(来源于TAKALA公司,CK5001BA)用1μl的T4 DNA连接酶(来源于TAKALA公司,CK5021B)于16℃连接过夜,连接产物通过热休克法转化用CaCl2制备的E.coli DH5α,涂布Amp+-LB平板,37℃培养16小时,挑取数个单菌落接种Amp+-LB培养液,37℃振荡培养12小时后抽提质粒。用NdeⅠ和XhoⅠ双酶切重组载体TB10.4(N/X)/pMD18T(重组质粒:700ng;酶:各1μl;20μl总体系,置于37℃作用3h)后,可以得到大小为310bp的目的片段,230bp的条带是从pMD18T上切下的一段(见附图2 A);用NdeⅠ和BamHⅠ双酶切重组载体TB10.4(N/B)/pMD18T(重组质粒:700ng;酶:各1μl;20μl总体系,置于37℃作用3h)后,可以得到大小为310bp的目的片段,同样也从pMD18T上切下230bp大小的片段(见附图2 B);用BamHⅠ和XhoⅠ双酶切重组载体F1(B/X)/pMD18T(重组质粒:600ng;酶:各1μl;30μl总体系,置于37℃作用5h)后,可以得到大小为1340bp的目的片段及其相应载体片段(见附图2 C),重组亚克隆载体构建成功。
实施例3:重组表达载体TB10.4/pET28a,TB10.4(N/B)/pET30a,TB10.4-F1/pET30a的构建。
重组表达载体TB10.4/pET28a的构建:将构建正确的TB10.4(N/X) /pMD18T及pET28a质粒用NdeⅠ和XhoⅠ于37℃酶切3小时,取胶回收纯化的TB10.4(N/X)片段50ng与纯化的表达载体pET28a双酶切产物150ng用1μl的T4 DNA连接酶于16℃连接过夜,通过热休克法转化用CaCl2制备的E.coli DH5α,涂布Kan+-LB平板,37℃培养16小时,挑取数个单菌落接种Kan+-LB培养液,37℃振荡培养12小时后抽提质粒。用NdeⅠ和XhoⅠ双酶切(重组质粒2μg;酶:各2μl;50μl总体系置于37℃作用5h)后,可以得到大小为310bp的目的片段及其相应载体片段(见附图3 A),说明重组表达载体TB10.4/pET28a构建成功。构建好的重组质粒采用上述方法再次转化E.coli BL21(DE3)用于诱导表达TB10.4蛋白。
重组表达载体TB10.4(N/B)/pET30a的构建:将构建正确的TB10.4(N/B)/pMD18T及pET30a用NdeⅠ和BamHⅠ于37℃酶切3小时,胶回收纯化TB10.4(N/B)/片段50ng与纯化的表达载体pET30a双酶切产物150ng用1μl的T4 DNA连接酶于16℃连接过夜,通过热休克法转化用CaCl2制备的E.coli DH5α,涂布Kan+-LB平板,37℃培养16小时,挑取数个单菌落接种Kan+-LB培养液,37℃振荡培养12小时后抽提质粒。用NdeⅠ和BamHⅠ双酶切(重组质粒:2μg;酶:各2μl;50μl总体系置于37℃作用5h)后,可以得到大小为310bp的目的片段及其相应载体片段(见附图3 B),说明重组表达载体TB10.4(N/B)/pET30a构建成功,构建好的重组质粒用于构建TB10.4-F1融合基因。
重组表达载体TB10.4-F1/pET30a的构建:将构建正确的TB10.4(N/B)/pET30a及F1(B/X)/pMD18T用BamHⅠ和XhoⅠ于37℃酶切3小时,胶回收纯化F1(B/X)片段100ng与纯化的TB10.4(N/B)/pET30a双酶切产物100ng用1μl的T4 DNA连接酶于16℃连接过夜,通过热休克法转化用CaCl2制备的E.coli DH5α,涂布Kan+-LB平板,37℃培养16小时,挑取数个单菌落接种Kan+-LB培养液,37℃振荡培养12小时后抽提质粒。用XbaⅠ(来源于TAKALA公司,CK3201B)和XhoⅠ双酶切(重组质粒:1.5μg;酶:各1.5μl;50μl总体系置于37℃作用7h)后,可以得到大小为1640bp的目的片段及其相应载体片段(见附图3 C),说明重组表达载体TB10.4-F1/pET30a构建成功。构建好的重组质粒采用上述方法再次转化E.coli BL21(DE3)用于诱导表达TB10.4-F1融合蛋白。
实施例4:TB10.4蛋白和TB10.4-F1蛋白的诱导表达。
分别挑取重组工程菌TB10.4/pET28a/BL21和TB10.4-F1/pET30a/BL21单菌落接种到Kan+-LB培养液中,37℃震荡培养过夜,第二天按1%分别接种到新鲜Kan+-LB培养液中,37℃震荡培养至OD600约为0.6时,取适当样品后分别加入终浓度为1mmol/L IPTG(来源于青岛生工公司,102189)在37℃下诱导表达4小时,诱导结束后取适量样品进行SDS-PAGE鉴定其大小是否正确,并用anti-His以1:1000稀释后作一抗(来源于中衫金桥公司,91123)Western blot鉴定。TB10.4蛋白和TB10.4-F1蛋白的表观分子量分别是13.3KDa(见附图4 A)和59.6KDa(见附图4 B),与理论计算值基本一致,均为包涵体表达形式;Western blot鉴定,目的蛋白能被anti-His特异性识别(见附图4 C)。说明目的片段即为重组蛋白。
实施例5:TB10.4及其TB10.4-F1蛋白的纯化。
TB10.4蛋白的纯化:诱导表达的重组菌液经过离心收集菌体,用TE缓冲液重悬,高压破菌,留沉淀弃上清,将沉淀溶于结合缓冲液(0.5mol/L NaCl,0.05mol/L Tris-Cl, 8mol/L 尿素,0.01mM咪唑,pH9.0)中,选择GE公司的His TrapTM HP凝胶柱进行镍离子亲和层析纯化,用洗涤缓冲液(0.1mol/L NaCl,0.05mol/L Tris-Cl,8mol/L 尿素,0.05mol/L咪唑,pH9.0)洗涤杂蛋白,使用洗脱缓冲液(0.1mol/L NaCl,0.05mol/L Tris-Cl,8mol/L 尿素,0.3mol/L咪唑,pH9.0)洗脱目的蛋白,SDS-PAGE检测并用软件BandScan分析,纯度为95%(见附图5 A)。
TB10.4-F1蛋白的纯化:诱导表达的重组菌液经过离心收集菌体,用TE缓冲液重悬,高压破菌,留沉淀弃上清,将沉淀溶于结合缓冲液(0.1mol/L NaCl,0.05mol/L Tris-Cl, 8mol/L 尿素,0.5%Triton X-100,pH9.0)中,选择GE公司的His TrapTM HP凝胶柱进行镍离子亲和层析纯化,用洗涤缓冲液1(0.1mol/L NaCl,0.05mol/L Tris-Cl,8mol/L尿素,0.05mol/L咪唑,pH9.0)和洗涤缓冲液2(0.1mol/L NaCl,0.05mol/L Tris-Cl,8mol/L 尿素,0.08mol/L咪唑,pH9.0)洗涤杂蛋白,使用洗脱缓冲液(0.5mol/L NaCl,0.05mol/L Tris-Cl,8mol/L 尿素,0.3mol/L咪唑,pH9.0)洗脱目的蛋白,SDS-PAGE检测并用软件BandScan分析,纯度为80%(见附图5 B)。
实施例6:动物的免疫及其免疫指标测定。
取生长健康的6-8周龄的雌性BALB/c小鼠若干,设置PBS(0.14mol/L NaCl,2.7mmol/L KCl,0.01mol/L Na2HPO4,1.8mmol/L KH2PO4,pH 7.3),TB10.4及其TB10.4-F1三组,每组5只。分别在0,2,4周免疫小鼠,每次抗原的使用剂量为30μg/只,PBS组以200μl的无菌PBS代替。每次免疫前一天尾部取血,用以监测血清中IgG的变化趋势。第3次免疫两周后摘眼球处死并取血制备血清。用TB10.4-F1融合蛋白(500ng/ml)包板,血清以1:1000稀释后作为一抗,HRP酶标兔抗鼠IgG(来源于KPL公司,100366);IgG1(来源于SouthernBiotech公司,D8105-MG86)和IgG2a(来源于SouthernBiotech公司,J5206-MQ17B)以1:4000稀释后作二抗,OPD显色,OD492读数。另外取PBS组小鼠血清直接包板,鉴定IgG1和IgG2a,用以作BALB/c小鼠背景对照。与TB10.4组相比,TB10.4-F1融合蛋白组滴度上升更加明显。说明融合蛋白可以产生两种蛋白的特异性IgG,引起有效地免疫应答(见附图6 A)。而对IgG两亚类作比值发现,融合蛋白免疫组IgG1/IgG2a值仍然大于1,但与TB10.4组相比,比值明显缩小(见附图6 B)。而在机体免疫反应中,IgG1代表Th2型体液免疫反应,而IgG2a代表Th1型细胞免疫反应。二者比值接近1,说明疫苗可以激发机体产生较为平衡的体液免疫和细胞免疫反应,同时也说明融合蛋白的设计对于平衡Th1/Th2反应方面很有意义。
序列表
SEQUENCE LISTING
<110> 昆明理工大学
<120> 一种结核分枝杆菌TB10.4-F1融合蛋白疫苗及其制备方法
<160> 2
<170> PatentIn version 3.5
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atgtcgcaaa tcatgtacaa ctaccccgcg atgttgggtc acgccgggga tatggccgga 60
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aaactgcaca catcccctct atgtacaacc aacacaaagg aagggtccaa catctgctta 900
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MET Ala MET MET Ala Arg Asp Thr Ala Glu Ala Ala Lys Trp Gly Gly
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Ser Leu Ile Asn Asp MET Pro Ile Thr Asn Asp Gln Lys Lys Leu MET
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Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile MET Ser
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Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr
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Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr MET
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Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val Asp Ile
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Phe Asn Pro Lys Tyr Asp Cys Lys Ile MET Thr Ser Lys Thr Asp Val
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Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly
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Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp Thr Val
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序列表
SEQUENCE LISTING
<110> 昆明理工大学
<120> 一种结核分枝杆菌TB10.4-F1融合蛋白疫苗及其制备方法
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<170> PatentIn version 3.5
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atgtcgcaaa tcatgtacaa ctaccccgcg atgttgggtc acgccgggga tatggccgga 60
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ttaccaagtg aggtaaatct ctgcaacgtt gacatattca accccaaata tgattgcaaa 1080
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tcatgctatg gcaaaaccaa atgtacagca tccaataaaa atcgtgggat cataaagaca 1200
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acattatatt atgtaaataa gcaagaaggc aaaaatctct atgtaaaagg tgaaccaata 1320
ataaatttct atgacccatt agtgttcccc tctgatgaat ttgatgcatc aatatctcaa 1380
gtcaatgaga agattaacca gagtctagca tttattcgta aatcagatga attattacat 1440
aatgtaaatg ctggtaaatc cactacaaat atcatgataa ctactataat tatagtgatt 1500
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MET Ser Gln Ile MET Tyr Asn Tyr Pro Ala MET Leu Gly His Ala Gly
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Asp MET Ala Gly Tyr Ala Gly Thr Leu Gln Ser Leu Gly Ala Glu Ile
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Ala Val Glu Gln Ala Ala Leu Gln Ser Ala Trp Gln Gly Asp Thr Gly
35 40 45
Ile Thr Tyr Gln Ala Trp Gln Ala Gln Trp Asn Gln Ala MET Glu Asp
50 55 60
Leu Val Arg Ala Tyr His Ala MET Ser Ser Thr His Glu Ala Asn Thr
65 70 75 80
MET Ala MET MET Ala Arg Asp Thr Ala Glu Ala Ala Lys Trp Gly Gly
85 90 95
Gly Ala Gly Ser Gly Ala Phe Leu Gly Phe Leu Leu Gly Val Gly Ser
100 105 110
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115 120 125
Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val
130 135 140
Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Arg Lys Val Leu Asp
145 150 155 160
Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn Lys Gln
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Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys
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Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn Ala Gly
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Val Thr Thr Pro Val Ser Thr Tyr MET Leu Thr Asn Ser Glu Leu Leu
210 215 220
Ser Leu Ile Asn Asp MET Pro Ile Thr Asn Asp Gln Lys Lys Leu MET
225 230 235 240
Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile MET Ser
245 250 255
Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr
260 265 270
Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys
275 280 285
Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp
290 295 300
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305 310 315 320
Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr MET
325 330 335
Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val Asp Ile
340 345 350
Phe Asn Pro Lys Tyr Asp Cys Lys Ile MET Thr Ser Lys Thr Asp Val
355 360 365
Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly
370 375 380
Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr
385 390 395 400
Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp Thr Val
405 410 415
Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly Lys Asn
420 425 430
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Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn Glu Lys
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Asn Val Asn Ala Gly Lys Ser Thr Thr Asn Ile MET Ile Thr Thr Ile
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530 535 540
His His
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Claims (4)
1.一种结核分枝杆菌TB10.4-F1融合蛋白疫苗,其特征在于其碱基序列如SEQ ID NO.1所示,氨基酸序列如SEQ ID NO.2所示。
2.一种结核分枝杆菌TB10.4-F1融合蛋白疫苗的制备方法,其特征是包括以下具体步骤:
(1)重组亚克隆载体TB10.4(N/B)/pMD18T和F1(B/X)/pMD18T的构建
以结核分枝杆菌标准菌株H37Rv全基因组为模板,PCR扩增获得TB10.4(N/B)基因,并引入NdeⅠ和BamHⅠ酶切位点,以F/pMD18T质粒为模板,PCR扩增获得F1(B/X)基因,并引入BamHⅠ和XhoⅠ酶切位点和6×His标签,扩增产物胶回收纯化后与pMD18T载体连接,获得重组亚克隆载体;
(2)重组表达载体TB10.4-F1/pET30a的构建
TB10.4(N/B)/pMD18T质粒经NdeⅠ和BamHⅠ双酶切后得到带有粘性末端的目的基因TB10.4(N/B),并与同样双酶切过的表达载体pET30a相连接,形成重组表达载体TB10.4(N/B)/pET30a;
F1(B/X)/pMD18T质粒经BamHⅠ和XhoⅠ双酶切后得到带有粘性末端的目的基因F1(B/X),并与同样双酶切过的重组载体TB10.4(N/B)/pET30a相连接,即形成重组表达载体TB10.4-F1/pET30a,而TB10.4(N/B)和F1(B/X)之间则以Linker相连,重组融合蛋白基因碱基序列以SEQ ID NO.1所示;
(3)TB10.4-F1蛋白的诱导表达及其纯化
重组表达载体TB10.4-F1/pET30a转化到大肠表达菌株中,形成重组工程菌;重组工程菌经过发酵培养,IPTG诱导表达TB10.4-F1融合蛋白,采用超声破菌,离心,最后通过金属离子亲合层析纯化法纯化,4℃下,采用梯度透析复性的方法对已纯化好变性蛋白复性即得到疫苗蛋白,氨基酸序列以SEQ ID NO.2所示;
所述大肠表达菌株为E.coli BL21(DE3);
所述转化大肠杆菌表达菌株的方法为热休克转化法。
3.根据权利要求2所述的制备方法,其特征在于大肠杆菌表达载体为质粒pET30a。
4.根据权利要求3所述的制备方法,其特征在于金属离子亲合层析纯化法为镍离子亲和层析法。
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