Human antibody response to the nucleoside triphosphate hydrolase of Toxoplasma gondii

An enzyme-linked immunosorbent assay (ELISA) that uses a recombinant protein of Toxoplasma gondii as antigen was used for the detection of specific antibodies in human sera. An antigenic portion of the T. gondii nucleoside triphosphate hydrolase (NTPase) was expressed in Escherichia coli as a glutathione S-transferase fusion protein with an apparent molecular mass of about 5000. A total of 118 T. gondii positive and 63 negative sera were examined. Seven % of the T. gondii positive sera, but none of the negative sera, reacted with the recombinant NTPase. Advantages and disadvantages that are associated with the use of fusion proteins as antigens in ELISA are discussed.     

Expression, characterization, and serologic reactivity of recombinant surface antigen P22 of Toxoplasma gondii.

The utility of recombinant Toxoplasma gondii surface antigen P22 for the detection of specific T. gondii antibodies in human sera was evaluated. Polymerase chain reaction was used to produce a 438-bp fragment of the P22 gene; the fragment corresponded to the amino acids predicted to be in the processed, native antigen. The fragment was subcloned into pGEX-2T and was expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. The fusion protein was purified in a soluble form and was found to be recognized by sera from infected individuals in immunoblots and an enzyme-linked immunosorbent assay. Immunoglobulin G antibodies in sera from 31 acutely infected patients in general reacted more strongly to the fusion protein than did those in sera from 31 patients with the chronic infection. None of the sera from a panel of 26 seronegative controls reacted with the fusion protein was separated from the GST partner by cleavage with thrombin, it retained its immunoreactivity and its electrophoretic mobility in polyacrylamide gels was found to be similar to that of native P22. By a modification of the published method for purification of the foreign polypeptide from the GST carrier, the recombinant P22 was readily purified to homogeneity by thrombin cleavage of the fusion protein while it was adsorbed to glutathione agarose.     

肠毒素大肠杆菌耐热肠毒素与谷胱甘肽巯基转移酶融合蛋白的制备和耐热肠毒素抗体测定

目的：弥补ST的弱抗原性，实现ST抗体测定。方法：采用生物工程技术，融合表达GST和带有前导序列的突变耐热肠毒素proSTm，制备ST融合蛋白，测定ST抗体。结果：测到了抗ST的血清IgG和黏膜IgA。结论：融合蛋白GST／proSTm能有效提高ST的抗原活性，可直接用于ST抗体测定。     

Expression and serologic activity of a soluble recombinant Plasmodium vivax Duffy binding protein.

Plasmodium vivax Duffy binding protein (DBP) is a conserved functionally important protein. P. vivax DBP is an asexual blood-stage malaria vaccine candidate because adhesion of P. vivax DBP to its erythrocyte receptor is essential for the parasite to continue development in human blood. We developed a soluble recombinant protein of P. vivax DBP (rDBP) and examined serologic activity to it in residents of a region of high endemicity. This soluble rDBP product contained the cysteine-rich ligand domain and most of the contiguous proline-rich hydrophilic region. rDBP was expressed as a glutathione S-transferase (GST) fusion protein and was isolated from GST by thrombin treatment of the purified fusion protein bound on glutathione agarose beads. P. vivax rDBP was immunogenic in rabbits and induced antibodies that reacted with P. vivax and Plasmodium knowlesi merozoites. Human sera from adult residents of a region of Papua New Guinea where malaria is highly endemic or P. vivax-infected North American residents reacted with rDBP in an immunoblot and an enzyme-linked immunosorbent assay. The reactivity to reduced, denatured P. vivax rDBP and the cross-reactivity with P. knowlesi indicated the presence of immunogenic conserved linear B-cell epitopes. A more extensive serologic survey of Papua New Guinea residents showed that antibody response to P. vivax DBP is common and increases with age, suggesting a possible boosting of the antibody response in some by repeated exposure to P. vivax. A positive humoral response to P. vivax DBP correlated with a significantly higher response to P. vivax MSP-1(19). The natural immunogenicity of this DBP should strengthen its usefulness as a vaccine.     

抗重组GST单克隆抗体的制备及其在融合蛋白纯化中的应用

目的 制备抗重组GST的单克隆抗体（mAb),并用来纯化重组GST融合蛋白,方法 用含重组GST融合蛋白基因的pGEX4T-1质粒转化E.coliBL21,IPTG诱导GST融合蛋白表达,亲和层析和凝胶过滤法分离表达的重组GST融合蛋白,以此蛋白作为抗原,免疫Balb/c小鼠,按传统的杂交瘤技术制备mAb。将抗GSTmAb经ProteinA纯化后,与Sepharose4B偶联。结果 经3次亚克隆后,获得两株分泌抗GST载体特异性mAb的杂交瘤,采用该mAb对两种不同的GST融合蛋白进行亲和层析纯化后,经SDS-PAGE鉴定达到了商品化Glutathione-Resin的亲和层析纯化效果。结论 用抗GST蛋白特异性mAb亲和层析纯化融合蛋白是一种经济、实用的方法,且可用于Glutathione-Resin亲和层析纯化后的二次纯化。     

丙型肝炎病毒基因组部分E2／NS1蛋白基因在大肠杆菌中的表达

在大肠杆菌中表达了丙型肝炎病毒基因组的部分Ｅ２／ＮＳ１蛋白（氨基酸３６４－５１２）。表达蛋白经ＳＤＳ－ＰＡＧＥ分析，主要表达带的分子量在～４３０００左右。表达蛋白用于检测已用Ｃ３３ｃ及Ｑ２２检测过的血清，发现Ｅ２ＮＳ１引抗体阳性率占Ｃ３３ｃ及Ｑ２２抗体阳性血清的２０％，尚没有发现单独Ｅ２／ＮＳ１抗体阳性的血清。     

Polymorphisms in biotransformation enzymes and the risk for recurrent early pregnancy loss

An imbalance between phase I drug metabolizing enzymes and phase II detoxification enzymes may contribute to the development of pre-eclampsia. Polymorphic variants in the phase I enzyme, cytochrome P450 genes may lead to increased toxification, whereas polymorphisms in the phase II enzyme, glutathione S-transferase genes may result in impaired detoxification. Most abundant in placenta and decidua is glutathione S-transferase P1-1, which may therefore be of particular importance in reproduction. We studied the frequencies of polymorphic variants in those enzymes in 187 women with recurrent early pregnancy loss and in 109 women with an uncomplicated obstetric history. DNA was extracted and subsequently polymerase chain reaction based genotyping assays were used. chi(2)-Analysis and Fisher's exact test were used for statistical evaluation. The glutathione S-transferase P1b-1b genotype was found significantly more often in women with recurrent early pregnancy loss than in controls (12% versus 5%, P = 0.03), in particular in those who consumed coffee (P = 0.02) or smoked cigarettes (P = 0.04). Polymorphisms in other glutathione S-transferase and cytochrome P450 genes occurred equally frequently in cases and controls. In conclusion, the occurrence of the glutathione S-transferase P1b-1b genotype, leading to lower glutathione S-transferase Pi enzyme activity and consequently to impaired placental detoxification, may represent a risk factor for recurrent early pregnancy loss.     

Expression and characterization of glutathione peroxidase activity in the human blood fluke Schistosoma mansoni.

Antioxidants may play an important role in immune evasion by schistosome parasites. Previous studies have focused on the roles of superoxide dismutase and glutathione S-transferase. In the present study, glutathione peroxidase (GPX) activity was measured in different fractions of worm extracts from several developmental stages of Schistosoma mansoni. The enzyme activity was shown to be developmentally regulated, with higher specific activities being found in the tegument-enriched Nonidet P-40 extract of adult worms (the stage least susceptible to immune killing) than in the larval stages (which are most susceptible to immune elimination). In all extracts tested, the activity against cumene hydroperoxide, even when glutathione S-transferase activity was removed, was higher than that for hydrogen peroxide. The expression of GPX cDNA in pGEX-2T by bacteria produced a 50-kDa fusion protein and a 32-kDa truncated protein. The latter was due to termination at the internal UGA codon that codes for selenocysteine. GPX activity was detected in the recombinantly produced GPX but not with Sj26-glutathione S-transferase from the vector. Mutating the TGA codon to TGT produced a full-length product, GPXm (19 kDa), that was used to produce 19 monoclonal antibodies. Anti-GPXm monoclonal antibodies recognized a 19-kDa molecule in adult-worm extract which, upon removal by immunoprecipitation, resulted in the loss of over 90% of the GPX activity, suggesting that a single form of GPX exists in the schistosome.     

Detection of antibodies against equine herpesvirus types 1 and 4 by using recombinant protein derived from an immunodominant region of glycoprotein B.

The N-terminal fragment comprising residues +1 to +50 (gB1-50) of equine herpesvirus type 1 (EHV-1) glycoprotein B was expressed as a glutathione S-transferase fusion protein in Escherichia coli. Recombinant gB1-50 (rgB1-50) was recognized in immunoblots by sera from rabbits immunized with EHV-1 and by convalescent-phase sera from horses with natural EHV-1 infections. An enzyme-linked immunosorbent assay (ELISA) for monitoring antibody levels against EHV-1 was developed by using rgB1-50, and its specificity was assessed with a panel of reference antisera against other equine viruses. A specific cross-reaction was detected with EHV-4, which was confirmed by inhibition ELISA. Convalescent-phase sera from horses with natural EHV-1 or EHV-4 infections possessed antibody titers against rgB1-50 ranging from 1:2,000 to 1:64,000, indicating the presence of an immunodominant antigenic site. The study demonstrated the potential application of rgB1-50 as a diagnostic antigen and highlights the glutathione S-transferase fusion system as a simple and effective method of producing purified milligram quantities of antigen.     

Determination of bovine adenovirus-3 titer based on immunohistochemical detection of DNA binding protein in infected cells

DNA sequence coding for a portion of DNA binding protein (amino acids 3-58) of bovine adenovirus type-3 (BAV-3) was cloned and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase. The fusion protein was affinity purified and used to immunize rabbits. Immunoprecipitation and Western blot analysis showed that the antiserum could specifically recognize a protein of 48 kDa in BAV-3-infected cells, which was produced both in early and late phases of BAV-3 life cycle. Based on the ability of antiserum to recognize DNA binding protein, a novel assay for BAV-3 quantitation was established. The assay is less time consuming and can be performed on a wide variety of bovine cells. In addition, virus titers determined by this assay are comparable to the standard plaque assay.     

Active immunization with recombinant V antigen from Yersinia pestis protects mice against plague.

The gene encoding V antigen from Yersinia pestis was cloned into the plasmid expression vector pGEX-5X-2. When electroporated into Escherichia coli JM109, the recombinant expressed V antigen as a stable fusion protein with glutathione S-transferase. The glutathione S-transferase-V fusion protein was isolated from recombinant E. coli and cleaved with factor Xa to yield purified V antigen as a stable product. Recombinant V antigen was inoculated intraperitoneally into mice and shown to induce a protective immune response against a subcutaneous challenge with 3.74 x 10(6) CFU of virulent Y. pestis. Protection correlated with the induction of a high titer of serum antibodies and a T-cell response specific for recombinant V antigen. These results indicate that V antigen should be a major component of an improved vaccine for plague.     

Construction and expression of hTNF-alpha fusion protein mediated by MMP1

This paper is aimed to present a research on fusion protein of human tumor necrosis factor-alpha (hTNF-alpha), matrix metalloproteinase 1 (MMP1), and foldon sequence using the methord of gene engineering. We transformed the recombinant plasmid, which contains the DNA sequences of hTNF-alpha, MMP1, and foldon sequence, into Rosetta2, and successfully induced the fusion protein to express under given conditions by isopropyl beta-D-1-Thiogalactopyranoside (IPTG). Then we purified the expression product through a glutathione S-transferase (GST) resin and collected the interested protein. This research may lay the groundwork for scientific research and clinical application.     

Identification of heparin as a ligand for the A-domain of Plasmodium falciparum thrombospondin-related adhesion protein.

Thrombospondin-related adhesion protein (TRAP) is a Plasmodium falciparum transmembrane protein that is expressed within the micronemes of sporozoites, and is implicated in host cell invasion and motility. Contained within the extracellular region of TRAP is an A-domain, a module found in a number of membrane, plasma and matrix proteins, that is often involved in ligand recognition. In order to determine the role of the TRAP A-domain, it has been expressed as a glutathione S-transferase fusion protein and its ligand binding compared with that of other characterised glutathione S-transferase A-domain fusion proteins. Using a solid phase assay to screen for binding to known A-domain ligands, the TRAP A-domain was found to bind heparin. Binding to heparin appeared to be specific as it was saturable, and was inhibited by soluble heparin, fucoidan and dextran sulfate, but not by other negatively charged sulfated glycosaminoglycans such as chondroitin sulfates. Furthermore, unlike some A-domain ligand interactions, the A-domain of both TRAP and the leukocyte integrin, Mac-1, bound to heparin in the absence of divalent cations. It has been shown previously that another domain within TRAP, which is homologous to region II-plus of circumsporozoite protein, binds to sulfatide and to heparan sulfate on the immortalised hepatocyte line HepG2. The TRAP A-domain also bound to sulfatide and to HepG2 cells. Thus the A-domain shares certain binding properties already attributed to the region II-plus-like domain of TRAP, and may contribute to the binding of TRAP to heparan sulfate on hepatocytes.     

High-level expression and purification of a truncated merozoite antigen-2 of Babesia equi in Escherichia coli and its potential for immunodiagnosis

The gene encoding a truncated merozoite antigen-2 (EMA-2t) of Babesia equi was cloned and highly expressed in Escherichia coli as a glutathione S-transferase fusion protein (G-rEMA-2t). Both G-rEMA-2t and rEMA-2t (after the removal of glutathione S-transferase) had good antigenicity. Either Western blot analysis with rEMA-2t or enzyme-linked immunosorbent assay (ELISA) with G-rEMA-2t clearly discriminated the sera of horses experimentally infected with B. equi from sera of horses infected with Babesia caballi and healthy horses, although rEMA-2t was not suitable for ELISA, probably owing to its poor absorbability to the plates. The specific antibodies in B. equi-infected horses were detectable during both acute and latent infection (6 to 244 days postinfection). Horse sera from Jilin Province, China, were examined by the two tests. The seroprevalence of B. equi was 49.2% (31 of 63 sera) by Western blot analysis with rEMA-2t and 47.6% (30 of 63 sera) by ELISA with G-rEMA-2t. The correspondence was 98.4% (62 of 63 sera) between the two tests. The results indicate that G-rEMA-2t and rEMA-2t proteins should be suitable antigens for the development of an effective immunodiagnostic assay due to their high sensitivity, specificity, and great yield.     

Expression and thrombin cleavage of Poa p IX recombinant allergens fused to glutathione S-transferase.

The high-level expression and purification of Poa p IX recombinant grass pollen allergens were examined utilizing a modified pGEX plasmid, designated as pGEX 2T-1. This vector permits frame-1 ligation of lambda gt11 cDNA inserts and cleavage of the recombinant allergenic protein from the fusion partner glutathione S-transferase. The expression of the fusion proteins in water-soluble form varied among the transformants of the same bacterial strain and also between different host strains. Purification of the fusion proteins by affinity chromatography employing glutathione agarose gel revealed that proteases in the bacterial lysate bound to the gel and were co-eluted with the fusion proteins. These proteases, which specifically degraded the recombinant proteins to varying degrees, were inhibited by both of the inhibitors, phenylmethylsulfonyl fluoride and aprotinin. Cleavage by thrombin of the fusion proteins indicated that the structure of the individual protein affected the thrombin accessibility to the cleavage site. Increased concentration of thrombin partly compensated this effect, but resulted in a broader specificity of the enzyme. By contrast, cleavage of the fusion protein when it was still attached to the glutathione gel was convenient and led to purification of the product devoid of proteolytic activity. Since almost all the recombinant allergens have been cloned in lambda gt11 vector, the pGEX 2T-1 vector reported herein will facilitate the synthesis, purification of the corresponding allergenic proteins or their peptides in soluble and biologically active forms.     

Echovirus 9 strain barty non-structural protein 2C has NTPase activity

Non-structural protein 2C is known to play a fundamental role in the replication of picornaviruses. Sequence analyses revealed that 2C belongs to a rapidly expanding group of proteins containing a consensus sequence for nucleotide binding (NTB). We report that echovirus 9 polypeptide 2C displays NTPase activity in vitro. In our experiments, several P2 genes were expressed in Escherichia coli as fusion proteins linked to glutathione S-transferase (GST) prior to purification close to homogeneity. In contrast to GST-2B, both GST-2C and GST-2BC showed ATPase as well as GTPase activity indicating that the site for NTB binding and splitting is located in 2C.     

Identification of a collagen-like protein gene from white spot syndrome virus

Summary. The most unique feature of white spot syndrome virus (WSSV) is the presence of a collagen-like protein (termed as WSSV-CLP). In this report, the N-terminal fragment of WSSV-CLP (CLPn) was expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli and purified. Specific antibody was then raised against the purified fusion protein (GST-CLPn). Temporal analysis showed that the WSSV collagen gene was an early viral gene. Immunogold localization using specific antibody revealed that the gold particles, under a transmission electron microscope, were presented along the outer envelope of WSSV virions. This experiment suggested that the collagen gene encoded an envelope protein of WSSV. Using immuno-affinity chromatography, the WSSV-CLP was purified from crudely purified WSSV virions. The WSSV-CLP was N-glycosylated, as indicated by the increased migration in SDS-PAGE after treatment with N-linked glycosidase F.     

Recombinant fusion proteins of protein A and protein G with glutathione S-transferase as reporter molecules

The regions encoding the IgG-binding domains of protein A (PA) and protein G (PG) were cloned into the bacterial expression vector pGEX. Both proteins were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (PA-GST and PG-GST) and were found to be soluble, abundant and easily purified in one step from the bacterial lysate by affinity chromatography on immobilized glutathione. Yields of 50 mg/litre of cultures were obtained. Both purified fusion proteins were shown to be functional in a variety of immunochemical procedures. In radial diffusion tests, PA-GST precipitated IgG from human, squirrel monkey, rabbit, dog, cat and pig but not mouse, sheep, goat, cow, horse or chicken. PG-GST formed precipitin bands with IgG from human, rabbit, mouse, pig, sheep, goat, cow and horse but not squirrel monkey, dog, cat and chicken IgG. The fusion proteins were shown to function as effective detection reagents in ELISA and Western blotting. Glutathione agarose beads with bound fusion protein were shown to be useful for immunoprecipitation.