基于IgY抗体检测日本血吸虫循环抗原的间接红细胞凝集试验的建立

目的 建立基于IgY的血吸虫循环抗原的间接红细胞凝集试验(indirect hemagglutination test,IHA)并初步探讨IgY在诊断日本血吸虫病中的应用价值.方法 以日本血吸虫卵可溶性虫抗原(soluble egg antigen,SEA)免疫海蓝蛋鸡,获得抗SEA的IgY抗体.取人O型红细胞醛化、鞣...     

Methanococcus vannielii selenium-binding protein (SeBP): chemical reactivity of recombinant SeBP produced in Escherichia coli

A selenium-binding protein (SeBP) from Methanococcus vannielii was recently identified, and its gene was isolated and overexpressed in Escherichia coli [Self, W. T., Pierce, R. & Stadtman, T. C. (2004) IUBMB Life 56, 501-507]. SeBP and recombinant SeBP (rSeBP) migrated as approximately 42-kDa species on native gels and as approximately 33-kDa species on SDS gels. rSeBP consists of identical 8.8-kDa subunits, each containing a single cysteine residue. rSeBP isolated in the absence of reducing agents contained oxidized cysteine (89%) and very little bound selenium (0.05 eq or less per subunit). Complete reduction of the oxidized cysteine residues in rSeBP with Tris(2-carboxyethyl)phosphine required addition of a denaturant, such as 1 M guanidine-hydrochloride. With selenite as the selenium source and the isolated reduced protein as sole reductant, binding of one selenium per tetramer under anaerobic conditions required four cysteine thiol groups, one on each subunit. In the corresponding reaction, with reduced glutathione (GSH), equimolar amounts of selenodiglutathione (GSSeSG) and glutathione disulfide are formed from selenite and 4 GSH. At GSH-to-selenite ratios >4:1, conversion of GSSeSG to a perselenide derivative, GSSe(-), occurs. However, with the reduced rSeBP as sole electron donor in the reaction with selenite, further conversion of the R-SSeS-R product apparently did not occur. Prior alkylation of the cysteine thiol groups in reduced rSeBP prevented selenite reduction and selenium binding under comparable conditions.     

抗大肠杆菌O157鸡卵黄抗体的初步研究

目的以煮沸15min灭活后的大肠杆菌O157为抗原,分4次免疫产蛋鸡,获取高免卵黄。经水稀释法去脂、超滤、盐析等粗分离,DEAESephadexA-50离子交换层析纯化,得到产物IgY为电泳单点纯,且仍保持效价在1∶104以上。从分离纯化过程中的跟踪检测表明,其卵黄制备的IgY抗体产量多、效价高、特异性强,可进一步用于大肠杆菌感染的检测研究。     

Identification of antigens by monoclonal antibody PD4 and its expression in Escherichia coli

AIM: To clone and express the antigen of monoclonal antibody (MAb) PD4 for further investigation of its function. METHODS: MGC803 cDNA expression library was constructed and screened with PD4 as probes to clone the antigen. After failed in the library screening, immunoprecipitation and SDS-polyacrylamide gel electrophoresis were applied to purify the antigen for sequence analysis. The antigen coming from Mycoplasma hyorhinis (M. hyorhinis) was further confirmed with Western blot analysis by infecting M. hyorhinis -free HeLa cells and eliminating the M. hyorhinis from MGC803 cells. The full p37 gene was cloned by PCR and expressed successfully in Escherichia coli after site-directed mutations. Immunofluorescence assay was used to demonstrate if p37 protein could directly bind to gastric tumor cell AGS. RESULTS: The cDNA library constructed with MGC803 cells was screened by MAb PD4 as probes. Unfortunately, the positive clones identified with MAb PD4 were also reacted with unrelated antibodies. Then, immunoprecipitation was performed and the purified antigen was identified to be a membrane protein of Mycoplasma hyorhinis (M. hyorhinis) by sequencing of N-terminal amino acid residues. The membrane protein was intensively verified with Western blot by eliminating M. hyorhinis from MGC803 cells and by infecting M. hyorhinis-free HeLa cells. The full p37 gene was cloned and expressed successfully in Escherichia coli after site-directed mutations. Immunofluorescence demonstrated that p37 protein could directly bind to gastric tumor cell AGS. CONCLUSION: The antigen recognized by MAb PD4 is from M. hyorhinis, which suggests the actions involved in MAb PD4 is possibly mediated by p37 protein or M. hyorhinis. As p37 protein can bind directly to tumor cells, the pathogenic role of p37 involved in tumorigenesis justifies further investigation.     

Detection of proteins like human gamma and beta globins in Escherichia coli carrying recombinant DNA plasmids.

Escherichia coli strain chi 1776 carrying recombinant DNA plasmids containing cDNA copies of human beta or gamma globin mRNAs has been shown by radioimmunoassay to synthesize polypeptides antigenically related to the beta and gamma chains of human hemoglobin. The gamma and beta polypeptides have been enriched from lysates on immunoabsorbent columns containing hemoglobin antibodies and shown to specifically inhibit the antigen-antibody binding between 125I-labeled hemoglobin and the homologous antibody but not other hemoglobin-antihemoglobin reactions. Clone JW151, which is known to contain a complete copy of the coding portion of the gamma globin mRNA, has been shown to produce a protein that reacts specifically with antibody to the chain of fetal hemoglobin, hemoglobin Kenya, and hemoglobin Bart's.     

Production of immunologically active surface antigens of hepatitis B virus by Escherichia coli.

Several plasmids have been constructed which direct the synthesis of hepatitis B virus surface antigens in Escherichia coli either as the native polypeptide or fused to other plasmid encoded polypeptides. When injected into rabbits, extracts from bacteria carrying some of these plasmids induced the synthesis of antibodies to the antigens even though the extracts did not give satisfactory positive results in radioimmunoassay for them. Either the NH2-terminal segment or the COOH-terminal segment of the surface antigens alone was sufficient to elicit the immune response, but antibodies against the two segments showed different specificities. The results emphasize the value of an in vivo assay for the presence of antigens in crude cell extracts and illustrate the feasibility of this type of screening with laboratory animals.     

Generation of antibody activity from immunoglobulin polypeptide chains produced in Escherichia coli.

Plasmids have been constructed that direct the synthesis in Escherichia coli of heavy chains and/or light chains of an anti-carcinoembryonic antigen (CEA) antibody. Another plasmid was constructed for expression of a truncated form of heavy chain (Fd' fragment) in E. coli. Functional CEA-binding activity was obtained by in vitro reconstitution in E. coli extracts of heavy chain or Fd' fragment mixed with extracts containing light chain.     

Intervention with Shiga toxin (Stx) antibody after infection by Stx-producing Escherichia coli

Shiga toxins (Stxs) produced by Escherichia coli (STEC) cause systemic vascular damage, manifested as hemolytic uremic syndrome in humans and as edema disease in pigs. Edema disease, a naturally occurring disease of pigs, was used to determine whether Stx antibodies, administered after infection and after the onset of Stx production, could prevent the systemic vascular damage and clinical disease caused by Stxs. A total of 119 STEC-infected pigs were treated with low, medium, or high doses of Stx antibody or with placebo. After inoculation with STEC, antibodies or placebo was injected intraperitoneally at 2 days postinoculation (DPI; low dose) or 4 DPI (medium and high doses). Edema disease was prevented with the low- and high-dose Stx antibody treatments administered at 2 and 4 DPI, respectively. High-dose antibody treatment also reduced the incidence and extent of vascular lesions. The degree of protection depended on the dose of antibody and the time of administration.     

Immunogenicity of recombinant fragments of Plasmodium falciparum acidic basic repeat antigen produced in Escherichia coli

The acidic basic repeat antigen (ABRA) of Plasmodium falciparum is a potential vaccine candidate against erythrocytic stages of malaria. We report, for the first time, the immunological characteristics of recombinant ABRA constructs. The recombinant proteins representing different fragments of ABRA were expressed in Escherichia coli, either as fusions with maltose binding protein or as 6X histidine tagged molecules, and purified by affinity chromatography. Immunogenicity studies with these constructs in rabbits and mice indicated that the N-terminal region is the least immunogenic part of ABRA. T-cell proliferation experiments in mice immunized with these constructs revealed that the T-cell epitopes were localized in the middle portion of the protein. More importantly, the purified immunoglobulin G specific to middle and C-terminal fragments prevented parasite growth at levels approaching 80-90%. We found that these proteins were also recognized by sera from P. falciparum-infected patients from Rourkela, a malaria endemic zone of India. Our immunogenicity results suggest that potential of ABRA as a vaccine candidate antigen should be investigated further.     

Detection of antibody to envelope (E2) antigen of hepatitis C virus

One hundred and four clinical specimens from provincial public health laboratories were tested for antibody to hepatitis C virus (HCV) envelope protein (anti-E2). To evaluate the effect of hypervariability of E2 region on anti-E2 assay, 49 recombinant immunoblot assay (RIBA) 3.0 positive samples were genotyped. All 49 genotyped samples were positive for anti-E2. Eight of 12 (67%) indeterminate, HCV RNA positive samples were anti-E2 reactive. Nine of 30 (30%) indeterminate, HCV RNA negative samples were also positive for anti-E2. Anti-E2 was detected in two of 13 (15%) RIBA-negative and enzyme immunoassays-positive samples. Although small number of samples were tested, the results showed that it may be possible to resolve indeterminate samples with the anti-E2 assay.     

Detection of antibodies to Epstein-Barr virus antigens by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) is described for the serologic analysis of antibodies to Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA), viral capsid antigen (VCA), and early antigen (EA). The specificity of each of the ELISAs was demonstrated by the use of well-characterized human sera shown by immunofluorescence assay to be variously reactive for antibodies to one or more of the three viral antigens studied. The ELISA for EBNA was four to 256 times more sensitive than immunofluorescence assays with all 33 EBNA-positive sera tested. The ELISAs for VCA and EA were also more sensitive than immunofluorescence assays: approximately 50% of the sera tested showed higher antibody titers. Sera that were negative for all three antigens by immunofluorescence assay were also negative by ELISA for each antigen. These ELISAs for EBV are rapid, sensitive, and objective and thus provide new and valuable methods for the detection of antibodies to EBV-related antigens.     

Efficient expression of influenza virus NS1 nonstructural proteins in Escherichia coli.

RNA segment 8 of the influenza A virus genome codes for two nonstructural proteins, NS1 and NS2, for which the functions are unknown. Cloned cDNA copies of this gene from three different influenza A virus strains were inserted into an Escherichia coli plasmid expression vector, pAS1, carrying the strong regulatable lambda phage promoter, PL. After induction, the NS1 proteins were overproduced to levels of 20-25% of total cellular protein. This was surprising in that the codon composition for these eukaryotic genes is similar to that for weakly expressed proteins in E. coli. Thus, under the appropriate conditions, it appears that high level expression of genes containing a relatively large proportion of minor codons can be obtained. The NS1 protein produced in bacteria from a cloned cDNA copy of the A/PR/8/34 virus NS gene was purified to apparent homogeneity and used to generate a high-titer monospecific rabbit antiserum. Immunoprecipitation studies showed this antibody to be crossreactive against the NS1 proteins produced by several different influenza A virus strains. Immunofluorescence experiments in Madin-Darby canine kidney cells showed the NS1 proteins to be located in the nucleoplasm early in infection for all strains examined. With some of the strains, NS1-specific immunofluorescence was observed predominantly in the nucleoli later in infection. This technology can be used to obtain other viral proteins in pure form for structural, functional, and immunological studies.     

Analysis of human serum antibodies to human immunodeficiency virus (HIV) using recombinant ENV and GAG antigens

Recombinant proteins representing gag and env amino acid sequences of the Human Immunodeficiency Virus (HIV) (HTLV-IIIb) were produced in Escherichia coli and used to analyze sera for the presence of antibodies to HIV. ENV-9 is a protein representing the carboxy terminus of gp120 and part of gp41 which is highly immunoreactive. GAG-1 represents 83% and GAG-55 100% of the amino acids of the gag open reading frame. The purified proteins allow sensitive detection by enzyme linked immunosorbent assay (ELISA) of antibodies directed against either env or gag of HIV. We have determined the reactivity of sera from several HIV exposed individuals, either form high risk populations or with clinically defined conditions, in the ENV-9, GAG-55, and GAG-1 assays and found that two major seropositive groups are observed. The quantitative analysis of sera with env and gag antigens by ELISA showed AIDS patients had very low gag reactivity while retaining high env reactivity. Results obtained with authentic p24 viral protein in both ELISA and radioimmunoassay correlated to those from the GAG-55 ELISA. This correlation and the analysis of sera with both the ENV and GAG ELISAs indicate that the antibodies reactive to gag are specifically affected relative to env reactivity and that different levels of antibodies to separate viral components in these sera may correlate with disease state.     

Protection against Escherichia coli infection by antibody to the Staphylococcus aureus poly-N-acetylglucosamine surface polysaccharide

Poly-N-acetylglucosamine (PNAG) is a surface polysaccharide produced by Staphylococcus aureus and Staphylococcus epidermidis and is an effective target for opsonic and protective Ab for these two organisms. Recently, it has been found that Escherichia coli produces an exo-polysaccharide, designated polyglucosamine, that is biochemically indistinguishable from PNAG. We analyzed 30 E. coli strains isolated from urinary tract and neonatal bloodstream infections for the pga locus, PNAG antigen production, and susceptibility to opsonic killing and protection from lethal infection by Ab to PNAG. Twenty-six of 30 strains carried the pga locus, 25 of 30 expressed immunologically detectable PNAG, and 21 of 30 could be killed by rabbit IgG specific for the deacetylated form of the staphylococcal PNAG. Ab to staphylococcal PNAG protected mice against lethality from five different E. coli strains expressing PNAG. PNAG expression by both Gram-negative and Gram-positive organisms could make this antigen a conserved vaccine target for multiple pathogenic species of bacteria.     

Immunological properties of recombinant proteins of the transmission blocking vaccine candidate, Pfs48/45, of the human malaria parasite Plasmodium falciparum produced in Escherichia coli

A precondition for the development of a transmission blocking vaccine based on the sexual stage-specific surface antigen Pfs48/45 of Plasmodium falciparum is its heterologous synthesis in a native state. Here we describe the production of recombinant Pfs48/45 in Escherichia coli . Two recombinant proteins, of which one is a glutathione-S-transferase fusion protein, were produced. Enzyme-linked immunosorbent assays showed that at least a subfraction of the recombinant proteins had a conformation capable of binding transmission blocking monoclonal antibodies. However, despite the fact that both proteins were very immunogenic, they did not induce transmission blocking immunity in mice or rabbits. Immunological studies with congenic mouse strains demonstrated that immune responses could be boosted with gametocyte extracts and were not restricted to a particular class II major histocompatibility complex haplotype .