枯草芽胞杆菌芽胞表面展示研究进展

枯草芽胞杆菌芽胞表面展示技术是把枯草芽胞杆菌作为芽胞表面展示的宿主来展示目的蛋白的一种技术。该技术不仅具备芽胞表面展示技术可展示分子量较大的目的蛋白、目的蛋白无需跨膜及芽胞的极强抗逆性等特点外,同时由于该技术的宿主菌--枯草芽胞杆菌的分子生物学信息研究得比较清楚、安全性高而被广泛应用。介绍了枯草芽胞杆菌表面展示近10年在生产疫苗和固定化酶方面的进展,并对如何提高表面展示目的蛋白的产量做了简要概述。     

枯草芽胞杆菌孢子表面展示外源蛋白的研究

枯草芽胞杆菌孢子表面展示技术是最近十几年新兴的一种外源蛋白固定方法,已在酶学、疫苗学、靶向药物制备、金属污染治理等领域获得了广泛应用。以孢子衣壳蛋白为载体蛋白,已经成功地把许多抗原、酶和其他蛋白展示在孢子外表面。枯草芽胞杆菌孢子衣壳由多种衣壳蛋白组成,但可用做载体蛋白的并不多,且它们的特性不同。综合介绍了枯草芽胞杆菌孢子表面展示外源蛋白这种新型技术的具体机理,及其在国内外各领域应用的研究进展。     

苏云金芽胞杆菌营养期杀虫蛋白基因vip3A在枯草芽胞杆菌中的表达

枯草芽胞杆菌Bacillus subtilis常被用于表达杀虫和抗菌蛋白.为了探讨苏云金芽胞杆菌B. thuringiensis营养期杀虫蛋白基因（vip3A）在枯草芽胞杆菌中的表达情况,促进杀虫防病工程菌构建,将枯草芽胞杆菌168菌株核糖体小亚基S4蛋白基因的启动子与苏云金芽胞杆菌WB7菌株vip3A基因的编码序列连接,插入大肠杆菌Escherichia coli与枯草芽胞杆菌穿梭载体pAD123,得到重组原核表达质粒pADpvip,将重组质粒转化枯草芽胞杆菌标准菌株168和分离自辣椒体内的生防内生枯草芽胞杆菌BS-2菌株中,获得工程菌株.SDS-PAGE分析表明在枯草芽胞杆菌168菌株的部分工程菌株中有约88 kDa大小的VIP条带,而BS-2的工程菌株中未见相应的条带,表明Vip3A蛋白仅在168菌株中表达.生物测定表明有5株168的工程菌株（168vip1-4,6）表现较高的杀虫活性,工程菌株发酵稀释液（约10⁷CFU/mL）处理的小白菜叶片饲喂斜纹夜蛾2龄幼虫72 h的杀虫效果可达87.64%~92.13%,但vip3A基因转入内生枯草芽胞杆菌BS-2中不表现杀虫作用.毒力测定表明168vip2菌株对斜纹夜蛾2龄幼虫72 h的LC₅₀为0.0194 mL/mL.这些结果为进一步研究基因在枯草芽胞杆菌中的表达构建杀虫防病工程菌打下了基础.     

外源木聚糖酶在枯草芽胞杆菌中信号肽筛选

[目的]本试验旨在筛选引导表达外源木聚糖酶基因高效分泌的信号肽,为枯草芽胞杆菌木聚糖酶高效分泌表达系统提供元件.[方法]构建信号肽筛选载体,载体是以含壮观霉素抗性基因的大肠-枯草穿梭载体为基本骨架,目标蛋白为耐碱性木聚糖酶,可在麦芽糖启动子Pglv诱导下表达.从枯草芽胞杆菌A1747基因组中扩增获得24个Sec途径信号肽,并将其全部链接到至筛选载体上,并在枯草芽胞杆菌WB700中实现表达分泌.重组菌在3%麦芽糖诱导下培养24h后用DNS法测定上清酶活.[结果]成功构建信号肽筛选载体pGPSX及24个表达载体,实现木聚糖酶表达分泌.且不同信号肽对于引导外源木聚糖酶分泌能力不同,其中YnfF信号肽引导分泌目标蛋白效率最高,上清酶活为37.2IU/mL.[结论]试验证明在枯草杆菌中对外源蛋白进行信号肽筛选是提高其分泌的有效途径,并获得了针对木聚糖酶高效分泌信号肽YnfF.     

地衣芽胞杆菌dif序列的功能鉴定

地衣芽胞杆菌是重要的工业菌株,如何为其建立一种有效的基因删除技术是对该菌株进行遗传改良的基础。枯草芽胞杆菌difB8序列已经被成功用于枯草芽胞杆菌多基因的删除。在分析地衣芽胞杆菌基因组序列并获得与枯草芽胞杆菌difB8以。序列十分相似的一段序列difBLi的基础上,构建了在庆大霉素抗性基因两侧具有difBLi的重组质粒pMD19-difGm和pHY-XI’：：difGm,通过电击转化法将质粒pHY-XI’：：difGm导入B．1icheniformis ATCC14580中,筛选获得了具有庆大霉素抗性的转化子。在转化子的传代过程中,重组质粒的庆大霉素抗性基因在体内Xer／dif／f位点特异性重组系统的介导下通过其侧翼的dif位点进行同源重组而被准确删除。确证了地衣芽胞杆菌中dif序列的功能,为地衣芽胞杆菌基因组中多基因的删除提供了一种新的实验途径。     

蛋白激发子PeaT1在枯草芽胞杆菌中的分泌表达及重组菌株提高小麦抗旱和促生的作用

PeaT1是从极细链格孢菌Alternaria tenuissima中分离的一种蛋白激发子,具有促进植物生长和诱导植物产生系统获得抗性的功能,为了实现peaT1基因在枯草芽胞杆菌Bacillus subtilis中的分泌表达,增加其应用途径,从枯草芽胞杆菌基因组DNA中分别扩增获得P43启动子和nprB基因的信号肽序列,并用SOE (Splicing by over lapping extension) 方法与peaT1基因连接,将连接产物克隆到大肠杆菌-枯草芽胞杆菌穿梭表达载体pHY300-PLK上,构建了重组表达载体pHY43N-peaT1。将重组载体转化枯草芽胞杆菌WB800菌株,SDS-PAGE和Western blotting分析证实,在NprB信号肽的引导下,枯草芽胞杆菌成功分泌表达了PeaT1蛋白。构建的重组菌株能够显著增强幼苗抗旱性,提高小麦株高。     

重组枯草芽胞杆菌不对称还原产d-伪麻黄碱

为了实现羰基还原酶基因mldh在枯草芽胞杆菌Bacillus subtilis中的表达并通过细胞内的葡萄糖脱氢酶完成辅酶的再生,以枯草芽胞杆菌rpsD基因的启动子PrpsD和终止子TrpsD为表达元件,将羰基还原酶基因mldh连接至构建好的质粒(pHY300plk-PrpsD-TrpsD上,得到质粒pHY300plk-PrpsD-mldh-TrpsD;进一步将重组质粒转化入B. subtilis Wb600中获得重组菌B. subtilis Wb600 (pHY300plk-PrpsD-mldh-Trps     

苏云金芽胞杆菌G03 spoIVF操纵子敲除对芽胞和晶体形成的影响

spoIVF是一个普遍存在于芽胞杆菌中的操纵子。在枯草芽胞杆菌中,它编码的两个蛋白是芽胞形成所必需的。采用基因重组技术敲除了苏云金芽胞杆菌G03菌株中的spoIVF操纵子,构建了spoIVF缺失株G03(spoIVF-)。研究表明:该突变株丧失了形成芽胞和晶体的能力。lacZ基因与cry1Aa基因的启动子融合表达分析发现:突变株中的cry1Aa基因的活性严重降低。利用载体pSTK携带spoIVF操纵子在突变株中的表达,使突变株部分恢复了产胞和形成杀虫晶体蛋白的能力。这说明spoIVF操纵子是所必需的,同时该操纵子还影响σE因子控制的cry1Aa基因表达。     

在枯草杆菌芽胞表面展示P74蛋白膜外肽段

目的:对家蚕核型多角体病毒(BmNPV)囊膜蛋白P74膜外肽段与枯草芽胞杆菌(Bacillus subtilis)CotC与进行融合表达,制备表面展示有P74蛋白的重组芽孢,为深入研究该重组芽孢的功能提供基础.方法:将CotC基因与BmNPV P74膜外编码序列进行融合,构建表达CotC-P74融合蛋白的重组质粒pJS700-p74.通过双交换使该重组质粒中的CotC-P74表达盒整合到枯草芽胞杆菌染色体淀粉酶基因位点,并对发生同源重组后的菌株进行筛选和PCR鉴定.结果:PCR结果表明CotC-P74成功地整合到枯草芽孢杆菌基因组上,通过作者实验室制备的P74多抗对诱导后的重组芽孢衣壳总蛋白进行Western blot分析,结果在49 kDa位置处能杂交到一条特异的蛋白带.结论:P74蛋白胞外肽段成功地展示在枯草芽胞杆菌芽胞表面.     

地衣芽胞杆菌FLP/FRT基因编辑系统的构建及验证

地衣芽胞杆菌有效的基因编辑工具有限,为了拓展和丰富其基因编辑手段,在地衣芽胞杆菌中构建一个抗性标记可重复使用的FLP/FRT基因编辑系统,并通过敲除α-淀粉酶基因amyL、蛋白酶基因aprE及敲入外源透明颤菌血红蛋白基因vgb对该系统进行初步验证。首先以温敏质粒pNZT1为载体分别构建amyL和aprE基因敲除质粒pNZTT-AFKF和pNZTT-EFKF,两个敲除质粒各自包含针对目标基因的同源臂、抗性基因及同向的FRT位点;将敲除质粒转化地衣芽胞杆菌并经过两次同源交换过程实现目标基因的敲除;最后导入一个FLP重组酶表达质粒通过FLP/FRT系统的重组作用介导抗性基因的回收。为进一步验证本系统的实用性及编辑效率,构建了包含透明颤菌血红蛋白编码基因vgb表达盒及基因组丙酮酸甲酸裂解酶编码基因pflB敲除盒的重组质粒pNZTK-PFTF-vgb,并以此进行外源基因vgb在基因组上的定向敲入。结果显示,成功敲除amyL及aprE并回收了抗性标记卡那霉素基因,敲除后淀粉酶活和蛋白酶活分别减少95.3%和80.4%;vgb基因成功整合入基因组pflB位点并回收了抗性标记四环素基因,并利用荧光定量PCR技术检测到vgb的整合表达。文中首次建立了一个适用于地衣芽胞杆菌的、抗性标记可重复使用的FLP/FRT基因编辑系统,并进行了基因敲除及基因敲入验证,为地衣芽胞杆菌遗传改造提供了良好的方法参考。     

Cloning and characterization of a cluster of genes encoding polypeptides present in the insoluble fraction of the spore coat of Bacillus subtilis.

The Bacillus subtilis spore coat is composed of at least 15 polypeptides plus an insoluble protein fraction arranged in three morphological layers. The insoluble fraction accounts for about 30% of the coat protein and is resistant to solubilization by a variety of reagents, implying extensive cross-linking. A dodecapeptide was purified from this fraction by formic acid hydrolysis and reverse-phase high-performance liquid chromatography. This peptide was sequenced, and a gene designated cotX was cloned by reverse genetics. The cotX gene encoding the dodecapeptide at its amino end was clustered with four other genes designated cotV, cotW, cotY, and cotZ. These genes were mapped to 107 degrees between thiB and metA on the B. subtilis chromosome. The deduced amino acid sequences of the cotY and cotZ genes are very similar. Both proteins are cysteine rich, and CotY antigen was present in spore coat extracts as disulfide cross-linked multimers. There was little CotX antigen in the spore coat soluble fraction, and deletion of this gene resulted in a 30% reduction in the spore coat insoluble fraction. Spores produced by strains with deletions of the cotX, cotYZ, or cotXYZ genes were heat and lysozyme resistant but readily clumped and responded more rapidly to germinants than did spores from the wild type. In electron micrographs, there was a less densely staining outer coat in spores produced by the cotX null mutant, and those produced by a strain with a deletion of the cotXYZ genes had an incomplete outer coat. These proteins, as part of the coat insoluble fraction, appear to be localized to the outer coat and influence spore hydrophobicity as well as the accessibility of germinants.     

Surface Display of Human Serum Albumin on Bacillus subtilis Spores for Oral Administration

Human serum albumin (HSA) is the major protein component of human plasma. To date, HSA for clinical uses is mostly produced by fractionation of human whole blood, which is accompanied by a lot of limitations. To obtain long-term bioactive albumin, we used hsa as a foreign gene and constructed a recombinant plasmid pJS700-HSA which carries a recombinant gene cotC-hsa under the control of cotC promoter. Plasmid pJS700-HSA was transformed into Bacillus subtilis by double cross-over and an amylase inactivated mutant was produced. After induction of spore formation, western blot and fluorescence immunoassay were used to monitor HSA surface expression on spores. We estimated that HSA displayed on the spore accounted for 0.135 % of the total spore proteins and about 0.023 fg HSA were exposed on the surface of each spore. Oral administration to mice with spores displaying HSA implied that the recombinant spores may have potential ability to increase the serum albumin level in vivo due to the resistant characters of spores.     

Spore-displayed streptavidin: a live diagnostic tool in biotechnology

Streptavidin, which is one of the most widely used proteins in biotechnological application field and is active only in tetrameric form, was surface expressed on the surface of Bacillus subtilis spore. Spore coat protein of B. subtilis, CotG, was used as an anchoring motif to display streptavidin. FACS using anti-streptavidin antibody was used for the verification of surface localization of expressed CotG-streptavidin fusion protein. FACS and dot-blot were used for the verification of biological activity of displayed streptavidin with FITC-labeled biotin.     

Display of Bombyx mori alcohol dehydrogenases on the Bacillus subtilis spore surface to enhance enzymatic activity under adverse conditions

Alcohol dehydrogenases (ADHs) are oxidoreductases catalyzing the reversible oxidation of alcohols to corresponding aldehydes or ketones accompanied by nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. ADHs attract major scientific and industrial interest for the evolutionary perspectives, afforded by their wide occurrence in nature, and for their use in industrial synthesis. However, the low activity of ADHs under extremes of pH and temperature often limits their application. To obtain ADH with high activity, in this study, we used Bombyx mori alcohol dehydrogenases (BmADH) as foreign gene and constructed a recombinant integrative plasmid pJS700-BmADH. This pJS700-BmADH was transformed into Bacillus subtilis by double cross-over and produced an amylase inactivated mutant. The fusion protein containing BmADH was expressed on the spore surface and recognized by BmADH-specific antibody. We also assayed the alcohol dehydrogenase activity of the fusion protein together with the native BmADH at different pH and temperature levels, which indicated the recombinant enzyme exhibits activity over wider ranges of temperature and pH than its native form, perhaps due to the resistance properties of B. subtilis spores against adverse conditions.     

Peptide ligands that bind selectively to spores of Bacillus subtilis and closely related species

As part of an effort to develop detectors for selected species of bacterial spores, we screened phage display peptide libraries for 7- and 12-mer peptides that bind tightly to spores of Bacillus subtilis. All of the peptides isolated contained the sequence Asn-His-Phe-Leu at the amino terminus and exhibited clear preferences for other amino acids, especially Pro, at positions 5 to 7. We demonstrated that the sequence Asn-His-Phe-Leu-Pro (but not Asn-His-Phe-Leu) was sufficient for tight spore binding. We observed equal 7-mer peptide binding to spores of B. subtilis and its most closely related species, Bacillus amyloliquefaciens, and slightly weaker binding to spores of the closely related species Bacillus globigii. These three species comprise one branch on the Bacillus phylogenetic tree. We did not detect peptide binding to spores of several Bacillus species located on adjacent and nearby branches of the phylogenetic tree nor to vegetative cells of B. subtilis. The sequence Asn-His-Phe-Leu-Pro was used to identify B. subtilis proteins that may employ this peptide for docking to the outer surface of the forespore during spore coat assembly and/or maturation. One such protein, SpsC, appears to be involved in the synthesis of polysaccharide on the spore coat. SpsC contains the Asn-His-Phe-Leu-Pro sequence at positions 6 to 10, and the first five residues of SpsC apparently must be removed to allow spore binding. Finally, we discuss the use of peptide ligands for bacterial detection and the use of short peptide sequences for targeting proteins during spore formation.     

The coat morphogenetic protein SpoVID is necessary for spore encasement in Bacillus subtilis

Endospores formed by Bacillus subtilis are encased in a tough protein shell known as the coat, which consists of at least 70 different proteins. We investigated the process of spore coat morphogenesis using a library of 40 coat proteins fused to green fluorescent protein and demonstrate that two successive steps can be distinguished in coat assembly. The first step, initial localization of proteins to the spore surface, is dependent on the coat morphogenetic proteins SpoIVA and SpoVM. The second step, spore encasement, requires a third protein, SpoVID. We show that in spoVID mutant cells, most coat proteins assembled into a cap at one side of the developing spore but failed to migrate around and encase it. We also found that SpoIVA directly interacts with SpoVID. A domain analysis revealed that the N-terminus of SpoVID is required for encasement and is a structural homologue of a virion protein, whereas the C-terminus is necessary for the interaction with SpoIVA. Thus, SpoVM, SpoIVA and SpoVID are recruited to the spore surface in a concerted manner and form a tripartite machine that drives coat formation and spore encasement.     

Morphogenesis of bacillus spore surfaces

Spores produced by bacilli are encased in a proteinaceous multilayered coat and, in some species (including Bacillus anthracis), further surrounded by a glycoprotein-containing exosporium. To characterize bacillus spore surface morphology and to identify proteins that direct formation of coat surface features, we used atomic-force microscopy (AFM) to image the surfaces of wild-type and mutant spores of Bacillus subtilis, as well as the spore surfaces of Bacillus cereus 569 and the Sterne strain of Bacillus anthracis. This analysis revealed that the coat surfaces in these strains are populated by a series of bumps ranging between 7 and 40 nm in diameter, depending on the species. Furthermore, a series of ridges encircled the spore, most of which were oriented along the long axis of the spore. The structures of these ridges differ sufficiently between species to permit species-specific identification. We propose that ridges are formed early in spore formation, when the spore volume likely decreases, and that when the spore swells during germination the ridges unfold. AFM analysis of a set of B. subtilis coat protein gene mutants revealed three coat proteins with roles in coat surface morphology: CotA, CotB, and CotE. Our data indicate novel roles for CotA and CotB in ridge pattern formation. Taken together, these results are consistent with the view that the coat is not inert. Rather, the coat is a dynamic structure that accommodates changes in spore volume.     

Surface display of recombinant proteins on Bacillus subtilis spores

We developed a novel surface display system based on the use of bacterial spores. A protein of the Bacillus subtilis spore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC). Western, dot blot and fluorescent-activated cell sorting analyses were used to monitor TTFC surface expression on purified spores. We estimated that more than 1.5 x 10(3) TTFC molecules were exposed on the surface of each spore and recognized by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record of B. subtilis spores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules.