Antigenicity analysis of Vibrio harveyi TS-628 strain

Vibrio harveyi, the major causative agent of vibriosis, affects a diverse range of marine cultured organisms over a wide geographical area. However, reports about screening the effective antigen and research on vaccines of V. harveyi are scarce. Flagellin, lipopolysaccharide (LPS) and outer membrane proteins (OMP) are major immunogenic antigens in many Gram-negative bacteria. In this study, the flagellin, OMP and LPS of the V. harveyi TS-628 strain isolated from infected groupers were extracted and Western blot analysis was used to detect the antigenicity of these extractions. Results of the Western blot assay reveal that there are four positive flagellin bands: 35 kDa, 38 kDa, 43 kDa, and 52 kDa, of which the 43 kDa and 52 kDa bands displayed the strongest positive reaction. There are five positive OMP bands about 35 kDa, 38 kDa, 43 kDa, 47 kDa, and 52 kDa, of which the 43 kDa appeared to have the strongest positive reaction although the other four proteins also displayed strong reactions. However, LPS is Western blot-negative. These results indicate that the 43 kDa and 52 kDa flagellin and OMP of size 43 kDa, 52 kDa can be candidates for developing vaccines against V. harveyi. Translated from Journal of Xiamen University (Natural Science), 2006, 45(3): 393–396 [译自: 厦门大学学报 (自然科学版)]     

大黄鱼三种病原弧菌外膜蛋白交叉保护性抗原筛选

弧菌是海水养殖环境中常见的条件性致病菌,弧菌病的暴发给水产养殖业造成了严重损失。鉴于水生动物尤其是鱼类弧菌病的发生常常是多种（血清型或亚种）弧菌的混合感染,筛选具有潜在的交叉保护性蛋白抗原,作为制备多价疫苗或联合疫苗的侯选成分具有重要意义。文中从患病大黄鱼中分离到8株弧菌,经生理生化和分子生物学鉴定分别为6株哈维氏弧菌Vibrio harveyi,1株溶藻弧菌Vibrio alginolyticus和1株副溶血弧菌Vibrio parahaemolyticus。选择典型的不同种的弧菌为代表,提取其外膜蛋白,经SDS-PAGE和Westernblotting分析,确定它们大约在45 kDa、35 kDa、22 kDa处出现了3条共同的免疫印迹条带,表明它们很有可能含有共同的能够彼此交叉保护的抗原。利用双向电泳和免疫印迹相结合的方法,借助于MALDI-TOF-MS质谱分析技术,发现溶藻弧菌V.alginolyticus的一种功能未知的孔蛋白（Porin,GenBank Accession No.ZP₀1260407）和副溶血弧菌V.parahaemolyticus的一种麦芽糖孔蛋白的前体蛋白（Maltoporin precursor,GenBank AccessionNo.NP₈01154）能够和哈维氏弧菌V.harveyi全菌多抗产生免疫反应,表明这两种蛋白可以作为3种弧菌的交叉保护性抗原,以此制备的疫苗可望对3种弧菌的感染产生交叉保护作用。     

Immune response induced by N-lauroylsarcosine extracted outer-membrane proteins of an isolate of Edwardsiella ictaluri in channel catfish

A virulent isolate of Edwardsiella ictaluri (AL-93-75), the causative agent of enteric septicaemia of catfish (ESC), was used to derive a lipopolysaccharide-reduced N-lauroylsarcosine outer-membrane protein (OMP) fraction vaccine. The OMP fraction was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and compared to whole-cell lysate, purified lipopolysaccharide (LPS) and a crude cell-wall fraction. The OMP fraction contained less than 2% (W/V) LPS. SDS-PAGE showed that whole cell lysates contained 27 proteins from 107 to 14.3 kDa, whereas OMP contained nine proteins from 97 to 14.3 kDa, LPS contained two proteins at 45 and 37 kDa bands and a smear of bands below 14.3 kDa, and cell wall fraction contained 21 proteins from 97 to 8 kDa. Channel catfish, Ictalurus punctatus, were vaccinated with 12.5 microg/100 microl OMP and immunogenicity was confirmed by subsequent Western blots. Blots showed that 97, 80, and 19 kDa proteins were immunogenic. Rapid enzyme-linked immunosorbent assays (ELISA) demonstrated that OMP produced a weak, but observable antibody response by 21 days post injection. OMP concentrations of 3.13, 6.25, 12.5, 25, and 50 microg/100 microl total protein were tested for protective immunity. Marginal protection by relative percent survival (RPS) was only seen for fish injected with 12.5 microg/100 microl with RPSs between 55-67.5%. A booster dose of 12.5 microg/100 microl OMP did not significantly enhance protection.     

Immunogenic outer-membrane proteins of Haemophilus influenzae type b in infection.

Outer-membrane proteins (OMPs) from Haemophilus influenzae type b (strain Eagan), grown both in vitro (broth) and in vivo (rat intra-peritoneal), were separated by SDS-PAGE. The major OMPs were present in both growth conditions although the amounts of OMP a and OMP d were reduced in rat-grown organisms. There were strong additional bands in in-vivo-grown organisms at 51 and 92 kDa. Antiserum was raised in rabbits against in-vivo-grown bacteria, and absorbed with lysates of in-vitro-grown bacteria. This serum was used in Western blot analysis of OMPs from in-vitro- and in-vivo-grown cells to identify immunogenic proteins present in infection. These infection-associated OMPs had apparent molecular masses of 43 kDa, 48 kDa, 81 kDa and greater than 200 kDa. Bands of reactivity, of the same molecular mass as some of these, were found on immunoblots when rat and human convalescent sera were used as the source of primary antibody. In particular, a band of 81 kDa was recognized by pooled rat and three human convalescent sera.     

Immunodominant antigens recognized by the human immune response to infection by organisms of the species Leptospira interrogans serogroup Australis

Abstract Serum samples from patients infected by organisms of Leptospira interrogans serogroup Australis were tested by Western blot to determine the nature of major antigens that are involved in the immune response. Although there was some patient-to-patient variability, immunodominant genus-specific antigens were found to be proteins of apparent molecular ratio 68, 46 and 35-kDa, and lipopolysaccharide (LPS) sub-units in the 35-14-kDa region. Serogroup epitopes specific for Australis were exclusively saccharides of about 32 and 24 kDa: a serovar-specific antigen for serovar lora was of 38–40 kDa and behaved like a protein. Antibodies to the LPS serogroup-specific antigens and to the 38–40 kDa protein were long-lasting and consequently suggest that these immunodominant epitopes are important in resistance to re-infection.     

Tyrosine phosphorylated proteins in the reproductive tract of the viviparous lizard Eulamprus tympanum and the oviparous lizard Lampropholis guichenoti

Plastic changes occur in the morphology of the uterus at various stages of the reproductive cycle in both oviparous and viviparous lizards and these may be influenced by estrogen. Estrogen driven phosphorylation of effector proteins on tyrosine residues plays a major role in the plastic modulation of uterine anatomy and physiology in vertebrates. We used electrophoresis and Western blotting to characterize the phosphotyrosine protein profiles at various stages of the reproductive pathway in an oviparous lizard Lampropholis guichenoti and a viviparous lizard Eulamprus tympanum. L. guichenoti displayed major bands in the 200-35 kDa range and a triplet of bands of molecular masses 61 kDa, 52 kDa and 48 kDa in 50% of specimens and a 38 kDa band in all specimens. In contrast, E. tympanum samples all displayed a single major band at 40 kDa, which was significantly elevated at the early pregnancy stage. Somewhat paradoxically, the viviparous species, which has the more complex uterine epithelial changes during pregnancy, has the fewest phosphotyrosine bands, so how tyrosine phosphorylation is affected during the evolution of viviparity is not clear.     

Mycoplasma penetrans infections and seroconversion in patients with AIDS: identification of major mycoplasmal antigens targeted by host antibody response

We examined Mycoplasma penetrans-specific antibodies in sera of five male homosexual AIDS patients from whom M. penetrans was isolated during the disease process. No consistent immune reaction pattern could be recognized in Western blot using whole cell proteins. Serum samples obtained prior to M. penetrans isolation reacted with a number of M. penetrans proteins, most likely due to non-specific cross-reactions. Further analysis revealed that patients produced prominent antibody reaction to lipid-associated membrane proteins (LAMPs) of M. penetrans at the time of mycoplasma isolation, which could not be observed for serum samples obtained prior to M. penetrans isolation. The positive antibody reaction was mainly directed against two major LAMPs of M. penetrans with molecular mass of 35 and 38 kDa and produced a distinctive pattern of positive immunoreaction bands. Our observation suggested that, comparing with whole mycoplasmal proteins, LAMPs were more specific target antigens in serological assays for M. penetrans infection.     

Characterization of OmpK, GAPDH and their fusion OmpK-GAPDH derived from Vibrio harveyi outer membrane proteins: their immunoprotective ability against vibriosis in large yellow croaker (Pseudosciaena crocea)

AIM: To investigate the immunoprotection of three recombinant proteins derived from the Vibrio harveyi outer membrane proteins (OMPs) OmpK, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and their fusion OmpK-GAPDH as vaccine candidates from vibriosis of large yellow croaker (Pseudosciaena crocea). METHODS: The ompK gene, of which the leader sequence was omitted, was fused with the gapdh gene. Three recombinant proteins r-OmpK, r-GAPDH and r-OmpK-GAPDH were expressed and purified. Western blots were carried out to detect the specificity of the antibodies raised against the recombinant proteins; Fish were immunized with recombinant proteins and challenged by native V. harveyi. The immunoresponse to the recombinant proteins were determined by ELISA and phagocytic activity assay. CONCLUSIONS: The fusion protein r-OmpK-GAPDH can afford greater protection against the wild V. harveyi than r-OmpK or r-GAPDH alone or their mixture in humoral and cellular immunity, indicating that OmpK and GAPDH could produce a synergistic immunoprotection against vibriosis of large yellow croaker (Pseudosciaena crocea) when fused into OmpK-GAPDH with a linker. SIGNIFICANCE AND IMPACT OF THE STUDY: It has been realized that a multi-component OMP antigen can induce a higher frequency of immune effectors than a single OMP. The results presented here bring forth a good suggestion for the subunit vaccine design based on the OMPs of gram-negative pathogens.     

Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development

Brucella abortus is the etiologic agent of bovine brucellosis and causes a chronic disease in humans known as undulant fever. In livestock the disease is characterized by abortion and sterility. Live, attenuated vaccines such as S19 and RB51 have been used to control the spread of the disease in animals; however, they are considered unsafe for human use and they induce abortion in pregnant cattle. For the development of a safer and equally efficacious vaccine, immunoproteomics was utilized to identify novel candidate proteins from B. abortus cell envelope (CE). A total of 163 proteins were identified using 2-DE with MALDI-TOF MS and LC-MS/MS. Some of the major protein components include outer-membrane protein (OMP) 25, OMP31, Omp2b porin, and 60 kDa chaperonin GroEL. 2-DE Western blot analyses probed with antiserum from bovine and a human patient infected with Brucella identified several new immunogenic proteins such as fumarate reductase flavoprotein subunit, F0F1-type ATP synthase alpha subunit, and cysteine synthase A. The elucidation of the immunome of B. abortus CE identified a number of candidate proteins for developing vaccines against Brucella infection in bovine and humans.     

Flagellar proteins and type III-exported virulence factors are the predominant proteins secreted into the culture media of Salmonella typhimurium

We analysed all major proteins secreted into culture media from Salmonella typhimurium. Proteins in culture supernatants were collected by trichloroacetic acid precipitation, separated in SDS-polyacrylamide gels and analysed by amino acid sequencing. Wild-type strain SJW1103 cells typically gave rise to nine bands in SDS gels: 89, 67, 58, 52, 50, 42, 40, 35 and (sometimes) 28 kDa. A search of the sequences in the available databases revealed that they were either flagellar proteins or virulence factors. Six of them were flagella specific: FlgK or HAP1 (58 kDa), FliC or flagellin (52 kDa), FliD or HAP2 (50 kDa), FlgE or hook protein (42 kDa), FlgL or HAP3 (35 kDa) and FlgD or hook-cap protein (28 kDa). The other four bands were specific for virulence factors: SipA (89 kDa), SipB (67 kDa), SipC (42 kDa) and InvJ (40 kDa). The 42 kDa band was a mixture of FlgE and SipC. We also analysed secreted proteins from more than 30 flagellar mutants, and they were categorized into four groups according to their band patterns: wild type, mot type, polyhook type and master gene type. Virulence factors were constantly secreted at a higher level in all flagellar mutants except a deltamot (motAB deletion) mutant, in which the amounts were greatly reduced. A new morphological pathway of flagellar biogenesis including protein secretion is presented.     

Utilization of hemin and hemoglobin by Vibrio vulnificus biotype 2.

The eel pathogen Vibrio vulnificus biotype 2 is able to use hemoglobin (Hb) and hemin (Hm) to reverse iron limitation. In this stud, the adjuvant effect of both compounds on eel pathogenicity has been evaluated and confirmed. Further, we have studied the heme-iron acquisition mechanism displayed by this bacterium. Whole cells were capable of binding Hb and Hm, independently of (i) iron levels in growth medium and (ii) the presence of polysaccharide capsules on bacterial surface. The Hb- and Hm-binding capacity was retained by the outer membrane protein (OMP) fraction and was abolished after proteolytic digestion of OMP samples. Western blotting (immunoblotting) of denatured OMPs revealed that two major protein bands of 36 and 32 kDa were involved in both Hm and Hb binding. The expression of these proteins was not affected by iron levels. In addition, V. vulnificus biotype 2 produced extracellular proteases, not regulated by iron, that were active against native Hb. In conclusion, the overall data suggest that the eel pathogen V. vulnificus biotype 2 can obtain iron by means of a mechanism which involves a direct interaction between the heme moiety and constitutive OMPs.     

Identification of cyst and trophozoite antigens from Colombian Giardia duodenalis isolates recognized by IgA

Little is known about the role of IgA in the immune response against Giardia duodenalis infection. The current study identified the antigens of Colombian G. duodenalis isolates which stimulate the production of IgA anti-G. dudoenalis. Cyst and trophozoite stage proteins were separated by SDS-PAGE and their antigenicity was determined by Western blot. Without 2-mercapto ethanol (2-ME), the protein profile of the cyst stage showed 24 proteins within a molecular weight range of 23-270 kDa; with 2-ME, 35 polypeptides ranging from 22 to 241 kDa were distinguished. The trophozoite stage protein profile without 2-ME was formed by 16 proteins within the range of 24-270 kDa; with 2-ME, 45 proteins were present between 18 and 241 kDa. The identification of 20 and 29 antigens from the cyst and trophozoite stage, respectively, suggested that G. duodenalis stimulates a specific humoral immune response in the human host. The antigens of 31, 57, 110, 133, and 170 kDa recognized by anti-G duodenalis IgA in both cysts and trophozoites corresponded with G. duodenalis isolates from other geographic regions, whereas those of 35, 38, 43, 45, 49, 52, 60, 62, 65, 72, 82, 99, 145, 155, and 185 kDa seemed specific to Colombian isolates. This indicated that antigens of 57, 65, 145, and 170 kDa, recognized by anti-G. duodenalis IgA antibodies in cysts (with frequencies between 82% and 96%) and trophozoites (with frequencies between 86% and 97%) can be considered identification markers for G. duodenalis infections.     

Characterization of body louse midgut proteins recognized by resistant hosts

Abstract. The human body louse, Pediculus humanus , showed eighteen midgut proteins ranging between 12 and 117 kDa, when analysed by SDS-PAGE electrophoresis. Seven of them (12 kDa, 17 kDa, 29 kDa, 35 kDa, 40 kDa, 55 kDa and 97 kDa) were major bands based on their intensity of staining. The immunization of rabbits with a midgut extract elicited the production of protective polyclonal antibodies. These antibodies reacted strongly with all major midgut proteins as well as with 63 kDa and 117 kDa proteins when tested by the Western blot technique. The analysis of the proteins revealed that the 12 kDa, 25 kDa, 29 kDa, 35 kDa, 45 kDa, 87 kDa and 97 kDa proteins are glycosylated and none of them contained a lipid moiety. By electroelution, the proteins of 35 kDa and 63 kDa were purified. On trypsinization, the proteins of 35 kDa and 63 kDa produced four major fragments (F, F₂, F₃, and F₄) when resolved on a 18% SDS-PAGE. The Fj fragment of the 35 kDa protein reacted with me polyclonal antibodies by the immunoblot technique.     

Cloning, protein expression and display of synthetic multi-epitope mycobacterial antigens on Salmonella typhi Ty21a cell surface

Expressing proteins of interest as fusion to proteins of bacterial envelope is a powerful technique for biotechnological and medical applications. The synthetic gene (VacII) encoding for T-cell epitopes of selected genes of Mycobacterium tuberculosis namely, ESAT6, MTP40, 38 kDa, and MPT64 was fused with N- terminus of Pseudomonas syringae ice nucleation protein (INP) outer membrane protein. The fused genes were cloned into a bacterial expression vector pKK223-3. The recombinant protein was purified by Ni-NAT column. VacII gene was displayed on the cell surface of Salmonella typhi Ty21a using N-terminal region of ice nucleation proteins (INP) as an anchoring motif. Glycine method confirmed that VacII was anchored on the cell surface. Western blot analysis further identified the synthesis of INP derivatives containing the N-terminal domain INP- VacII fusion protein of the expected size (52 kDa).     

Proteomic analysis of Trichinella spiralis proteins in intestinal epithelial cells after culture with their larvae by shotgun LC-MS/MS approach

Although it has been known for many years that Trichinella spiralis initiates infection by invading intestinal epithelium, the mechanisms by which the parasite invades the intestinal epithelium are unknown. The purpose of this study was to screen the invasion-related proteins among the increased proteins of intestinal epithelial cells after culture with T. spiralis and to study their molecular functions. The proteins of HCT-8 cells which cultured with T. spiralis infective larvae were analyzed by SDS-PAGE and Western blot. Results showed that compared with proteins of normal HCT-8 cells, four additional protein bands (115, 61, 35 and 24 kDa) of HCT-8 cells cultured with the infective larvae were recognized by sera of the mice infected with T. spiralis, which may be the invasion-related proteins released by the infective larvae. Three bands (61, 35 and 24 kDa) were studied employing shotgun LC-MS/MS. Total 64 proteins of T. spiralis were identified from T. spiralis protein database by using SEQUEST searches, of which 43 (67.2%) proteins were distributed in a range of 10-70 kDa, and 26 proteins (40.6%) were in the range of pI 5-6. Fifty-four proteins were annotated according to Gene Ontology Annotation in terms of molecular function, biological process, and cellular localization. Out of 54 annotated proteins, 43 proteins (79.6%) had binding activity and 23 proteins (42.6%) had catalytic activity (e.g. hydrolase, transferase, etc.), which might be related to the invasion of intestinal epithelial cells by T. spiralis. The protein profile provides a valuable basis for further studies of the invasion-related proteins of T. spiralis.     

Isolation of Vibrio harveyi acyl carrier protein and the fabG, acpP, and fabF genes involved in fatty acid biosynthesis.

We report the isolation of Vibrio harveyi acyl carrier protein (ACP) and cloning of a 3,973-bp region containing the fabG (encoding 3-ketoacyl-ACP reductase, 25.5 kDa), acpP (encoding ACP, 8.7 kDa), fabF (encoding 3-ketoacyl-ACP synthase II, 43.1 kDa), and pabC (encoding aminodeoxychorismate lyase, 29.9 kDa) genes. Predicted amino acid sequences were, respectively, 78, 86, 76, and 35% identical to those of the corresponding Escherichia coli proteins. Five of the 11 sequence differences between V. harveyi and E. coli ACP were nonconservative amino acid differences concentrated in a loop region between helices I and II.     

An outer membrane protein, OmpK, is an effective vaccine candidate for Vibrio harveyi in Orange-spotted grouper (Epinephelus coioides)

The outer membrane proteins of the fish pathogen, Vibrio harveyi, have a role in interaction between bacterium and host and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein, OmpK, which serves as the receptor for broad-host-range vibriophage KVP40 in V. harveyi, was isolated and characterized. Then the OmpK gene coding for mature peptide was subcloned into prokaryotic expression vector pBV220 and transformed into Escherichia coli DH5 alpha strain. After temperature induction, a recombinant protein was detected about 28 kDa in molecular weight and accounted for 24.8% of total proteins of whole cell as estimated by SDS-PAGE and scanning analysis of gel image. Polyclonal antibodies were raised in rabbits against the purified protein and the reaction of the antibody was confirmed by western blotting using the purified protein and crude extract of V. harveyi. Orange-spotted groupers (Epinephelus coioides) vaccinated with recombinant OmpK produced specific antibodies, and were highly resistant to infection by virulent V. harveyi. These results indicate that the OmpK is an effective vaccine candidate against V. harveyi in Orange-spotted groupers.     

Characterization of heat-shock response of the marine bacterium Vibrio harveyi

We have investigated heat-shock response in a marine bacterium Vibrio harveyi. We have found that 39 C was the highest tempature at which V. harveyi was able to grow steadily. A shift from 30° C to 39° C caused increased synthesis of at least 10 proteins, as judged by SDS-PAGE, with molecular masses of 90, 70, 58, 41, 31, 27, 22, 15, 14.5 and 14kDa. The 70, 58, 41 and 14.5 kDa proteins were immunologically homologous to DnaK, GroEL, DnaJ and GroES heat-shock proteins of Escherichia coli, respectively. V. harveyi GroES protein had a lower molecular mass (14.5 kDa) than E. coli GroES, migrating in SDS-PAGE as 15 kDa protein. We showed that a protein of ～43 kDa, immunologically reactive with antiserum against E. coli sigma 32 subunit (σ³²) of RNA polymerase, was induced by heat-shock and co-purified with V. harveyi RNA polymerase. These results suggest that the 43 kDa protein is a heat-shock sigma protein of V. harveyi. Preparation containing the V. harveyi sigma 32 homologue, supplemented with core RNA polymerase of E. coli, was able to transcribe heat-shock promoters of E. coli in vitro.     

35-37 kDa形式可溶性MHC I释放机制研究

目的:探讨35-37 k Da形式的可溶性MHC I释放机制,为开展造血系统恶性肿瘤免疫干预治疗研究奠定理论基础。方法:以细胞表面标记、免疫沉淀、免疫印迹和增强化学发光法探讨EGTA和蔗糖对THP1细胞释放43和35-37 k Da可溶性MHC I的影响;以超速离心法纯化外排小体,并用免疫沉淀、免疫印迹和增强化学发光法检测43和35-37 k Da可溶性MHC I;用Quantity One软件对43和35-37 k Da可溶性MHC I进行相对定量分析。结果:EGTA同时显著抑制43和35-37 k Da可溶性MHC I产生;蔗糖同时显著促进43和35-37 k Da可溶性MHC I产生;43 k Da可溶性MHC I存在于外排小体上,而35-37 k Da可溶性MHC I在外排小体上检测不到。结论:35-37 k Da可溶性MHC I与外排小体都来源于细胞内多泡小体同质膜的溶合后释放,但35-37 k Da可溶性MHC I并不包含在外排小体的泡囊中,而是独立于外排小体释放。     

Demonstration of species-specific and cross reactive components of Paragonimus westermani crude worm antigen by EITB

Enzyme-linked immunoelectrotransfer blot (EITB) using crude worm antigen of adult Paragonimus westermani was performed for human patients sera to identify the species-specific components. Crude antigen was obtained by homogenizing and centrifuging 24-week old adult worms at 10,000 rpm for 60 minutes in phosphate buffered saline (PBS, pH 7.2) containing phenyl methyl sulfonyl fluoride (PMSF). Gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed and blotted electrophoretically onto a sheet of nitrocellulose paper. The sheet was cut into strips and exposed to sera diluted 1: 200 with PBS. SDS-PAGE showed 26 protein bands ranging 229 to 10 kDa. Of them 229, 91, 60, 50, 35-31, 27, 25, 21, 17, 11 and 10 kDa components showed positive reaction with serum antibody of patients with P. westermani. Sera of patients infected with Clonorchis sinensis reacted with 35-31, 19, and 11 kDa bands. Human sera from cysticercosis and diphyllobothriasis cases showed non-specific cross reactions with 229, 35-31, 27, 25 and 17 kDa bands. Protein bands of 91, 60, 21 and 10 kDa showed strong positive reaction without cross reactions with sera from other helminthic infections.