重组百日咳毒素Sl亚单位的纯化及免疫原性

将重组百日咳毒素S1亚单位基因的质粒在DH5α和TOPl0两株大肠杆菌中进行表达,并对表达产物进行了初步纯化。粗制的rSl免疫家兔所得血清,用HP—PT及1B7-PT包被的ELISA测定效价,并做被动免疫保护试验。抗PT的抗体滴度为1：32000。该抗rSl血清在小鼠被动免疫保护试验中,对百日咳杆菌18323毒株进行脑腔攻击的被动免疫保护作用达到100％。证明rSl亚单位具有良好的抗原性和免疫原性,并与天然S1具有相同的抗原表位。     

重组百日咳毒素S1亚单位的纯化及免疫原性

将重组百日咳毒素S1亚单位基因的质粒在DH5α和TOP10两株大肠杆菌中进行表达,并对表达产物进行了初步纯化.粗制的rS1免疫家兔所得血清,用HP-PT及1B7-PT包被的ELISA测定效价,并做被动免疫保护试验.抗PT的抗体滴度为1:32 000.该抗rS1血清在小鼠被动免疫保护试验中,对百日咳杆菌18323毒株进行脑腔攻击的被动免疫保护作用达到100%.证明rS1亚单位具有良好的抗原性和免疫原性,并与天然S1具有相同的抗原表位.     

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     

应用纤维素结合域标签构建谷氨酸棒状杆菌表达系统

为优化谷氨酸棒状杆菌表达系统的纯化工艺,合成里氏木霉的CBD基因,将其与谷氨酸棒状杆菌分泌表达载体pXMJ19-sp连接,构建以CBD为纯化标签的重组载体pXMJ 19-sp-CBD.在该载体中插入GFP基因并转化至谷氨酸棒状杆菌,可获得分泌表达融合蛋白GFP-CBD的重组菌.该菌经IPTG诱导后的发酵液在紫外灯下显示强烈的绿色荧光,重组蛋白的分泌表达量达200 mg/L.利用CBD标签对纤维素柱的可逆性吸附,可直接对谷氨酸棒状杆菌分泌到培养基中的重组蛋白进行纯化,从而简化工艺和降低成本,为工业化大生产奠定基础.     

产气荚膜梭菌α毒素在干酪乳杆菌中的诱导表达及小鼠口服免疫保护效力的测定

摘要: 【目的】构建产气荚膜梭菌（Clostridium perfringens, C．perfringens）α 毒素基因的重组干酪乳杆菌口服疫苗,为产气荚膜梭菌毒素中毒的防治提供有效方法。【方法】将构建的重组产气荚膜梭菌α毒素基因细胞表面型载体pPG1及分泌表达载体pPG2电转化乳酸乳杆菌（Lactobacillus casei L.casei）,获得阳性重组菌pPG1-α/ L.casei 393 乳酸乳杆菌表面表达系统和pPG2-α/ L.casei393乳酸乳杆菌分泌表达系统。重组菌以1%乳糖为诱导物,在MRS培养基中进行诱导,通过Western-blot和间接免疫荧光方法鉴定,确定目的蛋白的表达。将重组菌口服免疫BALB/c小鼠,收集免疫小鼠粪便及眼冲洗液及外生殖道黏液样本测定小鼠产生抗α毒素的特异性sIgA 抗体水平,采集小鼠血液样本测定血清中抗α毒素的特异性IgG抗体水平。并对免疫小鼠进行α毒素的腹腔攻毒实验及对获得的抗血清进行α毒素中和试验测定。【结果】重组干酪乳杆菌pPG1-α/ L.casei 393及pPG2-/ L.casei 393免疫小鼠能够产生明显的抗α毒素的sIgA 和IgG 抗体水平,其对α毒素中和试验结果为完全保护。腹腔攻毒实验结果为能抵抗3倍最小致死剂量的α毒素攻击。【结论】表达产气荚膜梭菌α毒素免疫保护性抗原的重组乳酸乳杆菌口服免疫动物能够产生良好的局部和系统体液免疫应答和免疫中和效力。     

一株减毒百日咳鲍特菌的构建及生物学特性分析

百日咳是传染性强、感染率高的急性呼吸道传染病,主要感染婴幼儿,是婴儿死亡的主要原因之一。百日咳鲍特菌（Bordetella pertussis）是引起百日咳的最主要病原菌。近年来世界各地多次出现百日咳暴发,迫切需研制更加有效的新型百日咳疫苗。本研究构建了一株减毒百日咳活疫苗BPTM1,利用同源重组方法敲除编码百日咳鲍特菌主要毒力因子百日咳毒素（pertussis toxin,PTX）和皮肤坏死毒素（dermonecrotic toxin,DNT）的基因,并用大肠埃希菌的同源基因置换了负责气管细胞毒素（tracheal cytotoxin,TCT）转运的基因ampG。通过聚合酶链反应验证了毒素及相关基因的敲除和置换,蛋白免疫印迹法检测表明PTX的S1亚基未表达。体外生长曲线和体内定植曲线均表明,相比于野生型百日咳鲍特菌BPMM,减毒BPTM1的生长和定植能力未受影响,其所致肺部病理效应减轻,而所诱导的百日咳鲍特菌特异性IgG、IgG1、IgG2a抗体保持高水平。本研究表明,减毒百日咳鲍特菌BPTM1有可能成为百日咳疫苗的候选疫苗。     

表达ETEC F41重组干酪乳杆菌口服及滴鼻免疫效果的比较

摘要：【目的】比较小鼠滴鼻与口服接种ETEC F41重组干酪乳杆菌后,机体所产生的抗ETEC F41的黏膜免疫和系统免疫及免疫保护率的差异,为确定ETEC乳酸菌疫苗免疫程序奠定基础。【方法】将构建的重组质粒pLA-F41电转化入干酪乳杆菌,获得阳性重组菌。重组菌在MRS 培养基中进行表达,经Western blot检测目的蛋白的表达,间接免疫荧光及流式细胞术检测外源蛋白展示到菌体表面。SPF级BALB/c小鼠随机分成4组,每组40只,将重组菌以滴鼻和口服途径分别接种2组小鼠,对照组分别接种同剂量的空质粒菌     

产气荚膜梭菌α毒素在干酪乳杆菌中的诱导表达及其小鼠口服免疫力

[目的]构建产气荚膜梭菌(Clostridium perfringens,C.perfringents)α毒素基因的重组干酪乳杆菌口服疫苗,为产气荚膜梭菌毒素中毒的防治提供有效方法.[方法]将构建的重组产气荚膜梭菌α毒素基因细胞表面型载体pPG1及分泌表达载体pPG2电转化乳酸乳杆菌(Lactobacillus casei L.casei),获得阳性重组菌pPG1-α/L.case/393乳酸乳杆菌表面表达系统和pPG2-α/L.casei 393乳酸乳杆菌分泌表达系统.重组菌以1%乳糖为诱导物,在MRS培养基中进行诱导,通过Western-blot和间接免疫荧光方法鉴定,确定目的蛋白的表达.将重组菌口服免疫BALB/c小鼠,收集免疫小鼠粪便及眼冲洗液及外生殖道黏液样本测定小鼠产生抗α毒素的特异性slgA抗体水平,采集小鼠血液样本测定血清中抗α毒素的特异性lgG抗体水平.并对免疫小鼠进行α毒素的腹腔攻毒试验及对获得的抗血清进行α毒素中和试验测定.[结果]重组干酪乳杆菌pPG1-α/L.casei 393及pPG2-/L.casei 393免疫小鼠能够产生明显的抗α毒素的slgA和IgG抗体水平,其对α毒素中和试验结果为完全保护.腹腔攻毒实验结果为能抵抗3倍最小致死剂量(minimum lethal dose,MLD100)的α毒素攻击.[结论]表达产气荚膜梭菌α毒素免疫保护性抗原的重组乳酸乳杆菌口服免疫动物能够产生良好的局部和系统体液免疫应答和免疫中和效力.     

百日咳杆菌69KD外膜蛋白的研究进展

<正>百日咳是由百日咳杆菌引起的一种急性呼吸道传染病,严重威胁婴幼儿健康,对它的控制迄今主要依赖于菌苗预防。目前认为,百日咳杆菌有许多生活学活性成份,包括白细胞增多促进因子(LPF)[又称百日咳毒素(PT)]、丝状血凝素(FHA)、腺苷酸环化酶(AC)、外膜蛋白、脂多糖、不耐热毒素等。其中LPF、FHA、69KD外膜蛋白作为保护性抗原成份,是目前国内外学者研究的热点。     

兔多杀性巴氏杆菌C51-3株黏附蛋白的表达、纯化及其抗原性检测

应用PCR从兔多杀性巴氏杆菌C51-3株基因组DNA中扩增出编码36 kD黏附蛋白的cp36基因, 将其克隆到pMD18-T载体并对插入片段进行测序。以重组质粒pMD18-cp36为模板, 用PCR扩增得到编码信号肽除外的成熟黏附蛋白基因cpm36, 并克隆到原核表达质粒pQE30中, 得到重组质粒pQE30-cpm36, 转化大肠杆菌M15, 在IPTG诱导下表达融合蛋白CPM36, 经Ni2+-NTA亲和层析纯化。DNA测序结果表明cp36基因片段大小为1032 bp, 与已报道的16个血清型多杀性巴氏杆菌cp36基因的核苷酸序列比较, 同源性在76.9%~100%之间。SDS-PAGE结果显示, 表达分子量约为37 kD的带有6×His标签的CPM36蛋白, 与预期分子量相符。Western blotting结果表明, 抗重组蛋白抗体分别能与CPM36蛋白和多杀性巴氏杆菌36 kD蛋白发生特异性反应, 证明原核表达蛋白具有抗原性, 为进一步开展多杀性巴氏杆菌免疫保护性抗原的研究奠定了基础。     

Production and characterization of recombinant pertactin, fimbriae 2 and fimbriae 3 from Bordetella pertussis

Background  

Bordetella pertussis is a causative agent of pertussis or whooping cough in humans. Pertactin (Prn), fimbriae 2 (Fim2) and fimbriae 3 (Fim3) of B. pertussis are important virulence factors and immunogens which have been included in some acellular pertussis vaccines. In this present study, we cloned, expressed and purified Prn, Fim2 and Fim3, respectively. The immunogeniCity and protective efficacy of the three recombinant proteins (rPrn, rFim2 and rFim3) were investigated in mouse model.     

Engineering upstream transcriptional and translational signals of Bordetella pertussis serotype 2 fimbrial subunit protein for efficient expression in Escherichia coli: in vitro autoassembly of the expressed product into filamentous structures

Escherichia coli containing a cloned gene encoding the Bordetella pertussis serotype 2 fimbrial subunit failed to produce detectable levels of the gene product in whole-cell extracts. To engineer plasmids capable of directing the expression in E. coli of high levels of this product, both as a pre-protein and as a methionylated mature form the upstream signals of the fimbrial subunit gene were replaced by the lambda P(L) and P(R) promoters and the E. coli atpE translational initiation region. These constructs did not result in the expression of fimbrial subunit at detectable levels in several E. coli strains including DH5. However, they did in E. coli CAG629, which is lon protease and heat shock protein deficient. Both pre-protein and methionylated mature protein had molecular weights of 25.0 kD, which indicated that correct processing of the leader sequence had occurred and thus that it was transposed across the inner membrane. Electron microscopic investigation of the cell surface of E. coli cells expressing either form of the fimbrial gene failed to detect the presence of filamentous structures. The methionylated mature form of the recombinant fimbrial subunit was purified to apparent homogeneity. After dialysis in appropriate conditions it was seen to autoassemble into protein polymers. Antibodies raised against polymerized recombinant subunit reacted weakly with whole B. pertussis serotype 2 fimbriae in immunodot blot assays. However, such antibodies reacted in Western blots equally well with the recombinant and wild-type form of the fimbrial subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adjuvant and protective properties of native and recombinant Bordetella pertussis adenylate cyclase toxin preparations in mice

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin which is synthesised from the cyaA gene as an inactive protoxin that is post-translationally activated by the product of the cyaC gene. Purified active and inactive CyaA proteins were prepared from B. pertussis or from recombinant Escherichia coli expressing both cyaA and cyaC genes or the cyaA gene alone. respectively. In addition, a hybrid toxin (Hyb2) in which an internal region of CyaA had been replaced with the analogous region from the leukotoxin (LktA) of Pasteurella haemolytica, and which had low cell-invasive activity, was also prepared from E. coli expressing the cyaC gene. The CyaA preparations showed no evidence of toxicity in a mouse weight-gain test. Active toxin preparations were protective in mice against intranasal challenge with wild-type B. pertussis, as evidenced by lung:body weight ratios and bacterial numbers in the lungs, which were comparable to those in mice given whole-cell DPT vaccine. Hyb2 was not as protective as active CyaA and inactive CyaA preparations were not protective. Active CyaA, when co-administered with ovalbumin (OA), had a marked adjuvant effect on the anti-OA IgG antibody response which was not as apparent with inactive CyaA preparations. Similarly, active CyaA stimulated a greater anti-CyaA response than the inactive form.     

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.     

Two-step purification of Bacillus circulans chitinase A1 expressed in Escherichia coli periplasm

A protein purification procedure was developed to efficiently and effectively purify the target enzyme, chitinase A1 of Bacillus circulans WL-12, from Escherichia coli DH5alpha carrying the chiA gene with its natural promoter in the plasmid pNTU110. Chitinase A1 was purified to apparent homogeneity from E. coli periplasm with a final recovery of 90.6%. Two main steps were included in this protein purification procedure, ammonium sulfate precipitation (40% saturation) and anion-exchange chromatography at pH 6.0 using Q Ceramic HyperD column. The yield of chitinase A1 was estimated at 95 microg/L. A polyclonal antibody against chitinase A1 was raised by immunizing BALB/c mice with chitinase A1 purified from E. coli DH5alpha(pNTU110). As indicated by Western blot analysis, a 3000-fold diluted antibody detected purified chitinase A1 from E. coli DH5alpha(pNTU110) in an amount of at least 1 ng and specifically detected chitinase A1 produced by B. circulans WL-12.     

Inhibition of Bordetella pertussis filamentous hemagglutinin-mediated cell adherence with monoclonal antibodies

Abstract Filamentous hemagglutinin (FHA), a 220-kDa protein located on the surface of Bordetella pertussis , is one of the major cell adhesins of this bacterium. We have produced three hybridoma cell lines that express monoclonal antibodies (mAbs) against FHA: X3C, X3E and X4B. The anti-FHA mAbs X3C and X3E reacted with 220-kDa FHA protein bands on Western blots. The mAb X4B, which reacted with FHA in ELISA, did not bind to FHA in a Western blot assay. All three mAbs seemed to be directed to the same epitope or to epitopes in close proximity as suggested by competition ELISAs. All three mAbs were able to inhibit the adherence of Chinese hamster ovary cells to purified FHA, and they could also inhibit the FHA-mediated agglutination of goose red blood cells. The attachment of B. pertussis to epithelial cell monolayers was inhibited by the mAb X3C. These antibodies are very useful probes to identify the presence of FHA in bordetellae species and in clinical reagents such as pertussis vaccines, and to characterize the functional domains of this important bacterial adhesin.     

丙型肝炎病毒RNA多聚酶在昆虫细胞中的表达

ＨＣＶ ＮＳ５Ｂ基因片段克隆入ＢＡＣ－ＴＯ－ＢＡＣ＾ＴＭ重组杆状病毒表达系统的ｐＦＡＳＴＨＴｃ载体质粒,转化ＤＨ１０ＢＡＣ＾ＴＭ感受态细菌获得重组的Ｂａｃｍｉｄ质粒,将重组Ｂａｃｍｉｄ质粒转染Ｓｆ细胞,获得的重组杆状病毒可表达目的蛋白。免疫印迹和体外活性检测表明,所表达蛋白为ＨＣＶ ＮＳ５Ｂ蛋白,具有多聚酶活性。     

丙型肝炎病毒非结构3区基因片段的克隆表达及抗原纯化鉴定

通过逆转录ＰＣＲ技术,从中国江苏省丙型肝炎病人血清中扩增克隆了丙型肝炎病毒（ＨＣＶ）的非结构３区的部分基因片段（Ｃ３３）。ＤＮＡ序列分析证实,该片段全长８４２ｂｐ。在合成的一对逆转录ＰＣＲ引物上,上游引物增加了ＮｃｏⅠ酶切位点（内含起始密码子ＡＴＧ）,下游引物上增加了ＳａｌⅠ酶切位点及终止密码子ＴＡＡ,将克隆的Ｃ３３基因片段克隆至表达载体ｐＢＶ２２１内,获得了表达Ｃ３３抗原的工程菌,表达Ｃ３３抗原分子过为３０ｋＤ,经尿素裂解纯化、分子筛分离纯化及复性后进行离子交换和反相亲和层析纯化,获得的重组蛋白经ＥＬＢＡ和免疫印迹等证实有较好的抗原性和特异性。     

Export of Bordetella pertussis serotype 2 and 3 fimbrial subunits by Escherichia coli

Abstract Bordetella pertussis serotype 2 and 3 fimbrial subunits were expressed and exported in Escherichia coli using the recently described expression/secretion vector pCGV1. Two protease deficient E. coli strains (CAG629 and EC538) and two periplasmic-leaky mutants (AE84064 and A593) were transformed with the different constructs and, after thermal induction, proteins present in the various cellular compartments were analyzed by Western blot. The results obtained with the two types of fimbrial subunits were generally the same: a recombinant protein of the expected molecular mass (19.2 kDa) was present in the periplasm of the leaky mutants and of CAG629 strain (lon protease- and heat shock protease-deficient). Only the expression of the recombinant fimbrial subunits by the tolB A593 mutant resulted in protein release into the extracellular medium. These results indicate that the use of hybrid plasmids based on pCGV1 in combination with the tolB mutant constitute an efficient system for the export of recombinant proteins.     

Protective immunity against Bordetella pertussis by a recombinant DNA vaccine and the effect of coinjection with a granulocyte-macrophage colony stimulating factor gene

A recombinant pertussis DNA vaccine was described here with its immunogenicity and the ability to induce protection against B. pertussis infection in mice. Three immunodominant antigen gene fragments of pertussis, pertussis toxin subunit 1 (pts1), fragments of pertactin (prn) and filamentous hemagglutinin (fha), were recombined as fragment pts1-prn-fha named ppf, and it was cloned to plasmid pVAX1 as pVAX1/ppf. Compared to those injected with pVAX1, the mice injected with pVAX1/ppf significantly elicited more antigen specific antibody anti-PTS1, anti-PRN, anti-FHA and cytokine IL-10, IFN-gamma. When pGM-CSF was coinjected with pVAX1/ppf, the mice showed significantly increases of the three antibodies and cytokine IL-10, IL-4, IFN-gamma and TNF-alpha compared to those injected with pVAX1 only. The mice in group pVAX1/ppf & pGM-CSF, in particular; induced much more anti-PTS1, IL-4 and TNF-alpha than those in group pVAX1/ppf. In the intracerebral mouse protection test, the mice immunized with pVAX1/ppf or pVAX1/ppf & pGM-CSF induced protection to a lethal dose of B. pertussis. The results indicate that recombinant DNA vaccine and pGM-CSF coinjection can induce protective immunity against B. pertussis, demonstrating a valuable method to prevent pertussis.