CN105237632A - 阴道毛滴虫α-actinin蛋白免疫保护性抗原、疫苗和应用 - Google Patents
阴道毛滴虫α-actinin蛋白免疫保护性抗原、疫苗和应用 Download PDFInfo
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Abstract
本发明属于疫苗制备技术领域,具体公开了阴道毛滴虫α-actinin重组融合蛋白免疫保护性抗原、疫苗和应用;利用原核表达体系构建了可以大量稳定表达α-actinin亚片段的表达菌株,同时纯化并获得ACT-T蛋白,该蛋白作为抗原免疫感染阴道毛滴虫的小鼠后,小鼠在14天后基本痊愈,对小鼠的免疫保护作用接近100%,将ACT-T融合蛋白作为抗原制成亚单位疫苗,从而实现对小鼠的免疫保护,该疫苗具有广泛的开发应用价值。
Description
技术领域
本发明涉及疫苗制备技术领域,更具体地,涉及阴道毛滴虫α-actinin重组融合蛋白免疫保护性抗原、疫苗和应用。
背景技术
阴道毛滴虫是一种寄生于人体泌尿生殖系统的原虫。感染该虫之后会引起女性阴道炎、尿道炎、子宫颈炎;男性感染阴道毛滴虫后,发生前列腺癌的概率也比正常人群要高;阴道毛滴虫感染还会增加人群感染乙肝病毒、人型支原体以及艾滋病病毒的风险,也会引起孕妇早产以及新生儿体重偏轻,并且有可能在阴道分娩过程中传播给新生儿呼吸道。
阴道毛滴虫病呈全球分布,是最常见的非病毒性性传播疾病。然而由于缺乏对该病的充分认识和筛查计划,导致全球上百万病例未得报道及诊断,致使该病成为一种被忽视的性传播疾病。根据WHO评估,2008年全世界15至49岁年龄段的人群中,感染毛滴虫的患者约为2.764亿。相比2005年同年龄段的感染人数2.485亿,已上涨了11.2%。此外,虽然甲硝唑是目前治疗阴道毛滴虫感染的首选药物,然而,近年来除了逐渐发现甲硝唑有毒副作用以外,临床上还发现了不少甲硝唑的抗性株。由于阴道毛滴虫主要通过性接触直接传播,患者尤其是男性患者感染毛滴虫后又常为无临床症状的带虫者,可能导致其伴侣反复感染,导致交叉感染,扩大传播。因此,发展可持续控制的策略,例如疫苗的开发,就变得很有意义。而目前对于毛滴虫疫苗相关的研究非常有限。目前仅有研究人员用全虫为疫苗,或以阴道毛滴虫半胱氨酸蛋白CP62为疫苗,接种小鼠鼻腔,在小鼠的毛滴虫感染阴道模型中都有一定的保护作用。然而,与大分子抗原相比,在亚单位疫苗中,由于分子相似性而引起的自体免疫的可能性更小,因此开发亚单位疫苗更安全。
发明内容
本发明所要解决的技术问题是克服现有技术存在的上述缺陷,提供阴道毛滴虫α-actinin重组融合蛋白免疫保护性抗原。
本发明的第二个目的是提供阴道毛滴虫α-actinin重组融合蛋白免疫保护性疫苗。
本发明的第三个目的是提供上述免疫保护性抗原或疫苗的应用。
本发明的目的是通过以下技术方案予以实现的:
α-actinin重组融合蛋白ACT-T作为阴道毛滴虫病免疫保护性抗原的应用,其ACT-T的氨基酸序列如SEQIDNO:1所示。
阴道毛滴虫α-actinin蛋白是一种结合于actin,分子大小为115Kda的蛋白,也是阴道毛滴虫细胞骨架重排中比较活跃的蛋白之一。该蛋白参与毛滴虫阿米巴形变,被认为与虫株毒力相关;并且阴道毛滴虫中最常见的免疫原就是α-actinin,可以引起了强烈的体液免疫反应,经常被发现存在于裂解的靶细胞表面。由于该蛋白强的抗原性及免疫原性,而α-actinin全基因较长,那么包含蛋白中间区域的核心抗原决定簇的亚克隆,则成为设计毛滴虫诊断与疫苗的良好选择。为获取大量纯化的目的蛋白,本研究利用BL21原核表达体系构建了可以大量稳定表达α-actinin亚片段的表达菌株,进行疫苗相关的研究。
由于α-actinin全长序列较长,在进行原核表达时多数表达为包涵体,申请人前期研究中构建了3段序列,分别是α-actinin全长序列、涵盖中间核心抗原决定簇的α-actinin全长序列前半段(ACT-F)和涵盖中间核心抗原决定簇的α-actinin全长序列后半段(ACT-T);分别将这3段序列构建表达载体进行原核表达,虽然这三段序列获得的重组蛋白均具有免疫表现,但作为免疫原对动物进行免疫时,其免疫保护作于差别显著。
申请人研究发现适当浓度的重组融合蛋白免疫感染阴道毛滴虫的小鼠后,其中ACT-T的免疫保护作于尤其明显,接近100%,因此ACT-T是制备阴道毛滴虫病疫苗理想的候选抗原。
本发明还提供α-actinin重组融合蛋白ACT-T在制备治疗阴道毛滴虫病的制剂方面的应用,其ACT-T的氨基酸序列如SEQIDNO:1所示。
本发明还提供α-actinin重组融合蛋白ACT-T在制备预防阴道毛滴虫病的制剂方面的应用,其ACT-T的氨基酸序列如SEQIDNO:1所示。
优选地,本发明所述ACT-T的核苷酸序列如SEQIDNO:2所示。
本发明还提供一种阴道毛滴虫α-actinin重组融合蛋白疫苗,所述疫苗含有上述α-actinin重组融合蛋白ACT-T。
本发明所述ACT-T作为抗原免疫感染阴道毛滴虫的小鼠后,小鼠在14天后基本痊愈,本发明所述疫苗执行了主动免疫,抗原注入阴道毛滴虫的小鼠后,体内发生了复杂的免疫保护过程,从而最终实现对小鼠的免疫保护。
优选地,本发明所述疫苗还包括制备疫苗所需的辅剂。
更优选地,所述辅剂为免疫佐剂。
更优选地,所述α-actinin重组融合蛋白ACT-T的用量为20~100ug。
与现有技术相比,本发明具有以下有益效果:
本发明提供了阴道毛滴虫α-actinin重组融合蛋白免疫保护性抗原、疫苗和应用,利用原核表达体系构建了可以大量稳定表达α-actinin亚片段的表达菌株,同时纯化并获得ACT-T蛋白,该蛋白作为抗原免疫感染阴道毛滴虫的小鼠后,小鼠在14天后基本痊愈,对小鼠的免疫保护作用接近100%,将ACT-T融合蛋白作为抗原制成亚单位疫苗,从而实现对小鼠的免疫保护,该疫苗具有广泛的开发应用价值。
附图说明
图1为阴道毛滴虫α-actinin免疫保护性抗原诱导表达和纯化后的SDS-PAGE图,其中:M为蛋白maker;泳道1,2,3分别为全长序列后半段(ACT-T)表达菌株未经IPTG诱导的菌液电泳,经过IPTG诱导的菌液电泳以及蛋白纯化后的电泳条带;泳道5,6,7分别为全长序列前半段(ACT-F)表达菌株未经IPTG诱导的菌液电泳,经过IPTG诱导的菌液电泳以及蛋白纯化后的电泳条带。
图2为将纯化的蛋白用兔抗阴道毛滴虫α-actinin多克隆抗体进western-blot检测图;其中:M为蛋白maker;泳道1为ACT-T蛋白;泳道2为ACT-F蛋白。
图3为阴道毛滴虫α-actinin免疫保护性抗原babl/c后的相对免疫保护分析图。ACT-T(HC)和ACT-T(LC)分别代表ACT-T免疫的高浓度和低浓度组;ACT-F(HC)和ACT-F(LC)分别代表ACT-F免疫的高浓度和低浓度组;FA代表佐剂与PBS等体积混合后免疫组;Control代表仅注射PBS的对照组。
图4为阴道毛滴虫α-actinin免疫保护性抗原最后一次免疫babl/c两周后的血清中特异性抗体效价间接ELISA分析结果;其中,A图是分别以ACT-T和ACT-F纯化蛋白进行包被后,血清按照1:10000稀释后的总IgGOD450nm数值;B图是分别以ACT-T和ACT-F纯化蛋白进行包被后,免疫后的小鼠血清IgG抗体滴度;C图和D图是分别以ACT-F和ACT-T纯化蛋白进行包被,检测血清抗体亚型OD450nm数值(稀释度1:1000)。
具体实施方式
下面通过实施例,并结合说明书附图对本发明进一步具体描述。下述所使用的实验方法若无特殊说明,均为本技术领域现有常规的方法,所使用的配料或材料,如无特殊说明,均为通过商业途径可得到的配料或材料;本发明不应限制于实施例范围。
实施例1重组抗原蛋白基因序列扩增及表达载体构建
(1)将阴道毛滴虫培养液1000g离心5min,弃上清,收集约107虫体于1.5ml离心管中。加入1mlTrizol裂解液,用移液枪吹打混匀,室温静置5min后用氯仿/异丙醇进行提取RNA,然后用RNasefree水,55~60℃温浴10min溶解沉淀。
(2)除去RNA中的DNA后,用Nanodrop测RNA的A260/A280的值,确保其数值在1.8~2.0后,所测的浓度换算出RNA的原浓度。
(3)将RNA反转录为cDNA用于后续PCR扩增实验。
(4)引物设计
为达到更好的蛋白表达效果,本研究根据已公布的阴道毛滴虫a-actinin基因序列,设计两对引物,分别表达涵盖中间核心抗原决定簇的α-actinin全长序列前半段(ACT-F)和全长序列后半段(ACT-T)(表1)。
以RNA反转的cDNA为模板,以表1两对序列为引物,表2所示的条件进行PCR,得到目的基因片段,大小如表1所示。
PCR反应条件为:94℃,5min;94℃,5sec,55℃,30sec,72℃,2min,35个循环;最后72℃,10min,最后进行电泳鉴定。
(5)克隆菌株构建:将阴道毛滴虫a-actinin基因电泳鉴定以后,目的片段回收,用Takara公司的A-tailingkit加上A尾,再与PCR2.1载体连接后,导入DH5a大肠杆菌感受态细胞中。摇菌后提取质粒,克隆载体用两种限制性内切酶KpnⅠ和SalⅠ进行双酶切鉴定,并送至上海生工生物工程有限公司测序,无误后进行下一步操作。
(6)表达载体构建:用Takara公司的DNA胶回收试剂盒来回收克隆菌株中的目的基因,利用双酶切产生的粘性末端,将其与PET32a质粒连接,并将重组质粒转化入BL21菌株中。经双酶切和测序鉴定无误,即a-actinin表达菌株。测序鉴定的结果在NCBI上比对,与公布的序列匹配无误后进行诱导表达。
实施例2重组抗原蛋白的表达纯化
一、蛋白表达条件的优化
采用单因子变量,进行以下几项的优化。通过表达条件的优化,按照摸索出来的最佳表达条件:当温度为24℃,细菌OD600nm值达到0.8时,分别加入终浓度为0.6mmol/L的IPTG,持续诱导表达5h后,阴道毛滴虫α-actinin基因、全长序列前半段、全长序列后半段均能取得满意的表达效果。将重组蛋白诱导与未诱菌反复冻融破碎后,用CBA蛋白浓度检测试剂盒定量,然后直接与5Xloadingbuffer混匀煮沸后,进行SDS-PAGE电泳,结果如图1。
二、重组蛋白纯化
重组蛋白上携带His标签,所以早期用NTA-His树脂纯化(参考Novagen组氨酸标签纯化手册),所得蛋白用于免疫新西兰白兔,获得含多克隆抗体的血清。但是后续实验中,由于蛋白用量大,全长片段形成包涵体以及组氨酸标签可能丢失,导致纯化效率不够高,所以选用电洗脱法大量纯化重组蛋白,电洗脱法的过程如下:
(1)按最优条件诱导菌液后,4℃,10000g,10min离心,弃上清。加入适量的PBS,冰浴超声破碎,超声6S,停6S,200次。加入5x1oadingbuffer,沸水中煮5min,分装以后放置于-20℃保存。
(2)将蛋白样品跑SDS-PAGE电泳。上样量为每块胶200ul,浓缩胶恒压80V,分离胶恒压100V,跑至溴酚蓝到胶的底部。
(3)将胶取下,用预冷的0.5mol/LKCl染胶10min,目的条带会被染成较深的乳白色。根据分子大小,切下目的条带并切碎。用电洗脱液(50mMTrisbase;50mM甘氨酸;0.1%SDS;PH=8.9)将碎胶清洗至透明,然后装入清洗过的透析袋,在电洗脱液中进行电洗脱(恒压100V,4h~5h),再反向电泳(恒压100V,0.5h)。
(4)电洗脱之后将透析袋放入4℃预冷的0.1xPBS中透析,期间换液3~5次,时不时搅拌。
(5)将纯化好的蛋白溶液倒入到离心管内,-80℃冻过夜,然后放置于冷冻干燥机内进行冷冻干燥浓缩。
(6)浓缩后的蛋白用CBA蛋白浓度检测试剂盒进行蛋白定量。所有收集后的蛋白用混合在一起,用wetern-blot检测确定后,冻存于-80℃,用于后续试验。
将纯化后重组蛋白跑电泳,根据分子大小初步判断纯化后收集到的是目的蛋白,可以得到单一的条带,如图1所示,α-actinin全长序列前半段大小为70.33kDa,α-actinin全长序列后片段大小为61.7kDa。
上述SDS-PAGE电泳后的胶,冰浴中恒流300mA,1h转到硝酸纤维素膜上,用5%脱脂奶粉封闭过夜后,电洗脱后的蛋白用本实验室制备的兔来源多克隆抗体为一抗,37℃温育1h,TTBS清洗3次,10min/次;羊抗小鼠IgG-HRP为二抗37℃温育1h,TBST清洗3次,10min/次,DAB显色检测反应5~15min,置于蒸馏水中终止显色反应,抗体检测表明电洗脱方式纯化的蛋白为特异性目的蛋白,结果如图2所示。
实施例3动物免疫以及攻毒试验
将实施例2纯化获得的蛋白作为抗原和等体积的弗氏佐剂涡旋振荡进行乳化,制备免疫原。
购买体重为20±2g的健康雌性Babl/c小鼠60只,随机分为6组。每组免疫所用小鼠数量以及小鼠皮下多点接种的蛋白量如表3所示。
所有小鼠均免疫4次。第一次为抗原与等体积弗氏完全佐剂乳化后,皮下多点注射;第二次和第三次为抗原与等体积弗氏不完全佐剂乳化后,皮下多点注射;第4次为腹腔水剂注射;第4次免疫14天后,取小鼠尾血,分离血清。并收集对数生长期的阴道毛滴虫,1×107/0.5ml/只接种于babl/c小鼠腹腔。每日观察小鼠的情况,记录发病状况及死亡时间。
结果如图3,重组蛋白免疫后的四组小鼠与未用重组蛋白免疫的两组相比,均有一定的免疫保护作用,其中ACT-T(HC)组保护作用尤为明显,近乎达到100%。因此ACT-T是制备阴道毛滴虫病疫苗理想的候选抗原,即本发明的融合蛋白ACT-T具有很好的免疫原性及免疫保护作用。
实施例4a-actinin免疫小鼠抗体滴度情况
小鼠免疫四次后第13天取尾血0.1~0.2ml,全血置于4℃静止4~6h,离心后取上层血清,存储于-80℃;上述每组实验鼠10只按照3只、3只、4只取血清各5ul于0.5ml离心管内,轻轻混匀,制备3个间接ELISA检测抗体滴度的血清混合样品。
分别以2ug/ml纯化的重组ACT-F和ACT-T蛋白包被酶标板,以待测血清为一抗,羊抗小鼠HRP-IgG为二抗,检测各组血清抗体所产生总IgG抗体OD450nm数值及抗体滴度(图4A和B);在图中可看出血清样品稀释1:10000后,待测血清吸光值明显高于对照组,说明重组蛋白免疫小鼠后产生大量的特异性抗体,两种重组蛋白免疫产生的抗体滴度均很高。
血清样品稀释1:1000后特异抗体亚型OD450nm数值表明,特异性抗体中主要为IgG1,IgG2a亚型(图4C和D)。
抗体阳性判断根据P/N值来确定。本实验中待测血清OD450nm数值(P)与阴性血清OD450nm数值(N)的比值大于3,则在本实验中确定为阳性。
综上所述,本发明的阴道毛滴虫α-actinin免疫保护性、抗原免疫原性很好,为阴道毛滴虫病小鼠模型提供一种有效的免疫保护作用,可作为具有重要价值的抗阴道毛滴虫病候选疫苗用于后续开。
SEQUENCELISTING
<110>中山大学
<120>阴道毛滴虫α-actinin蛋白免疫保护性抗原、疫苗和应用
<130>
<160>6
<170>PatentInversion3.3
<210>1
<211>550
<212>PRT
<213>ACT-T氨基酸序列
<400>1
MetSerAspLysIleIleHisLeuThrAspAspSerPheAspThrAsp
151015
ValLeuLysAlaAspGlyAlaIleLeuValAspPheTrpAlaGluTrp
202530
CysGlyProCysLysMetIleAlaProIleLeuAspGluIleAlaAsp
354045
GluTyrGlnGlyLysLeuThrValAlaLysLeuAsnIleAspGlnAsn
505560
ProGlyThrAlaProLysTyrGlyIleArgGlyIleProThrLeuLeu
65707580
LeuPheLysAsnGlyGluValAlaAlaThrLysValGlyAlaLeuSer
859095
LysGlyGlnLeuLysGluPheLeuAspAlaAsnLeuAlaGlySerGly
100105110
SerGlyHisMetHisHisHisHisHisHisSerSerGlyLeuValPro
115120125
ArgGlySerGlyMetLysGluThrAlaAlaAlaLysPheGluArgGln
130135140
HisMetAspSerProAspLeuGlyThrThrTyrIleLysHisLeuLeu
145150155160
GluGlnLeuAsnGlyLysLeuPheGluGluThrAsnGluAlaArgIle
165170175
AsnGluTyrAsnAlaLeuAlaGlnProLeuTyrAspGluAlaIleAla
180185190
PheLysGluGluValLeuAlaIleSerGlyGluLeuArgGluArgArg
195200205
ThrGlnPheLeuAlaLysGlnAlaGluAlaProThrLysArgGluHis
210215220
ValAsnGluIleAspProIlePheAspGlyLeuGluLysAspSerLeu
225230235240
HisLeuArgValAsnHisSerProThrGluIleArgAsnValTyrAla
245250255
ValThrLeuGlnHisIleIleThrGluLeuAsnLysIlePheGluGlu
260265270
MetValAlaAsnPheAspAlaThrAlaValProIleIleAspGlyIle
275280285
ThrAlaLeuValThrSerSerHisGlnIleProGlyAspAlaAlaAla
290295300
ValLysAlaGlnValGluGluAsnLeuAlaSerLeuAspGlyPheAla
305310315320
GluLysIleGlnAlaLeuGlnAspProTyrAsnGluLeuValGluPhe
325330335
LysLeuAsnTyrLysValThrTyrThrTyrSerAspAlaThrGlyGlu
340345350
LeuAspGlnAlaArgLeuAspLeuLysGlnIleIleLeuAlaLysLys
355360365
ThrPheLeuGluGluGluGluArgLysAlaArgIleAsnAsnTyrThr
370375380
ValLysAlaAspGluHisMetAsnGluAlaHisAlaLeuAspGlyLys
385390395400
IleAsnSerValAspGlyGluLeuGluProLysArgGlnLysLeuTyr
405410415
GluValArgGluGluValAsnAlaLysLysGluLysAlaAlaGluGlu
420425430
LeuThrProIleTyrGluAspLeuGluLysAspGlnLeuHisLeuGlu
435440445
IleThrSerThrProAlaSerIleAsnIlePhePheGluAsnLeuIle
450455460
AlaHisIleAspThrLeuValLysGluIleAspAlaAlaIleAlaAla
465470475480
AlaLysGlyLeuGluIleSerGluGluGluLeuAsnGluPheLysAsp
485490495
ThrPheLysTyrPheAspLysAspLysSerAsnSerLeuGluTyrPhe
500505510
GluLeuLysAlaCysLeuThrAlaLeuGlyGluAspIleThrAspAsp
515520525
GlnAlaLysGluTyrCysLysValAspLysLeuAlaAlaAlaLeuGlu
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HisHisHisHisHisHis
545550
<210>2
<211>1653
<212>DNA
<213>ACT-T核苷酸序列
<400>2
atgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcg60
gacggggcgatcctcgtcgatttctgggcagagtggtgcggtccgtgcaaaatgatcgcc120
ccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaac180
atcgatcaaaaccctggcactgcgccgaaatatggcatccgtggtatcccgactctgctg240
ctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttg300
aaagagttcctcgacgctaacctggccggttctggttctggccatatgcaccatcatcat360
catcattcttctggtctggtgccacgcggttctggtatgaaagaaaccgctgctgctaaa420
ttcgaacgccagcacatggacagcccagatctgggtaccacatacatcaagcacctcctc480
gagcagctcaacggcaagctcttcgaagagacaaacgaggcccgcatcaacgagtacaac540
gctcttgcccagccactctacgacgaggccatcgccttcaaggaggaagtcttagccatc600
agcggcaaactccgcgagcgccgtacacagttcctcgccaagcaggctgaggctccaaca660
aagagagagcacgttaacgagatcgacccaatcttcgacggactcgagaaggattcactt720
cacctccgtgtcaaccaaagcccaacagaaatccgtaacgtttacgctgttacacttcag780
cacatcatcacagaactcaacaagatcttcgaggagatggttgccaacttcgatgccacc840
gctgtcccaatcatcgatggtatcacagccctcgtcacatcctcccaccagatcccagga900
gatgctgctgcagtcaaggctcaggttgaggagaacctcgcttccctcgatggcttcgct960
gaaaagatccaagccctccaggatccatacaacgagctcgttgaattcaagctcaactac1020
aaggtcacatacacatactctgatgctactggtgaactcgatcaggcacgtcttgacctc1080
aagcagatcatcctcgccaagaagacattcctcgaggaagaagagcgcaaggcccgcatc1140
aacaactacacagtcaaggctgacgagcacatgaacgaggctcatgctctcgatggcaag1200
atcaactccgtcgatggcgaactcgaaccaaagagacagaaactctacgaagtccgtgag1260
gaagtcaacgccaagaaggagaaggccgtcgaggaactcacaccaatctacgaagacctc1320
gaaaaggatcagctccatctcgaaatcacatccacaccagcttccatcaacatcttcttc1380
gagaacctcatcgcccacatcgatacactcgtcaaggaaatcgatgctgctatcgctgct1440
gctaagggtctcgagatctccgaagaagaactcaacgagttcaaggatacattcaagtac1500
ttcgacaaggataaatccaactccctcgagtacttcgaactcaaggcttgccttacagct1560
ctcggcgaagatatcacagatgaccaggctaaggaatactgcaaggtcgacaagcttgcg1620
gccgcactcgagcaccaccaccaccaccactga1653
<210>3
<211>30
<212>DNA
<213>ACT-F正向引物
<400>3
gacggtaccgagaagacccagatcaaggtt30
<210>4
<211>30
<212>DNA
<213>ACT-F反向引物
<400>4
agtgtcgacgaggaggtgcttgatgtatgt30
<210>5
<211>30
<212>DNA
<213>ACT-T正向引物
<400>5
gacggtaccacatacatcaagcacctcctc30
<210>6
<211>30
<212>DNA
<213>ACT-T反向引物
<400>6
agtgtcgaccttgcagtattccttagcctg30
Claims (8)
1.α-actinin重组融合蛋白ACT-T作为阴道毛滴虫病免疫保护性抗原的应用,其特征在于,其ACT-T的氨基酸序列如SEQIDNO:1所示。
2.α-actinin重组融合蛋白ACT-T在制备治疗阴道毛滴虫病的制剂方面的应用,其特征在于,其ACT-T的氨基酸序列如SEQIDNO:1所示。
3.α-actinin重组融合蛋白ACT-T在制备预防阴道毛滴虫病的制剂方面的应用,其特征在于,其ACT-T的氨基酸序列如SEQIDNO:1所示。
4.根据权利要求1至3任一项所述的应用,其特征在于,所述ACT-T的核苷酸序列如SEQIDNO:2所示。
5.一种阴道毛滴虫α-actinin重组融合蛋白疫苗,其特征在于,所述疫苗含有权利要求1所述α-actinin重组融合蛋白ACT-T。
6.根据权利要求5所述的疫苗,其特征在于,还包括制备疫苗所需的辅剂。
7.根据权利要求6所述的疫苗,其特征在于,所述辅剂为免疫佐剂。
8.根据权利要求6所述的疫苗,其特征在于,所述α-actinin重组融合蛋白ACT-T的用量为20~100ug。
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李玉峰: "阴道毛滴虫可溶性抗原组分分析", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
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