CN101613402A - 一种猪链球菌2型表面蛋白、其制备方法及用途 - Google Patents
一种猪链球菌2型表面蛋白、其制备方法及用途 Download PDFInfo
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Abstract
本发明公开了一种猪链球菌2型表面蛋白,还公开了该蛋白质的制备方法及用途。该蛋白质可用作诊断靶标,用于开发猪链球菌2型的诊断试剂。同时又是重要的保护性抗原,可用于制备疫苗。
Description
技术领域
本发明涉及一种细菌蛋白,具体地说涉及一种猪链球菌2型的表面蛋白,还涉及该蛋白质的制备方法以及用途。
背景技术
猪链球菌(Streptococcus suis),属于兰氏分类法的R群,有35个血清型,以猪链球菌2型流行最广,致病性最强。自丹麦在1968年最早报道人感染猪链球菌病后,20世纪70年代以来,欧美部分国家和香港地区均报道过猪链球菌病例,主要以脑膜炎为主,中毒性休克综合征(TSS)少见,未见暴发流行。我国于1998年和2005年两次在江苏和四川暴发人感染猪链球菌2型疫情,不同于国外人感染病例以脑膜炎为主的特点,临床表现为休克型和脑膜炎型。多数病例死于中毒性休克综合征(TSS),病理改变主要表现为全身多器官受损,败血症伴弥漫性血管内凝血(DIC)。人猪链球菌病已成为严重威胁我国人民健康的一种新的人畜共患病,如何有效地预防和控制猪链球菌感染已成为科学家们面临的重要研究难题。
由于对毒力因子和保护性抗原了解甚少,同一血清型的菌株在不同的地区毒力也不尽相同,高致病性猪链球菌感染缺乏有效的诊断靶标:对该类病原的分离鉴定主要依靠传统的分离培养和生化鉴定,然后用猪链球菌高免血清进行凝集试验分型。但家畜广泛携带链球菌,许多菌株毒力低,因此仅仅靠生化反应检测猪链球菌是远远不够的,必须建立针对特异毒力因子的检测手段,以区分弱毒株和强毒株。以上情况都阻碍了有效的猪链球菌病疫苗的研制及对感染的及时诊断与控制。
纵观国外猪链球菌病疫苗的研制情况,主要经历了全菌免疫和抗原蛋白免疫两个阶段。上世纪90年代,有研究者以福尔马林灭活的猪链球菌进行静脉注射能刺激被免疫猪产生调理化的抗体[Holt ME,Enright MR,AlexanderTJ.(1990)Immunisation of pigs with killed cultures of Streptococcus suis type 2.Res Vet Sci.48:23-7]。也有研究者通过筛选无毒力的猪链球菌突变体,如温度敏感性突变体、链霉素依赖性突变体等来获得具有保护性的全菌疫苗[KebedeM,Chengappa MM,Stuart JG.(1990)Isolation and characterization oftemperature-sensitive mutants of Streptococcus suis:efficacy trial of the mutantvaccine in mice.Vet Microbiol.22:249-57.Foster N,Staats JJ,Chengappa MM.(1994)Isolation,characterization and protection studies in mice of astreptomycin-dependent mutant of Streptococcus suis type 1/2.Vet Res Commun.18:155-63.],但是灭活的全菌免疫常会引起免疫综合征等副作用。后续有研究者利用猪链球菌相关毒力因子溶菌酶释放蛋白(MRP)、胞外因子(EF)结合油包水型乳剂(WO)和氢氧化铝佐剂(AH)作为疫苗,对其保护性和毒副作用作了评价,发现用MRP+EF/WO免疫的猪与用全菌/WO免疫的猪具有同等有效的抗-MRP和抗-EF滴度,而且在免疫后的存活猪中,没有观察到明显的临床表征[Wisselink HJ,Vecht U,Stockhofe-Zurwieden N,Smith HE.(2001)Protection of pigs against challenge with virulent Streptococcus suis serotype 2strains by a muramidase-released protein and extracellular factor vaccine.Vet Rec.148:473-7.]。也有人用纯化的溶血素(SLY)作为疫苗免疫小鼠,发现能对小鼠产生完全保护作用,免受毒力株的伤害[Jacobs AA,Loeffen PL,van den BergAJ,Storm PK.(1994)Identification,purification,and characterization of athiol-activated hemolysin(suilysin)of Streptococcus suis.Infect Immun.62:1742-8.]。但猪链球菌的毒力因子较常时间以来就呈现出时空上的相对多样性,虽然MRP、EF与毒力高度相关,但与欧洲致病株不同的是多数加拿大致病株不表达MRP和EF,且MRP和EF的2-型猪链球菌缺失突变体与野生型一样同样具备毒力[Gottshalk M,Lebrun A,Wisselink HJ,Dubreuil JD,SmithHE,Vecht U.(1998)Production of virulence-related proteins by Canadian strainsof Streptococcus suis capsular type 2.Can J Vet Res 62:75-79.]。我国流行株和非流行株都表达MRP,且动物实验显示流行株培养分泌的上清并不引起病变,单靠现了解的MRP、EF、SLY等毒力因子不足以解释我国2-型猪链球菌的致病机理,使用MRP作为人免疫疫苗产生抗体中和MRP可能难以达到理想的效果。而在国内,猪链球菌疫苗的研制还处于起步状态,即灭活的全菌疫苗[王卓,舒秀伟,王文成.猪链球菌病二价灭活疫苗候选株培养条件的优化及其安全性试验.(2004)中国药兽杂志.38:34-36.]。因此有必要根据我国实际情况,研制适合我国的2-型猪链球菌的新型疫苗,发掘新的具有免疫原性的毒力因子是寻找新型疫苗的重要途径。一般来说,表面暴露蛋白和细胞壁附着蛋白常被作为疫苗的候选靶标和血清学诊断试剂。近10年来,也有研究者试图寻找有免疫原性的猪链球菌表面细胞壁蛋白作为疫苗的候选分子,1996年Haataja S等分离并鉴定出一种半乳糖抑制型黏附素,在23种血清型的猪链球菌中,用免疫印迹的方法均能检测到这种黏附素的存在,发现具有很强的免疫原性和调理功能,适合作为疫苗候选分子[Haataja S,Tikkanen K,HytonenJ,Finne J.(1996)The Gal alpha 1-4 Gal-binding adhesin of Streptococcus suis,a gram-positive meningitis-associated bacterium.Adv Exp Med Biol.408:25-34.]。2005年Okwumabua O等人通过对2-型猪链球菌的全基因组进行筛选,发现一种分子量为38kDa蛋白,具有很好的免疫原性和保护性,认为这种蛋白是较好的疫苗候选分子并且适合用作诊断试剂的开发,这种蛋白的生物学功能以及与致病机理的关系正在进一步研究中[Okwumabua O,Chinnapapakkagari S.(2005)Identification of the gene encoding a 38-kilodalton immunogenic andprotective antigen of Streptococcus suis.Clin Diagn Lab Immunol.12:484-90]。目前在获得具有免疫原性的蛋白方面,还缺乏有力的手段,传统生物学方法鉴定免疫原性蛋白是困难的,需要构建基因文库,然后用相应血清筛选,该方法耗时较长,不能实现高通量筛选,并可能存在假阳性反应。免疫蛋白质组是一种快速、高效筛选候选诊断靶标的方法,通过将2D电泳分辨率高和质谱直接鉴定蛋白质的优势结合,直接选取2D电泳胶上有免疫反应的蛋白点,以质谱方法进行鉴定,因此,借助免疫蛋白质组学手段则完全可能在较短时间内快速高效的筛选并鉴定免疫原性蛋白,尤其对猪链球菌研究相对较少的病原微生物来说,更有可能短时间内鉴定多个全新的免疫原性蛋白。
发明内容
本发明公开了一种猪链球菌2型新型表面蛋白、其制备方法及用途。
本发明所公开的猪链球菌2型表面蛋白具有序列表中序列4或序列6所示的氨基酸序列,编码该表面蛋白的基因具有序列表中序列3或序列5所示的核苷酸序列。其中序列4和序列6前40个氨基酸为信号肽,序列5前120个核苷酸为信号肽编码基因。该蛋白命名为0186,编码该蛋白的基因在除无毒株外的猪链球菌2型菌株中广泛存在,呈现一定的多态性,在不同的菌株中表现为序列表中序列3或序列5两种长度不同的序列。
该表面蛋白是通过免疫蛋白质组鉴定的2型猪链球菌新的免疫原性抗原分子,目前根据猪链球菌基因组注释的结果,仅有该基因开放阅读框架的预测,尚未有该蛋白在猪链球菌中表达的报道,也未有该基因功能的线索。
免疫印迹实验证明该表面蛋白在不同菌株中其表达存在差异,在强毒株中高表达,而在国外弱毒株中不表达。
本发明还公开了上述表面蛋白的制备方法,该方法包括如下步骤:
首先克隆该表面蛋白基因:可通过PCR方法进行,首先设计合成引物,在引物两端可添加酶切位点和保护性碱基,然后按照PCR方法,用猪链球菌制备的模板,在一定条件下进行扩增,将扩增产物进行琼脂糖凝胶电泳,并回收产物进行酶切,确定表面蛋白基因。然后是表达该表面蛋白基因的重组质粒制备:将制备的表面蛋白基因与大肠杆菌表达载体相连接,表达载体可以为原核表达载体,可采用商业的原核表达载体,如大肠杆菌表达载体pET32a,制备表达载体,并转化大肠杆菌,制备重组菌,利用菌落PCR和限制性内切酶进行重组菌的筛选和鉴定;对重组菌进行IPTG诱导表达;超声波破碎重组菌,收集上清,以GE镍亲和层析柱进行纯化。
本发明还公开了上述表面蛋白的用途。该蛋白可用作诊断靶标,用于开发猪链球菌2型的诊断试剂,同时又是重要的保护性抗原,可用于制备疫苗。首先是该蛋白质在制备猪链球菌快速诊断试剂中的应用。如利用该表面蛋白免疫动物制备抗体,可用于制备猪链球菌2型诊断试剂。该抗体可为多克隆抗体或单克隆抗体。如采用该蛋白制备的多克隆抗体标记胶体金、猪链球菌全菌免疫制备的多克隆抗体喷膜,制成夹心法胶体金快检试剂。
本发明还公开了表面蛋白在制备猪链球菌2型疫苗中的应用。该蛋白制备的免疫血清调理的全血杀伤实验发现该蛋白具有与全菌免疫相当的保护性,可作为重点疫苗候选和诊断分子。
本发明首先将该表面蛋白鉴定为猪链球菌2型新的免疫原性抗原分子。已通过基因工程方法制备了该抗原并制备了相应抗体,通过免疫印迹验证了该抗原分子在强毒株中高表达,而在弱毒株中不表达。
在诊断试剂研制方面,传统的PCR方法并不能真正反映毒力因子的表达情况,操作也较为繁琐,在一些卫生技术条件差的地方根本就难以实现。生化鉴定虽然能较可靠的检测鉴定出2-型猪链球菌,但其必需以分离出的纯菌为基础,并且不能区分出强毒株和弱毒株。因此本研究通过基因组学、免疫蛋白质组学、抗原功能研究找到能代表我国2-型猪链球菌毒力因子以及在强弱毒株中存在差异的毒力因子来作为诊断试剂候选分子。在此基础上结合胶体金技术开发能快速检测2-型猪链球菌以及强弱毒株的试剂盒,不仅检测时间大大缩短,更难得的是该表面蛋白为靶标的检测方法用于快速检测猪链球菌2型强弱毒株的区别检测。同时通过抗体调理的全血杀菌试验证实:该蛋白制备的免疫血清可显著提高人全血的杀菌作用,与猪链球菌全菌免疫血清的调理杀菌作用相当,是新的高效保护性抗原,可作为重点疫苗候选分子。
附图说明
图1为猪链球菌2型表面蛋白0186的PCR结果图谱。
图2为重组质粒酶切图谱。其中1为重组质粒,2为空质粒,3为DL15000,4为载体,5为0186片段。
图3为重组质粒表达图谱,其中1为低分子量蛋白标准,从上至下依次为97.4kDa,66.2kDa,43.0kDa,31.0kDa,20.0kD,2为诱导后,3为诱导后,4为空质粒。
图4为猪链球菌2型表面蛋白0186的纯化蛋白电泳图。
图5为抗体介导的调理吞噬试验。
图6为胶体金试纸条检测图。其中检测样品:1为PBS,2,4,5为猪链球菌强毒株,3为猪链球菌弱毒株。
具体实施方式
实施例一猪链球菌2型表面蛋白0186的表达纯化,免疫原性及抗体介导的调理吞噬试验
1材料和方法
1.1菌株和质粒
猪链球菌2型98HAH12株[Chen C,Tang J,Dong W,Wang C,Feng Y,et al(2007)A Glimpse of Streptococcal Toxic Shock Syndrome from Comparative Genomicsof S.suis 2Chinese Isolates.PLoS ONE 2(3):e315];大肠杆菌DH5a,BL21,均为国际标准株;表达载体pET32a(+)为Novagen公司产品。
1.2酶和试剂
EcoRI和XhoI等限制性内切酶以及Taq酶、dNTPs、DL15000分子量标准等PCR所用试剂均为宝生物工程(大连)有限公司产品;DNA回收试剂盒为天为公司产品;镍亲和层析柱为GE公司产品;质粒提取盒为V-gene公司产品;弗氏完全佐剂及弗氏不完全佐剂为Sigma公司产品;Amp为中诺药业产品。
1.3PCR引物设计及0186核酸序列,蛋白序列
根据所要扩增的片段,设计一对引物,引物两端分别添加EcoRI和XhoI酶切位点和保护性碱基,引物P1和P2可扩增猪链球菌2型0186基因开放阅读框(ORF)全长1566bp的片断。引物序列分别为:
上游引物P1:见序列表中序列1;下游引物P2:见序列表中序列2。
核酸序列
>0186 putative surface anchored protein
见序列表中序列3。
蛋白序列
>0186 putative surface-anchored protein 182345:184030forward MW:61260
见序列表中序列4。
1.4PCR扩增
按下列顺序和条件依次加入各反应物并进行PCR扩增。猪链球菌2型模板DNA 2μL,10×buffer 5μL,2.5mmol/L dNTPs 3μL,引物P1和P2(5μmol/L)各1μL,ddH2O 37μL,LA-Taq酶(5U/μL)1μL。扩增的条件为:94℃7min;94℃1min,55℃1min,72℃2min,30个循环;72℃7min。
1.5PCR产物的回收和酶切
PCR产物在1%琼脂糖凝胶电泳,后以DNA回收试剂盒回收目的片段,并以EcoRI和XhoI 37℃酶切3h。
1.6重组质粒的构建和鉴定
在含有100μg/mL Amp的LB肉汤中接种携带pET32a空质粒的DH5α大肠杆菌单菌落,37℃摇振培养12~16h后,1%转接入100μg/mL Amp的新鲜LB肉汤37℃摇振培养6h,以质粒抽提试剂盒抽提质粒。EcoR I和Xho
I双酶切后,以DNA回收试剂盒回收酶切产物。将PCR双酶切回收产物与空质粒双酶切回收产物进行连接,并转化感受态的DH5α宿主菌。感受态细菌的制备、转化均按常规方法进行,利用菌落PCR和限制性内切酶酶切进行重组菌的筛选和鉴定。
1.7重组质粒的测序和序列分析
重组质粒的序列测定采用Sanger双脱氧末端终止法,由北京三博远志生物技术有限公司完成,以验证其阅读框架,并以BLAST软件进行同源性分析。
1.8重组质粒在大肠杆菌中的表达和纯化
将重组质粒按常规方法转化感受态的大肠杆菌BL21,利用Amp抗性筛选重组菌,挑单菌落接种LB肉汤中(含100μg/mL Amp),30℃剧烈摇振培养2~4h,当菌液浓度OD600达0.5~0.6时,加IPTG至终浓度1mmol/L,30℃继续剧烈摇振培养2~4h。6000r/min离心10min,沉淀以蒸馏水重悬,加等体积的2×电泳上样缓冲液煮沸10min,SDS-PAGE检测。含空pET32a(+)的大肠杆菌BL21亦同样经IPTG诱导处理后,SDS-PAGE检测表达情况。以超声波破碎诱导的重组菌,收集上清,以GE镍亲和层析柱进行亲和层析,具体步骤按使用说明书进行。
1.9重组蛋白动物免疫试验
将收集的蛋白以pH7.210mmol/L的PBS稀释至0.1mg/ml,加入等量的弗氏完全佐剂,皮下免疫Wistar大鼠(购自军事医学科学院试验动物中心),0.5ml/只;以后3-5周,分别以0.2,0.4,0.8,1.6mg/ml加弗氏不完全佐剂加强免疫,第7周断尾取血ELISA法测抗体效价。同时设立全菌免疫对照组和空白对照组(pET32a空质粒转化BL21,诱导表达Trx蛋白,以此蛋白免疫大鼠为空白对照)。以硫酸铵沉淀法制备抗血清。
1.10抗体介导的调理吞噬
血平板刮取猪链球菌2型单菌落,接入THB培养基,37℃,CO2孵箱培养8h后转接新鲜THB培养基培养8h至对数生长期。取1ml菌液(约1×108CFU/ml)13,000rpm,1.0min集菌,PBS洗菌并重悬,调整至106CFU/ml,涂血平板,记为初始菌量。取50μl(≈106CFU/ml)菌液加入100μ1抗体,37℃15min后冰上15min;加入350μl健康人全血37℃,3h;加入55μl 1%saponin,冰上20min;反复吹打后稀释涂血平板,16h后血平板记数单克隆。
1.11小鼠的免疫和攻毒
6周雌性Balb/C小鼠每次用20μg与弗氏佐剂混合的纯化蛋白皮下免疫,共免疫2次,间隔2周,第1次免疫采用弗氏完全佐剂,第2次采用弗氏不完全佐剂,以同等条件下免疫的硫氧还蛋白(Trx)为对照。第二次免疫一周后攻毒,每只小鼠腹腔接种悬浮于1ml THB培养基(Todd-Hewitt broth)猪链球菌5×108CFU,攻毒后观察7天记录发病率和死亡率。
2.结果
2.1PCR结果:PCR产物经电泳后,出现一条约1560bp的条带,大小与预期一致,见图1。
2.2重组质粒的鉴定
0186基因片段经回收,以EcoR I和Xho I酶切位点定向克隆至pET-32a中,获得重组质粒,命名为0186-pET-32a。重组质粒转化感受态的DH5α宿主菌,经Amp抗性筛选得到重组菌。提取重组质粒,经EcoR I和Xho I双酶切,得到4340bp和1560bp左右的片断,说明0186片断以成功克隆,见图2。
2.3重组质粒的表达
重组BL21转化菌经IPTG诱导后,菌体SDS-PAGE显示有一条97kD的蛋白带,表达量占菌体总蛋白的53%,而含pET32a(+)空载体的BL21菌在该处无特异条带。见图3。经镍柱纯化获得纯化的0186蛋白。见图4。
2.4测序结果和序列分析
克隆的序列长度经测序为1566bp,如序列表中序列3所示,翻译后为522个氨基酸残基,见序列表中序列4。
2.5重组蛋白抗体介导的调理吞噬作用
经GE镍亲和层析柱层析获得纯化的重组蛋白r0186,分别加弗氏完全和不完全佐剂免疫后,制备抗血清,进行间接杀菌试验。杀菌率计算:(CFUrTrx-CFUr0186)/CFUrTrx。Trx为从含pET32a(+)空载体的大肠杆菌BL21镍柱纯化的硫氧还蛋白。
抗体介导的调理吞噬试验:分别用猪链球菌2型全菌;猪链球菌2型分泌上清;0186重组蛋白;MRP重组蛋白免疫大鼠后的大鼠血清调理健康人血,进行人全血对猪链球菌2型活菌的杀伤试验。186重组蛋白的抗血清调理人全血对猪链球菌2型的杀伤率接近猪链球菌2型菌体免疫的抗血清,但略低于MRP蛋白的抗血清。见图5。
2.6重组蛋白对小鼠攻毒的保护作用
接种细菌10个小时后,对照组表现出临床症状,如发烧和对刺激反应缓慢,48小时内对照组死亡率为100%(10/10),而0186重组蛋白免疫组的动物临床症状轻微,持续时间短,死亡率为50%(5/10)。48小时后各组小鼠不再死亡。提示该重组蛋白对猪链球菌引起的小鼠致死具有显著保护作用。
实施例二186胶体金检测试纸条的制备过程
1.抗体的处理
用186免疫大鼠和猪链球菌免疫新西兰兔获得的免疫血清经辛酸-硫酸铵沉淀法或蛋白A柱纯化后用0.01M的PBS透析过夜,10,000r/min离心30min,收集上清测定浓度后备用。
2.胶体金的制备(柠檬酸钠还原法)
将HAuCl4先配成1%的水溶液,取100ml纯水加热至沸腾.搅动下同时加入1ml 1%的HAuCl4和1.5ml柠檬酸三钠水溶液。继续加热煮沸15min左右,观察到加入液体后颜色先变成黑,随后慢慢变成酒红色,看到酒红色后继续加热3-5min后停止加热,得到的即为直径约25nm的胶体金,冷却后4℃避光保存。
3.胶体金探针的最佳pH值
取1.5ml离心管加入1ml胶体金,将胶体金pH值调节为一系列从6到9的梯度,每两个梯度之间相差0.5。加入用0.01mol/L的PBS(pH7.2)稀释为0.2mg/ml的大鼠抗体100μl,混匀后静置2h。观察颜色变化,颜色开始发生变化的前一管的pH值再略提高一点即为胶体金的最佳pH值。结果为pH8.2。
4.标金抗体最低稳定量
在标记前,首先测定能稳定一定量胶体金所需要的最小抗体用量,测量其最小保护量必须在最佳pH值和离子浓度情况下进行。将胶体金调至pH8.2。用0.01mol/L的PBS(pH7.2)稀释大鼠抗体至0.2mg/ml。按下表操作。
表1试管观察法测定稳定胶体金最小标记量
静置后观察,含抗体量少的管呈现出由红变蓝的聚沉现象,而加入抗体达到或超过最低稳定量的试管中的溶液则保持红色不变。使红色保持不变的抗体含量最低的试管的抗体量就是抗体的最适保护量,在此基础上增加20%为稳定胶体金的抗体使用量。结果为5μg/ml。
5.胶体金探针的制备
取制备好调pH8.2的胶体金1ml置于1.5mlEP管中,加0.2mg/ml的大鼠抗体25μl,混匀5min后,加10%BSA100μl,混匀10min后12000r/min、4℃、30min离心两次,用重悬液重悬;再1000r/min、4℃、4min离心一次,上清及为胶体金探针,4℃保存。
6.胶体金试纸条的制备
胶体金试纸条由样品垫、胶金垫、NC膜和吸收垫四部分组成。样品垫和胶金垫用玻璃纤维,吸收垫用吸水滤纸。胶金垫上加胶体金探针,37℃干燥3h。NC膜上包被兔抗猪链球菌抗体(检测带)及兔抗大鼠免疫血清(质控带)。喷好膜后37℃干燥2h。将吸收垫、样品垫及处理后的胶金垫、NC膜叠加粘到PVC板上,组装成完整的试纸条,然后切割成4mm/条,干燥避光保存。
7.检测及结果判读
取40μl待测样品和40μl样品处理液加到制备好的试纸条样品垫上,10min后,检测带和质控带均出现红色的为阳性,只有质控带出现红色的为阴性,检测带和质控带均不显色则试纸条无效。见图6。
猪链表面蛋白2.SEQ
序列表
<110>中国人民解放军军事医学科学院微生物流行病研究所
<120>一种猪链球菌2型表面蛋白、其制备方法及用途
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ggggatttga atgattttaa atatcaaata aaagtggaaa actatatccg tcaggttgca 300
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gctccaactc cagtaccgga cactccggca ccgaaagagg acgaagttcc ggcaccaatt 720
ccagacgctc caacaccgaa agtagaagag gaaacacagg agccgaaaac agaagagaag 780
gcaccagaga cgaaagaaga aactccaact ccagtaccgg acactccggc accgaaagag 840
gacgaagttc cggcaccaat gccagatgct ccagcgccga aagcagagga agaagttcca 900
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gcagttccag aagtcgcacc aaaaacagcg gaaaaaccaa agctagactt cacaacaaaa 1140
gaacgcaagg tagaagaagc tctccctatc aaagaagaaa tcagatatga tgcaagccta 1200
ccgcttggca aatcatacct tcttcaagaa ggaaaagcag gtaaaaaagt atctgtttat 1260
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acaactcctt cagtacaatc aaatccgaca ctaagtcata agggcgcacc ttctgcaaac 1440
aaagcaaccc tacctgcaac aggtgaacag cgcaataacc tagccttagt aggccttggt 1500
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atctaa 1566
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atggaaactg caaataaaaa attcagatat agtattcgta aatttaaagt cggcgtaggc 60
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aagcttgtga aagaattaga aaaagagctg ggggaactag ataaggttcc aagttatggt 240
gatgctcaag attattctta tcagaaggct ttgtgggaag agtttttaag aattggaaaa 300
gataatatgg actatgcttc aaaaatgaaa gcagatgaca agtttttcca taaggttaaa 360
ggggatttga atgattttaa atatcaaata aaagtggaaa actatatccg tcaggttgca 420
gaattgcgaa agaaataccc tggtgataat acaattgagg aagaatataa tgcgcattta 480
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gatagagaag caagtgaggc catgggcaga attaagcaac gagttgctga actggaaaaa 600
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gcaccagaga cgaaagaaga aactccaact ccaacaccaa aagaggaagg gattccggca 780
ccaattccag acgctccagc gccgaaagca gaggacgaag ttccggcacc aaaagaggaa 840
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attccagacg ctccaacacc gaaagtagaa gaggaaacac aggagccgaa aacagaagag 1080
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actccggtac cggacactcc ggcaccgaaa gaggacgaag ttccggcacc aatgccagat 1200
gctccagcgc cgaaagcaga ggaagaagtt ccagccccaa ctccaatgcc agaaactcca 1260
atggacaaac cgaaaactga taaagtagag tctgacaaac agatgccaga ggctaagcaa 1320
ccagaaatgg agcaaccgaa agcagaagac atgccgaagg aggaaatgcc aaagtctgag 1380
caaccaaaag cggaagactc tgcacctaag acagcagttc cagaagtcgc accaaaaaca 1440
gcggaaaaac caaagctaga cttcacaaca aaagaacgca aggtagaaga agctctccct 1500
atcaaagaag aaatcagata tgatgcaagc ctaccgcttg gcaaatcata ccttcttcaa 1560
gaaggaaaag caggtaaaaa agtatctgtt tatcaagatg tcatagttga tggtaaagtt 1620
gtcgcaacca acctattatc agaaactgtt gttgaaggtc aaaatcgtat ccttgtaaaa 1680
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Gly Ile Pro Ala Pro Ile Pro Asp Ala Pro Ala Pro Lys Ala Glu Asp
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Gln Ser Asn Pro Thr Leu Ser His Lys Gly Ala Pro Ser Ala Asn Lys
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Ala Thr Leu Pro Ala Thr Gly Glu Gln Arg Asn Asn Leu Ala Leu Val
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Gly Leu Gly Leu Ala Gly Ile Ser Leu Ala Val Val Ala Thr Ala Ile
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Asn Lys Lys Ser Lys Asp Gln Ile
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Claims (9)
1.一种猪链球菌2型表面蛋白,具有序列表中序列4或序列6的氨基酸序列。
2.根据权利要求1的表面蛋白,其编码基因具有序列表中序列3或序列5的核苷酸序列。
3.含有权利要求1或2的表面蛋白编码基因的重组质粒或工程菌株。
4.根据权利要求3的重组质粒,其中所述重组质粒采用pET32a或pET28a。
5.权利要求1所述表面蛋白的制备方法,包括如下步骤:
(1)克隆该表面蛋白基因;
(2)将基因与大肠杆菌表达质粒相连接,转化大肠杆菌;
(3)诱导表达;
(4)对表达产物进行分离纯化。
6.根据权利要求3所述方法,其中大肠杆菌表达质粒为pET32a,大肠杆菌为E.coli BL21。
7.权利要求1所述的蛋白在制备猪链球菌2型诊断试剂中的应用。
8.根据权利要求5所述用途,其中所述诊断试剂为胶体金快速诊断试剂。
9.权利要求1所述的蛋白在制备猪链球菌2型疫苗中的应用。
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CN108671227A (zh) * | 2018-04-23 | 2018-10-19 | 中国科学院微生物研究所 | 一种预防猪链球菌感染的广谱多联亚单位疫苗 |
CN116281261A (zh) * | 2023-05-18 | 2023-06-23 | 眉山金豆智能科技有限公司 | 一种货物全自动装车机及其控制方法 |
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NL9100510A (nl) * | 1991-03-21 | 1992-10-16 | Centraal Diergeneeskundig Inst | Dna-sequenties die coderen voor kenmerken van virulentie van streptococcus suis en delen daarvan, daarvan afgeleide polypeptiden en antilichamen, alsmede toepassing daarvan bij de diagnose van en de bescherming tegen infectie met s. suis bij zoogdieren, waaronder de mens. |
CN101163499A (zh) * | 2005-02-11 | 2008-04-16 | Ace生物科学公司 | 表面定位的肺炎链球菌多肽 |
RS54963B1 (sr) * | 2005-09-02 | 2016-11-30 | Valorisation-Recherche Limited Partnership | Polipeptidi stereptococcus suis-a i polinukleotidi koji ih kodiraju i njihova primena u vakcinama i dijagnostici |
CN1955310B (zh) * | 2005-10-26 | 2010-11-24 | 中华人民共和国北京出入境检验检疫局 | 检测猪链球菌2型的核苷酸序列、检测试剂盒和方法 |
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2008
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CN103018440A (zh) * | 2012-11-23 | 2013-04-03 | 河南科技学院 | 一种氯羟吡啶胶体金层析检测试纸条或卡 |
CN108671227A (zh) * | 2018-04-23 | 2018-10-19 | 中国科学院微生物研究所 | 一种预防猪链球菌感染的广谱多联亚单位疫苗 |
CN108671227B (zh) * | 2018-04-23 | 2020-05-22 | 中国科学院微生物研究所 | 一种预防猪链球菌感染的广谱多联亚单位疫苗 |
CN116281261A (zh) * | 2023-05-18 | 2023-06-23 | 眉山金豆智能科技有限公司 | 一种货物全自动装车机及其控制方法 |
CN116281261B (zh) * | 2023-05-18 | 2023-09-26 | 眉山金豆智能科技有限公司 | 一种货物全自动装车机及其控制方法 |
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