Expression of protective and cardiac tissue cross-reactive epitopes of type 5 streptococcal M protein in Escherichia coli.

The immunochemical properties of type 5 M protein antigens that were expressed in Escherichia coli K-12 by recombinant lambda bacteriophages isolated from a gene bank of serotype 5 Streptococcus pyogenes have been analyzed in detail. M proteins from partially purified bacteriophage lysates displayed precipitin lines of identity with a purified peptic extract of type 5 M protein (pep M5) in immunodiffusion assays. Immunoblot analyses of the M protein-positive lysates demonstrated that the cloned M protein component resided in five polypeptides with relative molecular weights of 57,900 (57.9K), 55.4K, 52.9K, 40.0K, and 32.6K. The hybrid lambda phage (lambda M5)-produced M protein contained immunoprotective epitopes; lambda M5 protein inhibited opsonization of type 5 streptococci by pep M5 antibodies, and antiserum raised against lambda M5 lysates opsonized type 5 streptococci. Each of the five antigenic polypeptides of the recombinant phage M protein also shared epitopes with human heart tissue, as demonstrated by the reactivity of immunoblots of lambda M5 antigens separated on sodium dodecyl sulfate gels with anti-pep M5 antibodies absorbed to and eluted from human heart sarcolemmal membranes. Moreover, antiserum raised against the lambda M5 lysates reacted with sarcolemmal membrane proteins with relative molecular weights of 200K, 59K, 55K, 53K, and 27K as determined by immunoblot analyses. These results demonstrate that the structural gene coding for type 5 streptococcal M protein which was inserted into lambda DNA expresses immunoprotective epitopes, some of which are shared with human heart tissue.     

Cloning of the gene encoding streptococcal immunoglobulin A protease and its expression in Escherichia coli.

We have identified and cloned a 6-kilobase-pair segment of chromosomal DNA from Streptococcus sanguis ATCC 10556 that encodes immunoglobulin A (IgA) protease activity when cloned into Escherichia coli. The enzyme specified by the iga gene in plasmid pJG1 accumulates in the periplasm of E. coli MM294 cells and has a substrate specificity for human IgA1 identical to that of native S. sanguis protease. Hybridization experiments with probes from within the encoding DNA showed no detectable homology at the nucleotide sequence level with chromosomal DNA of gram-negative bacteria that excrete IgA protease. Moreover, the S. sanguis iga gene probes showed no detectable hybridization with chromosomal DNA of S. pneumoniae, although the IgA proteases of these two streptococcal species cleaved the identical peptide bond in the human IgA1 heavy-chain hinge region.     

Vimentin-cross-reactive epitope of type 12 streptococcal M protein.

The NH2-terminal amino acid sequence of type 12 M protein was determined by automated Edman degradation of a 38-kilodalton polypeptide fragment purified from a limited pepsin digest of intact type 12 streptococci. The sequence of the first 13 amino acid residues of the polypeptide confirmed that predicted by the nucleotide sequence of the mature type 12 M protein. A chemically synthesized peptide copying the NH2-terminal 25 residues, SM12(1-25)C, evoked opsonic antibodies against type 12 streptococci as well as renal glomerular cross-reactive antibodies. The serum from one of six rabbits reacted in immunofluorescence tests with human glomeruli in a mesangial staining pattern. The cross-reactive antibodies were completely inhibited by the immunizing peptide and absorption with type 12 streptococci. Subpeptides of the 25-residue synthetic peptide were without inhibitory effect, suggesting that the cross-reactive antibodies are directed against a conformational epitope of SM12(1-25)C. Anti-SM12(1-25)C antisera reacted specifically with the intermediate filament protein vimentin extracted from mesangial cells. None of the cross-reactions of anti-SM12(1-25)C were inhibited by a synthetic peptide SM1(1-26)C of type 1 M protein, which was previously shown to share a cross-reactive epitope with vimentin. These results indicate that type 12 M protein contains at least one vimentin cross-reactive epitope that is clearly distinct from the tetrapeptide epitope shared with vimentin by type 1 M protein.     

Cloning of the gene, speB, for streptococcal pyrogenic exotoxin type B in Escherichia coli.

The structural gene encoding streptococcal pyrogenic exotoxin type B, designated speB, was cloned in Escherichia coli and localized onto a 4.5-kilobase BamHI-BglII DNA fragment. Streptococcal pyrogenic exotoxin type B, partially purified from E. coli clones, was immunologically related to streptococcus-derived toxin. Also, toxin derived from either E. coli or Streptococcus pyogenes had similar lymphocyte mitogenic activity and molecular weight (29,300) and displayed comparable microheterogeneity when evaluated by isoelectric focusing.     

Cloning and expression in Escherichia coli of a Fasciola hepatica gene encoding a calcium-binding protein.

A Fasciola hepatica cDNA clone of 994 bp was isolated from an adult worm cDNA expression library using a rabbit serum against the excretory-secretory antigens. The nucleotide sequence of the cDNA clone revealed the presence of an open reading frame of 572 bp which encoded a 22 kDa polypeptide (Fh22) showing putative EF-hand domains. This gene was expressed in Escherichia coli and the recombinant protein used for the production of specific antibodies. Immunoblotting studies using the anti-Fh22 serum showed the presence of a polypeptide of similar molecular mass in the excretory-secretory extract of the adult parasite. The recombinant Fh22 polypeptide showed calcium-dependent electrophoretic mobility (decreased with Ca2(+)-ions and increased with EGTA). The observed behaviour of recombinant Fh22 in gel filtration experiments also suggested calcium-induced conformational changes.     

Extraction of streptococcal type 12 M protein by cyanogen bromide.

Conditions for the release of streptococcal type 12 M protein from whole cells by cyanogen bromide are described; they demonstrated that methionine is not essential to the structural arrangements which account for some of its immunological and biological properties. The released M protein was separated from other proteins by column chromatography with hydroxylapatite. The type-specific molecules which reacted with precipitating antibodies were found only in the 0.3 M eluate, formed zones with mobilities less than 12% of that of the dye front on electrophoresis in the standard acrylamide disc gel system, formed at least four bands in sodium dodecyl sulfate-acrylamide disc gels with molecular weights ranging from 12,000 to 23,000, and stimulated the formation of opsonic antibodies in rabbits. Cyanogen bromide provides a highly specific method for the release of M proteins which should prove particularly useful in analyses of structural-functional relationships among different M proteins.     

Cloning and expression of the leukotoxin gene of Pasteurella haemolytica A1 in Escherichia coli K-12.

A clone bank of Pasteurella haemolytica A1 was constructed by partial digestion of the genomic DNA with Sau3A and ligation of 5- to 10-kilobase-pair fragments into the BamHI site of the plasmid vector pBR322. After transformation into Escherichia coli K-12, a total of 4 X 10(3) recombinant clones was obtained. These were screened for the production of P. haemolytica soluble antigens by a colony enzyme-linked immunosorbent assay blot method with a rabbit antiserum raised against the soluble antigens. The clones producing P. haemolytica soluble antigens were then analyzed for the production of the leukotoxin by a cytotoxicity assay with cells from a bovine leukemia-derived B-lymphocyte cell line as the target cells. Positive clones were identified, and subsequent restriction analysis of the recombinant plasmids showed that the same 6.3 kilobase pairs of insert DNA was cloned in either of the two orientations into the plasmid vector pBR322. One of the clones was selected for further characterization of the leukotoxin as produced in E. coli. Tests for heat lability and target cell species specificity with canine, porcine, and human peripheral blood lymphocytes indicated that the activity of the cloned leukotoxin was identical to that of the P. haemolytica leukotoxin. Furthermore, the E. coli-produced leukotoxin was also neutralized by bovine or rabbit antiserum known to have antitoxic activity. When cellular proteins from the E. coli clones were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis, a 100,000-dalton protein was identified which corresponded to one of the soluble antigens found in the leukotoxic culture supernatant of P. haemolytica. These results demonstrated that the gene(s) for the P. haemolytica leukotoxin have been cloned and that the leukotoxin was expressed in E. coli.     

Molecular analysis of the M protein of Streptococcus equi and cloning and expression of the M protein gene in Escherichia coli.

A Streptococcus equi gene bank was constructed in the bacteriophage lambda gt11 cloning vector, and hybrid phage plaques were screened with S. equi M protein antiserum. A hybrid phage expressing the S. equi M protein (lambda gt11/SEM7) was identified and lysogenized into Escherichia coli Y1089. The cloned M protein appeared in immunoblots as three polypeptides with relative molecular weights of 58,000, 53,000, and 50,000. When reacted with S. equi M protein antiserum in an agar double-diffusion assay, the cloned M protein formed a line of identity with a protein in an acid extract of S. equi. Furthermore, lambda gt11/SEM7 protein inhibited opsonization of S. equi by antiserum to S. equi M protein. In addition, the recombinant protein expressed determinants of the antigen in the immune complexes of purpura hemorrhagica. Native M protein obtained from S. equi and recombinant M protein showed very similar molecular weight distributions on immunoblots, appearing as multiple closely spaced bands with molecular weights ranging from 52,000 to 60,000. Antisera prepared separately against each of the acid-extracted polypeptides shown to be important in serum bactericidal responses (molecular weight, 29,000) and nasopharyngeal local antibody responses (molecular weights, 41,000 and 46,000) of the horse each reacted with all three polypeptides in an acid extract. Moreover, antisera against protoplasts and against recombinant M protein of S. equi also reacted with these polypeptides. These results suggest that the entire M protein molecule of S. equi is present in these preparations and that the fragments in acid extracts carry overlapping segments.     

B族链球菌C5a肽酶蛋白表位的克隆、表达和免疫学分析

目的应用生物信息学方法预测B族链球菌C5a肽酶蛋白表位，结合基因工程手段进行表位重组、表达和免疫原性分析。方法用预测程序ProPred和ANTIGENIC预测B族链球菌C5a肽酶蛋白的表位，应用PCR技术扩增出编码该表位基因片段，克隆PCR产物构建重组质粒，测序验证。在大肠杆菌BL21（DE3）中诱导表达融合蛋白。表达的蛋白经质谱分析和Western blot鉴定。纯化该融合蛋白并免疫C57／BL小鼠，萃取GBS表面蛋白，双向琼脂扩散试验检测抗体水平。结果在SCPB中预测到1个既具有MHC结合肽特性又具有B细胞表位特征的肽段。重组和表达了这一肽段，质谱得出与SCPB蛋白的相似性分数为79，Western-blot证实能与抗SCPB的抗体反应，纯化后融合蛋白纯度〉90％。动物实验证实融合蛋白能产生特异性的抗GBS抗体。结论重组表位具有一定免疫原性。为相关蛋白的毒力机制研究和亚单位疫苗等方面的研究打下了良好的基础。     

Localization of the streptococcal C5a peptidase to the surface of group A streptococci.

Immunofluorescent staining was used to determine that the streptococcal C5a peptidase (SCP) exists as a cell surface antigen on group A streptococci. The ability of hyperimmune serum to neutralize cell-associated SCP activity provided further evidence for the location of SCP. Quantification of SCP during growth in vitro by indirect enzyme-linked immunosorbent assay showed that approximately 90% of the measurable antigen is cell bound.     

Interactions of the streptococcal C5a peptidase with human fibronectin

Group B Streptococci (GBS) is a leading cause of sepsis and meningitis in neonates and immunocompromised adults in western countries. GBS do not bind to fibronectin (Fn) in solution, but will bind to Fn adsorbed onto a solid surface. The reason for the specificity of this binding is unknown. Single molecule force spectroscopy was used to test the hypothesis that GBS, through streptococcal C5a peptidase (ScpB) molecules present on the surface of the bacteria, binds to a motif created by the juxtaposition of multiple adjacent Fn molecules. Atomic force microscopy (AFM) topographical images of adsorbed Fn deposited from various Fn coating concentrations were used to determine the Fn surface concentration. ScpB was tethered to an AFM tip with all surface modifications characterized by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. At the lowest Fn coverages the probability of observing a ScpB–Fn binding event increased linearly with Fn surface coverage. As an Fn monolayer was reached the probability of a ScpB–Fn binding event occurring increased markedly (50 fold), with a concomitant increase in the rupture force from 17 pN to 33 pN. These results are consistent with the hypothesis that ScpB binds to a motif created by the juxtaposition of multiple Fn molecules.     

Similarity between the group B and A streptococcal C5a peptidase genes.

Group B streptococci (GBS) and group A streptococci (GAS) are known to have surface-associated peptidase activity which specifically cleaves C5a (C5a-ase), the primary chemotaxin for polymorphonuclear leukocytes. Amplification products were obtained from GBS genomic DNA template by using the polymerase chain reaction and primers corresponding to the C5a peptidase gene of GAS (scpA12). A restriction map of the GBS full-length amplified gene (scpB) was developed. The scpB restriction map was found to be highly similar to that of the analogous gene in GAS. A 50-bp deletion was located near the 5' end of scpB in a region where repeat sequences are found in scpA12. Hybridization experiments confirmed that other serotypes of GBS also carry an scpB-like gene. These results show that GBS contain a gene, scpB, which is very similar to that harbored by M12 GAS. The probability that scpB encodes the C5a-ase protein is discussed.     

Mapping T-cell epitopes in group A streptococcal type 5 M protein.

Group A streptococcal cell surface M proteins elicit highly protective, serotype-specific opsonic antibodies and many serotypes also elicit host cross-reactive antibodies, which may contribute to the pathogenesis of poststreptococcal autoimmune disease. To date, studies aimed at designing safe (non-host-cross-reactive, defined-epitope) M vaccines have focused almost exclusively on antibody epitopes. Here we identify T-cell epitopes recognized by T cells from BALB/c, C57BL/6, and CBA/Ca mice immunized with purified, recombinant serotype 5 M protein (rM5). The responses of rM5-specific, major histocompatibility complex class II-restricted, T-cell clones to synthetic peptides representing most of the M5 sequence identified at least 13 distinct T-cell recognition sites, including sites recognized by more than one major histocompatibility complex haplotype of mice. Although none of these sites appeared to be strongly immunodominant, an N-terminal peptide, sM5[1-35], was recognized by lymph node T cells of rM5-immunized mice and by a larger proportion of rM5-specific T-cell clones than any other individual peptide. The fine specificity of these clones was mapped with subpeptides to a single site at or overlapping the sequence ELENHDL at residues 21 to 27, which is in close proximity to previously mapped protective antibody epitopes. Other T-cell recognition sites are distributed throughout the M protein and include several in the highly conserved C-terminal region of the molecule. One of these C-terminal sites, located within residues 300 to 319, was recognized by a significant proportion of T-cell clones from two strains of mice. Helper T-cell epitopes located in the C-terminal region of M5 are likely to be widely conserved between different M serotypes and could be particularly useful in designing multivalent, defined-epitope M vaccines.     

Cloning and expression of the phospholipase D gene from Corynebacterium pseudotuberculosis in Escherichia coli.

A toxic phospholipase D (PLD) is putatively involved in pathogenesis of Corynebacterium pseudotuberculosis infections. We report here the cloning and expression of the PLD gene (pld) in Escherichia coli. A cosmid library of DNA from C. pseudotuberculosis biovar ovis isolate Whetten 1 was constructed and screened for PLD-producing recombinants by plating them on LB agar containing sheep erythrocytes and equi factors. One recombinant, designated pCpO1, yielded a gene product which displayed synergistic hemolytic and sphingomyelinase D activities, both of which are characteristic of PLD. Subcloning into pUC19 yielded a recombinant, pCpO50, which contained a 1.8-kilobase insert. Analysis of supernatant fluids and cell extracts of cultures of E. coli(pCpO50) revealed sphingomyelinase activity and a protein of about 31,000 Mr, neither of which were detected in E. coli(pUC19). The 31-kilodalton protein also reacted with antibodies in serum from a sheep naturally infected with C. pseudotuberculosis, serum which also contained PLD-neutralizing antibodies. When Southern blots of BamHI digests of DNA from biovar ovis and biovar equi isolates of C. pseudotuberculosis were probed with pCpO50, bands of 4.8 and 1.9 kilobases, respectively, were seen, suggesting that the genome organization of pld is different for isolates from the two biovars.     

可溶性巨噬细胞集落刺激因子受体基因的克隆和原核表达

目的：克隆可溶性巨噬细胞集落刺激因子受体（sMR）基因，在大肠杆菌中表达，经纯化、复性后获得活性蛋白。 方法： 从人骨髓细胞中提取总RNA，在sMR基因引物两端分别加上NdeI和BamHI酶切位点，利用RT-PCR技术扩增sMR基因，经NdeⅠ和BamHⅠ酶切后连接到表达载体pET28a，测序证实克隆基因的正确性，并在大肠杆菌BL21菌株中诱导表达，采用his-tag纯化柱纯化目的蛋白，并经复性处理，配体结合实验证实蛋白活性。 结果： 限制性内切酶切出了预期条带，测序证实了克隆基因的正确性，sMR经诱导后在大肠杆菌中以包涵体形式表达，表达蛋白经纯化和复性处理，获得了具有活性的功能蛋白。 结论： 成功克隆与表达了sMR基因，得到了具有活性的功能蛋白，为进一步研究其生物学功能和应用奠定了基础。     

Global changes in gene expression in Escherichia coli K12 induced by bacteriophage Mu Gem protein.

We have studied the growth properties of some Mu lysogens with respect to the non-lysogenic strain and have observed that the division time in minimal medium was increased over 4-fold when the bacteria carried the prophage mutated in the gem gene (Mu gem3). Since this phage gene has previously been shown to be involved in modulation of expression of host genes, we have analysed the proteins extracted from lysogens and non-lysogens as a rapid assay of global gene expression. The pattern of proteins extracted showed marked quantitative variations between non-lysogens, lysogens for wild-type Mu and lysogens for phage Mu gem3. These effects were no longer as evident when the strains were grown in rich medium. This dramatic change in the physiological state of the lysogenic strain versus the non-lysogenic in particular growth conditions extends the concept of lysogeny. For many years, the prophage has been considered only as a potentially lethal factor, while here it also appears as a genetic element capable of profoundly modifying host biology.