百日咳杆菌69KDa外膜蛋白的分离纯化及生物学特性研究

本文发展了一种从百日咳杆菌Ⅰ相菌株中纯化６９ＫＤａ外膜蛋白的简易方法,将细菌体经加热浸提、乙醇沉淀蛋白、ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ５０柱层析精制而成。用ＳＤＳ－ＰＡＧＥ、免疫印迹、光密度仪扫描分析,证明纯化制剂为均一的、特异性的６９ＫＤａ外膜蛋白,其收率为５４．２％,纯度达９９．２％,每微克６９ＫＤａ蛋白制剂中的内毒素含量低于０．８５ＥＵ;ＰＴ残留量小于０．１０５ｎｇ。抗６９ＫＤａ蛋白抗血清能     

百日咳杆菌丝状血凝素的纯制及其生物学特性的研究

介绍了两种纯制百日咳杆菌丝状血凝素的方法，并对不同方法纯化的丝状血凝素的产量、纯度、生物学活性等方面进行了比较，从中选择出一种纯度高、活性好、可以大批量提纯的方法。     

家兔支气管败血波氏杆菌重组百日咳杆菌粘附素（PRN）蛋白的原核表达及其间接ELISA方法的建立

目的表达支气管败血波氏杆菌（Bordetella bronchiseptica,Bb）PRN蛋白,并以此建立检测Bb抗体的间接ELISA方法。方法参照GenBank公布的猪源支气管败血波氏杆菌prn基因序列（AY376325）设计了一对特异性引物,PCR扩增出相应的核苷酸片段。将PCR扩增产物连接至原核表达载体pGEX-4T-1中,以E.coli BL21（DE3）为表达菌株进行诱导表达,以纯化重组蛋白PRN作为诊断抗原,通过探索最佳抗原包被量和抗体血清稀释倍数等,建立检测支气管败血波氏杆菌抗体的间接ELISA方法。结果成功克隆了prn全基因序列,并在E.coliBL21（DE3）中获得高效表达,经SDS-PAGE、Western blot分析显示重组蛋白PRN具有良好的抗原性。应用重组蛋白PRN为抗原建立了检测Bb血清抗体的间接ELISA诊断方法。试验确定重组蛋白PRN抗原的包被浓度为500ng/mL,最适血清稀释度为1∶40。结论建立的ELISA检测方法,不仅为Bb抗体检测提供了实用的血清学检测手段,也为进一步开发Bb检测试剂盒奠定了基础。     

百日咳杆菌去内毒素及保护性抗原的纯化

内毒素(Endotoxin,简称ET)是百日咳全菌苗(Bordetellapertussis vaccine)产生副作用的主要毒素之一,且不易除去。现有的分离方法,如蔗糖密度梯度离心法,较繁琐,成本高。本文采用Sepha-cryl S-300凝胶层折法可以简便有效的去除大部分内毒素。初步毒素试验结果表明:已达到日本生物制品规格的要求。两种保护性抗原FHA和LPF-HA也得到进一步分离纯化,为今后研制高效的百日咳组分菌苗提供了实验条件。     

百日咳杆菌粘附素中针对支气管败血波氏杆菌的保护性抗原肽的筛选

【目的】本研究通过百日咳杆菌黏附素(PRN)基因的分段克隆表达及其在BALB/c小鼠的主动和被动免疫保护试验筛选PRN中的保护性抗原肽。【方法和结果】利用大肠杆菌进行PRN的完整蛋白、N端和C端多肽及其RⅠ和RⅡ区域多肽(双拷贝)的表达,命名为GST-PRN、GST-PN、GST-PC、GST-2PRⅠ和GST-2PRⅡ。Westernblot检测证实5种表达产物均具有良好的反应原性。在主动免疫保护试验中,5种表达产物均能诱导小鼠产生较高的PRN抗体水平;当使用3LD50的支气管败血波氏杆菌(Bb)强毒株HH0809进行鼻腔攻击后,GST-2PRⅠ的保护率为66.7%(6/9),其余4者的保护率均为100%(9/9);当使用10LD50的HH0809攻击时,其保护率分别为77.8%(7/9)、33.3%(3/9)、66.7%(6/9)、10%(1/10)和60%(6/10)。在被动免疫保护试验中,当使用10LD50的HH0809进行腹腔攻击时,GST-2PRⅠ抗血清对小鼠的保护率为20%(2/10),其余4者的保护率均为100%(10/10);但经天然PRN吸附后的5种兔抗血清均无任何保护力(0/5)。【结论】本研究表明PRN的N端和C端表达多肽均具有较强的针对Bb的保护力,且C端强于N端;而C端RⅡ区域的保护力也显著强于N端RⅠ区域。     

小麦类脱水素的表达、纯化及多克隆抗体的制备

脱水素在胚胎发育后期累积,外源脱落酸(ABA)、低温、干旱和其他一些环境条件下能诱导脱水素的产生,尽管植物在脱水条件下脱水素广泛存在于细胞中,但其生化功能仍不清楚.为研究小麦在不同时期脱水素基因的表达情况和生物学功能及抗体制备,以小麦幼芽为材料,经干旱胁迫处理后,提取总RNA,通过RT-PCR得到小麦类脱水素基因片段(WZY1-1),再连接至克隆载体PUCM-T,并成功构建重组表达质粒PET-32a( )-wzy1-1,将阳性重组质粒转化于受体菌BL21(DE3)感受态细胞中,经IPTG诱导表达,进行表达产物的聚丙烯酰胺凝胶电泳(SDS-PAGE)检测.结果表明,表达蛋白位于37ku处,小麦类脱水素基因获得高效表达.表达蛋白经Ni2 琼脂糖凝胶亲和层析和透析袋电洗脱法纯化后,对兔子进行免疫,制备的抗血清通过ELISA检测到较高的多克隆抗体效价.蛋白质印迹结果显示,利用纯化的蛋白质制备的兔抗血清可以很好地和所表达的蛋白质带特异性结合,且郑引1号小麦幼苗进行干旱处理,提取粗蛋白,SDS-PAGE,蛋白质印迹检测显示,在分子质量28ku处出现特异的蛋白质条带,这说明所制备的抗血清可以与小麦叶片所表达的dehydrin蛋白特异性结合,证明其具有良好的免疫原性.     

巨大芽孢杆菌淀粉酶基因的克隆及其在枯草杆菌中的表达

以λ噬菌体为载体,采用鸟枪法由B.megaterium基因组克隆得到了1个淀粉酶基因,并已被亚克隆到E.coli和B.subtilis中,其表达水平较B.megaterium高250倍。克隆株产生的淀粉酶对直链淀粉的早期水解产物主要为麦芽三糖和麦芽糖,随着水解时间的延长,又将它们转变为葡萄糖。同时能以麦芽三糖为底物水解为麦芽糖和葡萄糖。受体菌的平行提取物无上述水解活性。因而该酶被确定为糖化型α-淀粉酶。SDS-凝胶电泳法确定酶分子量为58000道尔顿。     

猪胸膜肺炎放线杆菌毒素ⅢA基因的克隆、序列分析及原核表达

因胸膜肺炎放线杆菌的致病性主要是由毒素决定的,故参照猪胸膜肺炎放线杆血清2型菌株的序列（GenBank L12145）设计了一对特异性引物,用PCR的方法扩增apxⅢA基因并得到了长3 466bp的片段,然后将其克隆到pMD18T中,经酶切鉴定和序列分析表明克隆是成功的;再将apxⅢA插入到原核表达载体pET28b后,转化BL21(DE3),在IPTG诱导下获得高效表达,经Western blotting检测证实表达产物有活性。以表达产物包被ELISA板,建立了特异、敏感的ELISA诊断方法。     

百日咳杆菌粘附素对猪源支气管败血波氏杆菌的保护性抗原的筛选

摘要：【目的】本研究通过百日咳杆菌黏附素(PRN)基因的分段克隆表达及其在BALB/c小鼠的主动和被动免疫保护试验筛选PRN中的保护性抗原肽。【方法和结果】利用大肠杆菌进行PRN的完整蛋白、N端和C端多肽及其RI和RII区域多肽（双拷贝）的表达,命名为GST-PRN、GST-PN、GST-PC、GST-2PRI和GST-2PRII。Western blot检测证实5种表达产物均具有良好的反应原性。在主动免疫保护试验中,5种表达产物均能诱导小鼠产生较高的PRN抗体水平;当使用3 LD50的支气管败血波氏杆菌     

Pertactin antigens extracted from Bordetella pertussis and Bordetella bronchiseptica differ in the isoelectric point

Pertactin, which is a membrane-associated antigen of Bordetella pertussis and which is present in many acellular vaccines against whooping cough, has been reported to be similar to the homologous protein in Bordetella bronchiseptica. By running parallel experiments using proteins derived from the two species, we show that the isoelectric point of pertactin from B. pertussis is lower than reported and clearly distinguishable from the homologous protein of B. bronchiseptica. Received: 9 April 1997 / Accepted: 20 May 1997     

溶液环境对百日咳疫苗分离纯化的影响

针对百日咳疫苗在低盐条件下不稳定, 容易聚集而导致层析过程收率低、分离度低的难题, 实验中选择脲作为稳定剂来改善百日咳疫苗所处的溶液环境, 并采用离子交换层析和凝胶过滤层析进行百日咳疫苗的分离纯化, 通过ELISA抗原活性测定和还原性SDS-PAGE等方法研究了脲对百日咳疫苗分离纯化的影响。结果表明, 在流动相中加入 2 mol/L脲作为稳定剂, 能显著提高离子交换层析和凝胶过滤层析中的PT和FHA活性回收率、凝胶过滤层析的分离度、PT和FHA的纯度。这些结果对百日咳疫苗的分离纯化和层析工艺优化提供了重要的依据和参考。     

Variability in LPS composition, antigenicity and reactogenicity of phase variants of Bordetella pertussis

Comparison of lipopolysaccharides (LPS) from phase variants of different strains of Bordetella phase variants of different strains of Bordetella pertussis has shown a difference in their composition, antigenicity and reactogenicity. Phase I variants of B. pertussis, with the exception of strain 134, contain a preponderance of LPS I whereas the major component of LPS of phase IV variants is LPS II. Sera raised to LPSs of phase I strains, other than 134, cross-react with each other but not with phase IV LPSs; and similarly all sera raised to phase IV LPSs cross-react with each other and with LPS from 134 phase I. The LPSs of all phase I variants, including that of 134, are approximately ten-fold or more reactive in the limulus amoebocyte lysate assay (LAL) than phase IV LPSs. In the human mononuclear cell pyrogen assay phase IV LPSs also stimulated a lower response than phase I LPSs. The B. pertussis phase I LPSs are 10-times more reactive than Escherichia coli standard endotoxin in the LAL assay but 100-times less reactive than E. coli LPS in the monocyte test for pyrogen. The SDS-PAGE profiles of B. pertussis LPSs are quite different from those of B. parapertussis and B. bronchiseptica strains. B. pertussis LPSs produced a typical lipo-oligosaccharide (LOS) pattern. B. bronchiseptica LPS produced a similar pattern but was antigenically distinct from B. pertussis LPSs I and II. B. parapertussis in contrast produced a ladder pattern typical of smooth type LPS.     

Purification and analysis of the antigenicity of a 69000 Da protein from Bordetella pertussis

Abstract A purification scheme was devised for a 69-kDa outer membrane protein of Bordetella pertussis , a virulence-associated protein which may play a role in the pathogenesis of the organism. The protein was purified to apparent homogeneity by heating B. pertussis cells for 1 h at 60°C followed by DEAE-Sepharose and Affi-Gel Blue chromatography. Antibodies found in sera obtained from patients diagnosed as having pertussis reacted with this protein. This purification scheme should be useful for the production of the 69 kDa protein which is currently being evaluated as a pertussis vaccine candidate.     

Proteomic Adaptation of Australian Epidemic Bordetella pertussis

Bordetella pertussis causes whooping cough. The predominant strains in Australia changed to single nucleotide polymorphism (SNP) cluster I (pertussis toxin promoter allele ptxP3/pertactin gene allele prn2) from cluster II (non‐ptxP3/non‐prn2). Cluster I was mostly responsible for the 2008–2012 Australian epidemic and was found to have higher fitness compared to cluster II using an in vivo mouse competition assay, regardless of host's immunization status. This study aimed to identify proteomic differences that explain higher fitness in cluster I using isobaric tags for relative and absolute quantification (iTRAQ), and high‐resolution multiple reaction monitoring (MRM‐hr). A few key differences in the whole cell and secretome were identified between the cluster I and II strains tested. In the whole cell, nine proteins were upregulated (>1.2 fold change, q < 0.05) and three were downregulated (<0.8 fold change, q < 0.05) in cluster I. One downregulated protein was BP1569, a TLR2 agonist for Th1 immunity. In the secretome, 12 proteins were upregulated and 1 was downregulated which was Bsp22, a type III secretion system (T3SS) protein. Furthermore, there was a trend of downregulation in three T3SS effectors and other virulence factors. Three proteins were upregulated in both whole cell and supernatant: BP0200, molybdate ABC transporter (ModB), and tracheal colonization factor A (TcfA). Important expression differences in lipoprotein, T3SS, and transport proteins between the cluster I and II strains were identified. These differences may affect immune evasion, virulence and metabolism, and play a role in increased fitness of cluster I.     

Protective effects of in vivo‐expressed autotransporters against Bordetella pertussis infection

Bordetella pertussis causes whooping cough, a severe and prolonged respiratory disease that results inhas high morbidity and mortality rates, particularly in developing countries. The number incidence of whooping cough cases is increasing in many countries despite high vaccine coverage. Causes for the re‐emergence of the disease include the limited duration of protection conferred by the acellular pertussis vaccines (aP)s and pathogenic adaptations that involve antigenic divergence from vaccine strains. Therefore, current vaccines therefore need to be improved. In the present study, we focused on five autotransporters: namely SphB1, BatB, SphB2, Phg, and Vag8, which were previously found to be expressed by B. bronchiseptica during the course of infection in rats and examined their protective efficiencies as vaccine antigens. The passenger domains of these proteins were produced in recombinant forms and used as antigens. An intranasal murine challenge assay showed that immunization with a mixture of SphB1 and Vag8 (SV) significantly reduced bacterial load in the lower respiratory tract and a combination of aP and SV acts synergistically in effects of conferring protection against B. pertussis infection, implying that these antigens have potential as components to for improvinge th the currently available acellular pertussis vaccine.

     

Cloning of the virulence regulatory (vir) locus of Bordetella pertussis and its expression in B. bronchiseptica

The gene coding for a thermostable alpha-amylase from Clostridium thermosulfurogenes (DSM 3896) was cloned in Escherichia coli using pUC18 as a vector. The recombinant plasmid pCT2 of an amylolytic positive transformant of E. coli contained a 2.9 kbp fragment of chromosomal DNA of C. thermosulfurogenes carrying the alpha-amylase gene. In E. coli the gene was apparently transcribed by its own promoter. Comparative studies showed no difference between the original and the heterologously in E. coli expressed enzyme. The latter was not secreted into the medium.     

Bordetella pertussis respiratory infection in C57B1/6 and Balb/c mice: Pathophysiology and immune responses

A virulent clone of Bordetella pertussis, injected intranasally into C57B1/6 or Balb/c mice, induced a respiratory tract infection that mimicked the infectious process of whooping cough. The density of the inoculum influenced the kinetics of in vivo bacterial growth, as well as the associated leucocytosis, which were of equivalent intensity in both strains of mice. Convalescing mice became resistant to re-infection, but not to the effects of the leucocytosis-promoting factor of the pertussis toxin. Prominent immune response, associated with acquired resistance, was a delayed type hypersensitivity (DTH) reaction, the intensity of which depended upon the genotype of the mice when the eliciting antigen contained the pertussis toxin in a biologically active form. Intranasal infection of congenic mice may represent an improved quantitative test for reproducible measurement of virulence and immunogenicity of B. pertussis.