CN116063582A - 血清型b型类鼻疽菌的特异性o-抗原及其提取方法和应用 - Google Patents
血清型b型类鼻疽菌的特异性o-抗原及其提取方法和应用 Download PDFInfo
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Abstract
本发明提供了一种血清型B型类鼻疽菌的特异性O‑抗原及其提取方法和应用,本发明提供的一种由鼠李糖、半乳糖和木糖构成的多糖抗原可作为B型血清型类鼻疽菌的诊断候选抗原以及多糖结合疫苗候选抗原方面的应用,弥补当前只有基于A型类鼻疽菌的诊断抗原和多糖结合的疫苗,导致诊断和防治范围有限,防护力度不足的问题。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及一种血清型B型类鼻疽菌中脂多糖(Lipopolysaccharide,LPS)的特异性O-抗原及其提取方法和应用。
背景技术
类鼻疽伯克霍尔德菌(Burkholderia pseudomallei,BP)简称类鼻疽菌,引起以肝、肺等脏器多发性脓肿为特点的类鼻疽疾病。类鼻疽菌可以通过破损皮肤、吸入或摄入等方式感染人和动物,导致从亚临床感染到重症败血症的多种临床表现,死亡率较高(泰国约为40%,在澳大利亚约为15%)。类鼻疽菌具有广泛耐药性,目前尚无有效疫苗,已被WHO列为B 类生物恐怖剂。
类鼻疽菌在入侵宿主细胞的过程中,会通过多种复杂机制抵御宿主的免疫清除作用,而脂多糖(Lipopolysaccharide,LPS)作为类鼻疽菌外膜的主要成分,在其刺激宿主产生先天免疫应答的过程发挥重要作用。LPS由三部分构成:脂质A(细菌内毒素)、核心寡糖(外核和内核)以及特异性O-抗原。一般脂质 A在不同菌株中是保守的,但O-抗原在物种内结构上具有多样性,并可基于此对细菌按血清型进行分类。O-抗原的分子结构和组成对于类鼻疽菌的血清型分类、诊断和流行病学监测非常重要。前期的研究报道揭示了包括A、B和B2在内的多种类鼻疽菌LPS基因型,并确定了已知或预测参与O-抗原生物合成的基因多样性。文献报道显示,在东南亚和澳大利亚,大多数类鼻疽病是由A型类鼻疽菌引起,进而推测基于A型LPS的多糖结合疫苗可能具有潜在的免疫保护效果。然而,研究表明由于O-抗原的结构不同,来自不同LPS类型(A型和B型)的抗原与抗体之间不存在交叉反应,对于A型和B型类鼻疽菌同时存在的国家和地区,例如澳大利亚阿纳姆和凯瑟琳地区,以及我国海南等地区,仅基于A型LPS 的多糖结合疫苗无法提供有效的免疫保护效果。迄今为止,仅鉴定得知了A型 O-抗原的分子结构为[→3-β-D-Glc-1→3-α-L-6-脱氧-Tal-1→]的重复单元组成的线性聚合物,其中部分6-脱氧-Tal残基存在4-O-乙酰基、2-O-甲基和/ 或2-O-乙酰基修饰基团。截止目前,尚无其他血清型类鼻疽菌O-抗原具体结构信息的相关报道报道,尤其对于我国类鼻疽主要流行区域海南等地类鼻疽菌血清型的分布情况尚不清楚,因此,当前亟需建立纯化获得类鼻疽菌特异性O-抗原的方法,并明确A型O-抗原之外的其他血清型O-抗原的结构特征究。
发明内容
本发明提供了一种类鼻疽菌脂多糖特异性O-抗原的提取、纯化方法,鉴定出一种由鼠李糖、半乳糖和木糖构成的多糖抗原可作为类鼻疽的血清学诊断和候选疫苗抗原方面的应用。
本发明首先提供了一种血清型B型类鼻疽菌的特异性O-抗原,其包含由 [→4)-α-L-Rhap(1→4)-α-L-Rhap(1→2)-α-L-Rhap(1→2)-α-L- Rhap(1→3)-α-L-Rhap(1→3)-α-L-Rhap(1→4)-α-L-Rhap(1→6)-α-D- Galp(1→]n构成的主链,其中,Rhap的O-2或O-3位链接t-β-D-Xylp侧链, Galp的O-3位链接t-β-D-Galp侧链。
本发明的另一方面提供了上述O-抗原的提取方法,其包括:
一种血清型B型类鼻疽菌的特异性O-抗原的提取方法,其特征在于,包括:
1)将血清型B型类鼻疽菌于LB液体培养基中培养增殖,然后,取菌液离心后得到菌体沉淀;
2)加入浓度不低于2%的苯酚水溶液将菌体灭活,得到灭活后的菌体溶液;
3)向所述灭活后的菌体溶液中加入苯酚,至苯酚终浓度为50%,然后于65℃磁力搅拌提取,得到初提混合液;
4)将所述初提混合液离心,收集上清液;
5)将所述上清液置于截流量为3500Mw透析袋中,透析除去苯酚,得到透析液;
6)将所述透析液浓缩后冻干,得到粗提物;
7)将所述粗提物复溶,然后先加入DNaseI和RNaseA使其终浓度为50 μg/mL,于37℃进行酶解,再加入Proteinase K于60℃进行酶解;
8)酶解处理后,冷却至室温后离心,将上清以截留分子量为3500Da的透析袋进行透析处理;
9)透析完成后,回收袋内液体经浓缩、冻干即得脂多糖样品。
在根据本发明的一个实施方案中,步骤2)中所述的灭活是通过包括下述步骤的方法实现的:
向菌体沉淀中加入浓度为2%的苯酚溶液,充分混匀后于室温处理30min。
在根据本发明的一个实施方案中,步骤7)中加入DNaseI和RNaseAg进行的酶解是在37℃搅拌2h。
在根据本发明的一个实施方案中,步骤7)中加入ProteinaseK进行的酶解是于60℃搅拌3h。
在根据本发明的一个实施方案中,酶解后于80℃处理30min使酶失活。
在根据本发明的一个实施方案中,还包括:
10)将所述脂多糖样品溶于2%乙酸溶液中,至终浓度10mg/mL,然后,加热处理2h,得到悬浊液;
11)将所述悬浊液离心,得到上清液,将所述上清液经G-10脱盐柱纯化,即得多糖组分;
12)将所述多糖组分经分子筛层析,即得O-抗原纯品。
在根据本发明的一个实施方案中,所述分子筛层析是通过包括下述条件的方法实现的:
以0.15M NaCl溶液为洗脱液,以流速0.15mL/min过夜洗脱,收集洗脱液。采用苯酚-硫酸法测定并绘制洗脱曲线,收集主要洗脱峰,透析浓缩、冻干,即得O-抗原纯品。
本发明进一步提供了上述的O-抗原在制备用于诊断、预防或治疗血清型B 型类鼻疽菌感染中的试剂或药物中的应用。
本发明的上述技术方案的有益效果如下:
本发明基于我国类鼻疽菌临床分离株获得了血清型B型类鼻疽菌的特异性 O-抗原,并提供了类鼻疽菌的特异性O-抗原的纯化方法,首次鉴定出主链由 [→4)-α-L-Rhap(1→4)-α-L-Rhap(1→2)-α-L-Rhap(1→2)-α-L- Rhap(1→3)-α-L-Rhap(1→3)-α-L-Rhap(1→4)-α-L-Rhap(1→6)-α-D- Galp(1→]n构成,其中Rhap的O-2或O-3位链接t-β-D-Xylp侧链,Galp的O- 3位链接t-β-D-Galp侧链的B型O-抗原。该特异性O-抗原制备的抗血清能够识别B型血清型类鼻疽菌,可用于研发类鼻疽菌血清学诊断试剂盒或作为疫苗研发候选抗原。
附图说明
图1为本发明提供的BPC004菌株脂多糖特异性O-抗原基因型鉴定结果,为 B型;
图2为本发明提供的BPC004-OPS经分子筛层析纯化后的洗脱曲线图;
图3为本发明提供的BPC004-OPS-b的高效凝胶渗透色谱图;
图4为本发明提供的BPC004-OPS-b的甲基化分析GC-MS谱图;
图5为根据本发明实施例得到的BPC004-OPS-b的1D-NMR图谱;其中,A为1H NMR图谱,B为13C-NMR图谱;
图6为根据本发明实施例得到的BPC004-OPS-b的COSY图谱;
图7为根据本发明实施例得到的BPC004-OPS-b的TOCSY图谱;
图8为根据本发明实施例得到的BPC004-OPS-b的HSQC图谱;
图9为根据本发明实施例得到的BPC004-OPS-b的2D-NMR图谱;其中,A为 NOESY图谱;B为HMBC图谱;
图10为根据本发明实施例得到的BPC004-OPS-b(B型O-抗原)的抗血清效价图;
图11为根据本发明实施例得到的BPC004-OPS-b(B型O-抗原)抗血清的特异性及交叉反应性结果图,其中,A为BPC004-OPS-b(B型O-抗原)抗血清的特异性检测结果图;B为BPC004-OPS-b(B型O-抗原)抗血清与不同类鼻疽菌临床菌株的交叉反应结果图。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合附图及具体实施例进行详细描述。
实施例1类鼻疽菌脂多糖基因型的鉴定
从超低温冰箱(-80℃)取出类鼻疽菌株BPC004(来自海南三亚市人民医院标本库,在本实验室之前的研究报道中,所有临床菌株均已使用VITEK-2鉴定系统进行了鉴定,并进行了MLST分型)的甘油菌种,在LB固体平板上复苏,然后挑取单菌落于10mL LB液体培养基中,37℃,200rpm增菌培养8h。离心 (8000rpm,5min),收集菌体。类鼻疽菌株BPC004基因组的提取使用TIANGEN 生化科技有限公司的试剂盒,按照参考说明书进行提取,提取后测定DNA含量及纯度。分段设计Type B型O-抗原基因簇引物(表1),以BPC004全基因组 DNA为模板,经PCR扩增得到目的基因(每段约4000bp、2500bp,图1),说明BPC004菌株O-抗原基因型为B型。
表1 BPC004菌株LPS基因型鉴定引物
实施例2类鼻疽菌B型脂多糖的提取
将-80℃保存的类鼻疽菌BPC004采用三线法复苏于LB固体平板,在恒温培养箱培养18h后,挑取单菌落于400mL LB液体培养基中过夜增菌培养,次日,将菌液离心(8000rpm,5min)弃上清,向菌体沉淀中加入PBS缓冲液(0.01 M,pH 7.4)重悬菌体后再次离心(8000rpm,5min)弃上清,最后向菌体沉淀中加入浓度2%的苯酚(阿拉丁生化科技有限公司)溶液,充分混匀后于室温处理30min使菌体灭活(取少量菌液涂布LB固体平板,培养48h乃至更长时间,发现平板上无细菌生长,说明菌体灭活处理成功)。将处理后的菌液转移至烧杯中,继续加入苯酚,使苯酚溶液终浓度为50%,于65℃磁力搅拌提取10min,离心(12000rpm,15min),收集上清液,转移至截流量为3500Mw透析袋,透析至袋内溶液中无苯酚为止,收集透析液浓缩后冻干。粗提物复溶后加入 DNaseI(美国Sigma)和RNaseA(美国Sigma)使其终浓度为50μg/mL,于37℃磁力搅拌2h,再加入ProteinaseK(美国Sigma)于60℃磁力搅拌3h,最后于80℃处理30min使酶失活,冷却至室温后离心(15000rpm,10min),收集上清转移至透析袋(截留分子量为3500Da)(北京索莱宝)进行透析处理,回收袋内液体经浓缩、冻干后得脂多糖(命名为BPC004-LPS)。
实施例3类鼻疽菌O-抗原的纯化
1)称取上一步提取的脂多糖样品BPC004-LPS溶于2%乙酸溶液(美国Sigma) 中,使其终浓度为10mg/mL,于100℃加热处理2h,冷却至室温后离心 (20,700×g,5min),除去沉淀部分类脂A,收集上清液经G-10脱盐柱(GE PD MidiTrap)(美国GE公司)纯化获得多糖部分(命名为BPC004-OPS)。
2)将多糖组分BPC004-OPS利用分子筛层析(Sepharose Cl-6B)(美国GE 公司)根据分子量的差异进行纯化,利用0.15M NaCl溶液过夜洗脱,流速设定为0.15mL/min,收集洗脱液。采用苯酚-硫酸法测定并绘制洗脱曲线,收集主要的洗脱峰BPC004-OPS-b(图2),经透析、浓缩后冻干备用。
3)称取1~2mg BPC004-OPS-b溶于0.15M NaCl溶液中,充分混匀后离心取上清,用0.22μm滤膜过滤后上TSK-gel G-3000PWXL(7.8×300mm,TOSOH, Japan)色谱柱,结合示差折射率检测器(RAD-10A),测定纯化多糖抗原的均一性和分子量分布,结果表明只出现一个对称分布的色谱峰(图3),说明纯化后的成分相对均一,经计算其分子量大小约为40.2kDa。
实施例4类鼻疽菌O-抗原的单糖组成分析
准确称取1mg BPC004-OPS-b于干净的样品瓶中,加入1mL 2mol/L醇制标准滴定液(厦门海标科技有限公司)后充N2封管,在80℃进行甲醇解反应8 h,待样品恢复至室温后用空气泵反复吹干,加入1mL 2mol/L三氟乙酸(TFA) (阿拉丁生化科技有限公司),在120℃进行水解反应1h,待样品恢复至室温后反复加入无水乙醇蒸除残余的TFA。加入1mL 20mg/mL的NaBH4(天津市科密欧化学试剂厂)溶液,在室温下搅拌还原8h,接着加入50μL 50%乙酸调节整个体系pH至中性,然后加入阳离子交换树脂(上海麦克林生化科技有限公司)除去整个体系中的钠离子,室温下搅拌处理2h,过滤(除去树脂),向滤液中反复加入无水甲醇(阿拉丁生化科技有限公司),用空气泵吹干以去除多余的硼酸。向样品中分别加入0.5mL无水吡啶(广东化学试剂开发中心)和乙酸酐(成都科隆化学品厂),充N2封管,在100℃乙酰化反应2h,冷却至室温后反复加无水乙醇用空气泵吹干以去除多余的残留试剂。加入800μL二氯甲烷 (成都科隆化学品厂)萃取样品瓶中的产物,萃取液过滤后进行GC-MS(AgilentTechnologies 7890B-5977B,美国安捷伦)上机分析。仪器设置梯度升温程序为: 80℃持续1min;5℃/min升温至210℃持续1min;10℃/min升温至260℃持续4min。
GC-MS分析结果表明BPC004-OPS-b主要由L-Rha,D-Gal和D-Xyl组成,其摩尔比为4.42:1.0:1.0,此外,还含有少量D-deoxy-manno-Hep。
实施例5类鼻疽菌0-抗原的甲基化分析
1)称取10mg纯化后的多糖组分BPC004-OPS-b,加入1mL DMSO,充N2封管,室温下磁力搅拌30min使之充分溶解。
2)NaOH-DMSO混悬液的制备:取100μL NaOH溶液(50%)、200μL甲醇和6mL二甲亚砜(DMSO)(美国Sigma),经混匀、离心、弃上清处理后,沉淀用6mL DMSO重悬再次离心处理(重复此步骤3次),最后取2mL DMSO重悬后的液体于4℃保存备用。
3)取等体积NaOH-DMSO混悬液加到样品溶液中,充N2封管,室温下磁力搅拌1h,然后在避光条件下逐滴加入2mL碘甲烷(美国Sigma),冰浴条件下准确反应30min,再加入3mL蒸馏水终止反应,最后用自来水、一级水分别透析 24h后再浓缩冻干。
4)重复步骤3),加入等体积的二氯甲烷剧烈搅拌30min,待静置分层后,水层在上,二氯甲烷层在下,甲基化后的多糖易溶于二氯甲烷层,收集下层溶液置于干净的小瓶中(重复该步骤三次),合并所得萃取液。再用蒸馏水反萃取二氯甲烷层三次,弃去水层(重复该步骤三次),将最后所得的萃取液用空气泵吹干,加入1mL蒸馏水溶解甲基化后的产物,冻干备用。
5)甲基化后的BPC004-OPS-b产物进行红外光谱(美国安捷伦)分析,检测甲基化后的产物在3400cm-1处是否有明显的羟基吸收峰。
6)甲基化多糖的水解、还原和乙酰化:向上述干燥的甲基化糖样中加入2 mL混合酸(HCOOH:CF3COOH:H2O=3:1:2),充N2密封,100℃水解6h,然后用无水乙醇蒸除混合酸至pH=7。加入35mg/mL NaBH4溶液1mL(现用现配) 室温搅拌还原12h,加入约100μL 50%冰乙酸中和至中性,加入适量强酸型阳离子交换树脂,磁力搅拌2h,过滤(以除去树脂),向滤液中加入甲醇蒸除硼酸。加乙酸酐、无水吡啶各0.5mL,N2密封,100℃反应2h,反复加无水乙醇蒸除乙酸酐,用800μL二氯甲烷溶解样品瓶中的产物后用0.22μm滤膜过滤后进行GC-MS上机分析。
红外光谱结果显示甲基化后的BPC004-OPS-b在3400cm-1处的羟基吸收峰消失,说明多糖被完全甲基化。接着,甲基化产物经混合酸水解、还原和乙酰化修饰后,进行GC-MS分析,结果如图4和表2所示。BPC004-OPS-b中糖链的连接方式主要为:1,4-连接的Rhap(19.8%)、1,2-连接的Rhap(16.4%)、1,3,4- 连接的Rhap(9.8%)、1,3-连接的Rhap(6.3%)、和1,2,3-连接的Rhap(3.4%);此外,还存在1,3-连接的6-deoxy-Hepp(19.7%),1,3,6-连接的Galp(8.8%),非还原末端Galp(6.3%)和Xylp(9.5%)。
表2 GC-MS分析BPC004-OPS-b的连接方式
实施例6类鼻疽菌0-抗原的核磁谱分析
称取20mg冻干的多糖样品BPC004-OPS-b溶于D2O中(美国Sigma,99.8%),使其终浓度为40mg/mL,进行1H-NMR、13C-NMR、COSY、TOCSY、HSQC、NOESY、 HMBC核磁谱(BrukerAvance 600MHz spectrometer,Germany)测定。
利用1D/2D NMR对BPC004-OPS-b的结构特征进行深入分析,结果如下:1H- NMR谱中显示了较复杂的异头质子信号,但有10个异头质子信号可以被明确识别(A-I',图5的A,表3)。位于高场的信号峰2.06ppm、1.25ppm和1.20 ppm归属为O-乙酰基和Rhap残基的甲基质子(H-6)。为了便于讨论,根据异头质子的化学位移递减顺序,将糖残基依次标记为A、B、C、C'、D、E、F、G、H、 I和I'。在13C-NMR谱中(图5的B),糖残基的几个异头碳信号出现在102.26ppm~94.96ppm范围内,位于高场的信号峰18.85ppm和15.48ppm归属为α-Rhap残基的C-6位(甲基),而位于19.06ppm(CH3CO)和172.19ppm(CH3 CO) 的信号归因于O-乙酰基。
COSY谱中,每个残基的H-1至H-2的交叉信号可以很容易地观察到,进而从H-2开始,其他的质子信号也可以根据交叉峰来定位(图6)。将TOCSY和 COSY光谱联合起来进行分析,对于残基A-G,在TOCSY谱中,异头质子信号显示出与H-2的强烈关联,而与其他质子的关联强度较低或未显示明显交叉信号峰(图7)。对于残基H,其H-1显示出与H-2的强烈关联,从H-2信号出发,可以观察到另外两个交叉峰。对于残基I和I',可以观察到其H-1分别与H-2、H-3和H-4的交叉信号峰。对BPC004-OPS-b的COSY、TOCSY和HSQC光谱进行进一步分析,可以确定每个单糖残基的化学位移(图8,表3)。
每个糖残基的构型可以根据3JH-1,H-2和1JH-1,C-1耦合常数值来确定。糖残基A (δH/δC,5.11/99.82ppm)和D(δH/δC,5.03/99.76ppm)被确定为1,2- Rhap,根据1JH-1,C-1值和NOESY光谱中H-1和H-2的关联性,该残基为α构型。在HSQC谱中可以明显观察到二者H-2/C-2位的化学位移分别为3.98/76.46ppm 和4.02/76.92ppm。糖残基B(δH/δC,5.07/99.23ppm)被确定为2,3-取代的L-Rhap,其C-2和C-3位的化学位移分别为77.45ppm和74.56ppm。残基E (δH/δC,4.96/101.06ppm)被确定为3,4-取代的L-Rhap,其C-3和C-4位的化学位移分别为76.11ppm和82.08ppm。残基F(δH/δC,4.92/101.33 ppm)被确定为4-取代的L-Rhap,其H-4/C-4的化学位移为3.53/82.08ppm。残基G(δH/δC,4.90/101.14ppm)被确定为3-取代的L-Rhap,其H-3/C-3 的化学位移为3.80/77.20ppm。残基C和C'的H-1/C-1信号均为5.04/101.16 ppm,其中残基C的C-3和C-6的化学位移分别位于低场3.92/76.86ppm和 3.87;3.78/66.17ppm,被确定为3,6-取代的D-Galp单元;而残基C'的C-3和 C-6信号分别在3.97/69.64ppm和3.82;3.63/59.92ppm,被确定为非还原末端 Galp残基。根据B、C/C'、E、F和G单元的1JH-1,C-1耦合常数分别为175.5、176.5、 175.7、175.5和175.3Hz,可以确定所有的糖类残基都具有α构型。根据位于高场的H-1信号峰(4.85ppm)和位于低场的C-3化学位移(3.91/77.65ppm),以及1JH-1,C-1耦合常数(165Hz),残基H(δH/δC,4.85/94.96ppm)被确定为 3-取代的β-D-6-脱氧-Hepp单元。COSY和TOCSY光谱显示,该区域位于低场的信号峰(5.23ppm)不是异头质子,而是被乙酰基取代的H-2。追踪TOCSY谱中这个自旋体系的交叉峰显示,其包含一个脱氧亚甲基(H6a;6b-δH/δC,2.09; 1.65/32.31ppm),相邻一个含氧亚甲基(H7-δH/δC,3.71/56.79ppm)。位于高场的信号峰2.06ppm归属为O-乙酰基的质子,在13C-NMR谱中,19.06ppm (CH3CO)和172.19ppm(CH3 CO)的信号被归属于O-乙酰基。HMBC中可观察到O- 乙酰基的H和H残基的C-2之间明显的耦合信号。自旋体系I(δH/δC, 4.44/102.26ppm)和I'(δH/δC,4.34/102.37ppm)被归属为非还原末端β- D-Xylp残基,其1JH-1,C-1耦合常数值为164.8Hz,H-5/C-5非对映的氧亚甲基信号分别位于3.86;3.19/63.81ppm和3.91;3.19/63.98ppm。
根据NOESY和1H-13C HMBC光谱(图9的A和B,表4)确定了BPC004-OPS- b重复单元中残基的连接序列。位于磁场的碳信号可以确定糖残基被取代的位置,包括:残基A和D的O-2位,残基F的O-4位,残基G的O-3位,残基B的O- 2位和O-3位,残基C的O-3位和O-6位,以及残基E的O-3位和O-4位,上述结果与甲基化分析结果一致。在NOESY光谱中,基于糖残基间的耦合关联信号 DH-1/AH-2以及HMBC长程耦合信号(DH-1/AC-2),证明自旋体系A在O-2位被残基D取代;基于长程耦合信号(AH-1/BC-3;I'H-1/BC-2)和NOE相关信号(AH- 1/BH-3;I'H-1/BH-2)证实,自旋体系B在O-2处被残基I'取代,O-3处被残基A取代;如NOE相关信号和HMBC长程耦合信号(BH-1/GH-3;BH-1/GC-3)所示,残基G在O-3处被残基B取代;基于长程耦合信号GH-1/FC-4和EH-1/FC- 4,以及相应的NOE关联信号,自旋体系F在O-4处被残基G和E取代;如NOE 相关信号和长程耦合信号(FH-1/CH-6a;FH-1/CC-6)所示,自旋体系C在O-6 处被残基F取代。此外,观察到位于低场的C-3信号峰(3.92/76.86ppm),以及C/C'残基的NOE相关信号和HMBC长程耦合信号(C'H-1/CH-3;C'H-1/CC-3),证实糖残基C在O-3处被残基C'取代。NOE相关信号(CH-1/EH4;IH-1/EH-3) 和HMBC谱的长程耦合信号(IH-1/EC-3)证明,自旋体系E在O-3处被残基I取代,O-4处被残基C取代。上述结果与甲基化分析结果一致。基于上述结果, BPC004-OPS-b结构中序列重复单元连接方式如下图所示,基于甲基化分析结果和BPC004-OPS-b分子量的综合分析,BPC004-OPS-b的重复单元数量约为27个。
表3 BPC004-OPS-b的HSQC谱化学位移
表4 BPC004-OPS-b的HMBC和NOESY谱信号关联
实施例7类鼻疽菌B型O-抗原的免疫原性检测
将纯化的B型O-抗原BPC004-OPS-b溶液与等体积的无菌PBS溶液和2倍体积铝佐剂(奥默生物,Abmole,M9831)混合,在室温下震荡2h,使铝佐剂充分吸附多糖抗原。待离心(12000rpm,5min)后取上清液,经苯酚-硫酸法检测不含多糖后,取上述混悬液经皮下注射免疫小鼠(50μg/只)。初次免疫后第14、28天和35天分别进行第二次、第三次免疫和第四次免疫(初次实施免疫的时间记为第0天),免疫完成后,采用眼球取血的方式收集小鼠全血,分离血清备用。O-抗原抗血清效价检测(间接ELISA法):将BPC004-OPS-b以5 μg/mL的浓度包被96孔板(美国Costa公司),于4℃包被过夜,5%BSA(广州赛国生物科技有限公司)溶液37℃封闭2h,加入O-抗原抗血清,同时以 PBS组小鼠抗血清为阴性对照,包被液为空白对照,抗体稀释度分别设置为1: 1000,1:2000,1:4000,1:8000,1:16000,1:32000和1:64000,于37℃反应1h,加入1:5000稀释的山羊抗小鼠二抗(赛默飞Invitrogen),TMB(上海生工)显色后在450nm处检测其吸光度值。通过GraphPad Prism7.0软件作图并计算各稀释度下的P/N值,以P/N值大于2.0者为阳性,P/N值接近2.0的抗血清稀释度为该抗体效价。
结果表明多糖抗血清效价在1:32 000左右(图10)。
实施例8血清学交叉反应
将12株类鼻疽菌临床菌株(BPC003、BPC004、BPC006、BPC010、BPC013、 BPC014、BPC016、BPC018、BPC046、BPC080、BPC084和BPC158)(收集自海南省三亚市人民医院),以及泰国伯克霍尔德菌(Burkholderia thailandensis, Bt)、洋葱伯克霍尔德菌(Burkholderia cepacia,Bc)、大肠杆菌(Escherichia coli,Ec)、金黄色葡萄球菌(Staphylococcus aureus,Sa)和铜绿假单胞菌 (Pseudomonas aeruginosa,Pa)(均购自北京北纳创联生物技术研究院)于 37℃过夜培养,经超声破碎后收集上清液冻干。将上述冻干粉配置成5μg/mL 溶液,以100μL/孔加入96孔板(美国Costa公司)于4℃过夜包被,5%BSA溶液37℃封闭2h,加入1:800倍稀释的小鼠抗血清Anti-BPC004-OPS-b于 37℃反应1h,加入1:5000倍稀释的山羊抗小鼠二抗于37℃反应1h,TMB (上海生工)显色后在450nm处检测其吸光度值。
检测结果表明B型O-抗原的抗血清特异性较好,不与非类鼻疽菌发生交叉反应(图11的A),可以与少部分类鼻疽菌临床菌株发生交叉反应(图11的 B),结合前期利用PCR对菌株基因型的鉴定结果,可以推测我国海南流行的类鼻疽菌中存在血清型为B型的类鼻疽菌。
结论:本发明提供了一种由类鼻疽菌脂多糖中特异性O-抗原的提取、纯化以及结构鉴定等一系列方法,并通过对其免疫学性质进行研究,本申请所表征的 B型O-抗原可能在类鼻疽菌的血清学诊断和疫苗开发方面都具有较大的应用价值。
以上所述是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明所述原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (9)
1.一种血清型B型类鼻疽菌的特异性O-抗原,其特征在于,包含由[→4)-α-L-Rhap(1→4)-α-L-Rhap(1→2)-α-L-Rhap(1→2)-α-L-Rhap(1→3)-α-L-Rhap(1→3)-α-L-Rhap(1→4)-α-L-Rhap(1→6)-α-D-Galp(1→]n构成的主链,其中,Rhap的O-2或O-3位链接t-β-D-Xylp侧链,Galp的O-3位链接t-β-D-Galp侧链。
2.如权利要求1所述的O-抗原的提取方法,其特征在于,包括:
一种血清型B型类鼻疽菌的特异性O-抗原的提取方法,其特征在于,包括:
1)将血清型B型类鼻疽菌于LB液体培养基中培养增殖,然后,取菌液离心后得到菌体沉淀;
2)加入浓度不低于2%的苯酚水溶液将菌体灭活,得到灭活后的菌体溶液;
3)向所述灭活后的菌体溶液中加入苯酚,至苯酚终浓度为50%,然后于65℃磁力搅拌提取,得到初提混合液;
4)将所述初提混合液离心,收集上清液;
5)将所述上清液置于截流量为3500Mw透析袋中,透析除去苯酚,得到透析液;
6)将所述透析液浓缩后冻干,得到粗提物;
7)将所述粗提物复溶,然后先加入DNaseI和RNaseA使其终浓度为50μg/mL,于37℃进行酶解,再加入ProteinaseK于60℃进行酶解;
8)酶解处理后,冷却至室温后离心,将上清以截留分子量为3500Da的透析袋进行透析处理;
9)透析完成后,回收袋内液体经浓缩、冻干即得脂多糖样品。
3.如权利要求2所述的提取方法,其特征在于,步骤2)中所述的灭活是通过包括下述步骤的方法实现的:
向菌体沉淀中加入浓度为2%的苯酚溶液,充分混匀后于室温处理30min。
4.如权利要求2所述的提取方法,其特征在于,步骤7)中加入DNaseI和RNaseAg进行的酶解是在37℃搅拌2h。
5.如权利要求2所述的提取方法,其特征在于,步骤7)中加入ProteinaseK进行的酶解是于60℃搅拌3h。
6.如权利要求4或5所述的提取方法,其特征在于,酶解后于80℃处理30min使酶失活。
7.如权利要求2所述的提取方法,其特征在于,还包括:
10)将所述脂多糖样品溶于2%乙酸溶液中,至终浓度10mg/mL,然后,加热处理2h,得到悬浊液;
11)将所述悬浊液离心,得上清液,将所述上清液经G-10脱盐柱纯化,即得多糖组分;
12)将所述多糖组分经分子筛层析,即得O-抗原纯品。
8.如权利要求7所述的提取方法,其特征在于,所述分子筛层析是通过包括下述条件的方法实现的:
以0.15M NaCl溶液为洗脱液,以流速0.15mL/min过夜洗脱,收集洗脱液。采用苯酚-硫酸法测定并绘制洗脱曲线,收集主要洗脱峰,透析浓缩、冻干,即得O-抗原纯品。
9.如权利要求1所述的O-抗原在制备用于诊断、预防或治疗血清型B型类鼻疽菌感染中的试剂或药物中的应用。
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