Recombinant Expression Product Research Articles

Biological activity and pathogenicity of M23 superfamily metalloendopeptidases of Leptospira interrogans

Objective To understand and determine the biological activity and pathogenicity of metalloendopeptidases encoded by LA2582 and LA2901 genes of Leptospira interrogans(L.interrogans) serogroup Icterohaemorrhagiaeserovar Lai strain Lai. Methods Structures and functions of LA2582 and LA2901 genes were analyzed by using bioinformatic software. Prokaryotic expression systems for expressing the extracellular regions of LA2582 and LA2901 genes were generated. The target recombinant expression products, rLA2582 and rLA2901, were extracted by Ni-NTA affinity chromatography. The Azo-casein-hydrolyzingactivity of rLA2582 and rLA2901 was detected by spectrophotometry. Activities of rLA2582 and rLA2901 in the hydrolysis of Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans, a fluorescence-labeling pentapeptide substrate, were detected by fluorospectrophotometry, and then the Km and Kcat values were determined. SDS-PAGE and spectrophotometry were performed to detect the activities of rLA2582 and rLA2901 in hydrolyzing extracellular matrix molecules such as collagen type-Ⅰ (COL1), fibronectin (FN) and Congo red-labeling elastin (ELN). Real-time fluorescent quantitative RT-PCR (qRT-PCR) and Western blot were respectively used to measure the expression of LA2582 and LA2901 genes at mRNA and protein levels after infecting human umbilical vein endothelial cells(HUVEC) with L.interrogans strain Lai. Results The gene products of LA2582 and LA2901 genes were identified as the signal peptide and matrix metalloproteinase motif HXH-containing Zn2+ -dependent Gly-Gly metalloendopeptidases belonging to the M23 superfamily. rLA2582 and rLA2901 did not hydrolyze Azo-casein (Km=126.54 μmol/L, Kcat=4.67/s), but could hydrolyze the pentapeptide substrate (Km=190.25 μmol/L, Kcat 4.86/s). rLA2582 and rLA2901 could hydrolyze COL1, FN and ELN. Expression of LA2582 and LA2901 genes at both mRNA and protein levels was significantly increased after infection of HUVEC with L.interrogans strain Lai (P<0.05). Conclusion The products of LA2582 and LA2901 genes of L.interrogans strain Lai are Zn2+ -dependent M23 metalloendopeptidases, which can hydrolyze multiple ECM molecules and are closely associated with the leptospiral invasiveness. Key words: Leptospira interrogans; M23 superfamily; Metalloendopeptidase; Biological activity; Pathogenecity

Development and validation of a genotype 3 recombinant protein-based immunoassay for hepatitis E virus serology in swine

Hepatitis E virus (HEV) is classified within the family Hepeviridae, genus Hepevirus. HEV genotype 3 (Gt3) infections are endemic in pigs in Western Europe and in North and South America and cause zoonotic infections in humans. Several serological assays to detect HEV antibodies in pigs have been developed, at first mainly based on HEV genotype 1 (Gt1) antigens. To develop a sensitive HEV Gt3 ELISA, a recombinant baculovirus expression product of HEV Gt3 open reading frame-2 was produced and coated onto polystyrene ELISA plates. After incubation of porcine sera, bound HEV antibodies were detected with anti-porcine anti-IgG and anti-IgM conjugates. For primary estimation of sensitivity and specificity of the assay, sets of sera were used from pigs experimentally infected with HEV Gt3. For further validation of the assay and to set the cutoff value, a batch of 1100 pig sera was used. All pig sera were tested using the developed HEV Gt3 assay and two other serologic assays based on HEV Gt1 antigens. Since there is no gold standard available for HEV antibody testing, further validation and a definite setting of the cutoff of the developed HEV Gt3 assay were performed using a statistical approach based on Bayes' theorem. The developed and validated HEV antibody assay showed effective detection of HEV-specific antibodies. This assay can contribute to an improved detection of HEV antibodies and enable more reliable estimates of the prevalence of HEV Gt3 in swine in different regions.

Distribution of Salmonella paratyphi A pagC gene and immunoprotective effect of its recombinant expressed products

To determine the distribution and sequence conservation of pagC gene in Salmonella paratyphi A isolates, and the immunogenicity and immunoprotection of its recombinant expression products (rPagC). The distribution of pagC gene in Salmonella paratyphi A isolates and its sequence conservation were examined by PCR and sequencing. A prokaryotic expression system of pagC gene was constructed and the expressed rPagC was extracted by Ni-NTA affinity chromatography. SDS-PAGE and Bio-Rad Gel Image Analyzer were applied to examine the expression and yield of rPagC. The antigenicity and immunoreactivity of rPagC were detected by immunodiffusion test, ELISA and Western Blot assay. The immunoprotective effect of rPagC against infection of Salmonella paratyphi A in mice was determined, while the agglutinative effect of sera from rPagC-immunized mice was measured by micro-Widal's test. All the Salmonella paratyphi A isolates tested had the pagC gene, the similarity of nucleotide and amino acid sequences was 99.1 %-100 % and 98.4 %-100 %, respectively. The constructed prokaryotic expression system expressed rPagC with high efficiency. The rPagC immunized rabbit produced a high level antibody and it also combined with antiserum against whole cell of S. paratyphi A to generate a positive Western hybridization signal. ELISA results indicated that 97.1 % (66/68) paratyphoid patients infected with Salmonella paratyphi A were positive for rPagC antibody in their serum specimens. When mice were immunized with 100 μg or 200 μg rPagC, the immunoprotective rates were 73.3 % (11/15) or 86.7 % (13/15), respectively. The sera from rPagC-immunized mice offered 1:10-1:40 agglutination titers with the H antigens of Salmonella paratyphi A and Salmonella typhi. PagC gene has an extensive distribution in Salmonella paratyphi A isolates. rPagC can be used as the candidate antigen in genetic engineering vaccine due to its fine immunogenicity and powerful immunoprotective effect.

Generation of spaO-ompA fusion gene of Salmonella paratyphi A and the immunoprotection of expression product of the fusion gene

Objective To generate the spaO-ompA fusion gene of Salmonella paratyphi A and its prokaryotic expression system,and to determine the immunoprotection of the recombinant expression product rSpaO-OmpA.Methods A flexible peptide sequence was used to link spaO and ompA genes and a prokaryotic expression system of spaO-ompA fusion gene was subsequently generated.SDS-PAGE and Bio-Rad Agarose Image Analyzer were applied to examine the expression as well as the yield of the target recombinant protein rSpaO-OmpA.The antigenicity and immunoreactivity of rSpaO-OmpA were determined using immunodiffusion test,Western Blot assay and micro-Widal's test.By a mouse infection model,the immunoprotection of rSpaO-OmpA against the lethal challenge of S.paratyphi A was determined.In the animal protective test,the recombinant expressed SpaO (rSpaO) and OmpA ( rOmpA ) were used as the controls.Results The generated spaO-ompA fusion gene had 100％ nucleotide and amino acid sequence identities compared to the single spaO or ompA gene.The constructed prokaryotic expression system IPTG E.coli BL21DE3pET42a-spaO-ompA expressed the recombinant protein rSpaO-OmpA.rSpaO-OmpA combined with the antiserum against wholecell of S.paratyphi A to present positive hybridization signal and induced specific antibody in the immunized rabbits.Immunization with 100 or 200 μg rSpaO-OmpA contributed 66.7％ (8/12) or 83.3％ (10/12) immunoprotective rates in mice when the animals were attacked with S.paratyphi A.The immunoprotective rates produced by rSpaO-OmpA were significantly higher than that of equal rSpaO or rOmpA( P＜0.05 ).The sera from rSpaO-OmpA immunized mice presented 1∶5-1∶40 agglutination titers to the H antigens of different S.paratyphi species,and 1∶1-1∶16 immunodiffusion titers to rSpaO,rOmpA and rSpaO-OmpA proteins,respectively.Conclusion The artificially fusion antigen,rSpaO-OmpA,has more powerful immunogenicity and immunoprotection that the equal rSpaO or rOmpA. Key words: Salmonella paratyphi A ; spaO-ompA fusion gene ; Recombinant expression ; Immunogenicity; Immunoprotection

Deletion of phoQ gene decreases the resistance of Pseudomonas aeruginosa to aminoglycoside antibiot-ics

Objective To analyze the sequences of two component signaling system PhoP/PhoQ encoding genes of Pseudomonas aeruginosa strains sensitive or resistant to aminoglycoside antibiotics and to determine the correlation between the PhoQ/PhoP and the resistance. Methods The segments of entire pimQ and phoP genes of P. aeruginosa were obtained by PCR and then sequenced after T-A cloning. Two prokaryotic expression systems of phoQ and phoP genes were constructed and the target recombinant expres-sion products rPhoQ and rPhoP were extracted by Ni-NTA chromatography. Rabbits were intracutaneoualy immunized with rPhoQ and rPhoP to obtain antisera and double immunodiffusion test was used to detect the titers of antisera. The phoQ genes of aminloglycoside antibiotics-resistant P. aeruginosa strains were knocked out by using Red recombination system, and phoQ mutants were identified by PCR plus sequencing and Western blot assay. Tube dilution method was applied to determine MIC values of wild and mutant strains of P. aeruginosa to four different aminoglycoside antibiotics. Results In comparison with the corresponding sequences in GenBank, the similarities of nueleotide and putative amino acid sequences of the cloned phop and phoQ genes were 98.7%-99.6% and 98.7%-100% , and 98.4%-99.8% and 99.1%-100%, respec-tively. Both rPhoQ and rPhoP were successfully expressed using pET-42a and E. coil BL21 DE3 system, and their rabbit antisera with 1 : 4 and 1 : 8 double immunodiffusion titers were also obtained. The deletion of phoQ genes and absence of the products in the two phoQ mutants were confirmed by PCR, sequencing and West-ern blot assay. MIC values of the four different aminoglycoside antibiotics to the two mutants were 1/512-1/2048 as those of their wild strains. Conclusion PboQ/PhoP is a sequence conserved two component sig-naling system of P. aeruginosa, and this system mediates resistance of the microbe to aminoglycoside antibiotics. Key words: Pseudomonas aeruginosa; phoQ/phoP genes; Gene knock out; Aminoglycoside anti-biotics

Analysis of Leptospira interrogans ompA gene and immunological identification of its recombinant expression product

Objective To investigate the distribution of ompA gnne in 15 Chinese reference standard strains belonging to 15 serogroups of Leptospira interrogate, and to express recombinant OmpA ( rOmpA ) and to identify immunogenicity and immunoprotection of rOmpA. Methods Genomic DNAs from different leptospiral strains were extracted by phenol-chloroform method. Entire ompA gene fragments from the strains were amplified by PCR and then sequenced after T-A cloning. A prokaryotic expression system of ompA gene from L. interrogans strain 56601 was constructed, and the expression and yield of rOmpA were determined by SOS-PAGE plus Bio-Rad Agarose Image Analyser. Rabbits were immunized with rOmpA for obtaining antiserum, and immunodiffusion test was used to measure the antiserum's titer. Western blot assay was performed to determine the immunoreaetivity of rOmpA with the antiserum against rOmpA and antiserum against whole cell of L. interrogans strain 56601, while mi-croscopic agglutination test (MAT) was applied to detect the cross agglutination to the 15 L. interrogans strains. A leptospire adhering cell model and a leptospire infecting guinea pigs model were used to determine the adhesion-bloc-king effect of rOmpA antiserum and immunoprotection of rOmpA. Results All the 15 L. interrogans strains, but not L. biflexa strain Patoe Ⅰ , had sequence conserved ompA genes. The yield of rOmpA was approximate 20% of the total bacterial proteins, rOmpA could induce rabbits to produce antibody and immunodiffusion titer of the anti-serum was 1:4. Both antisera against rOmpA and against whole cell of L. interrogans strain 56601 were able to pro-duce positive Western blot signs to rOmpA, and the former offered 1 : 20-1 : 320 MAT titers to the 15 L. interrogans strains. 1: 10-1:160 dilutions of rOmpA antiserum could efficiently block L. interrogans strain 56601 adhering to J774A. 1 cells, and 100 μg and 200 μg rOmpA displayed 50.0% and 75.0% immunoprotective rates in the infee-ted guinea pigs. Conclusion ompA gene only exists in genomes of different pathogenic L. interrogans serogroups. rOmpA has relatively stronger antigenicity, cross immunoreactivity and certain immunoprotection, implying that this recombinant protein may be used as a candidate antigen for developing universal genetic engineering vaccine of L. interrogans. Key words: Leptospira interrogans; ompA gene; Recombinant expression; Immunogenicity; Immunopro-teetion

Synthesis of the Ostreococcus tauri ADP‐glucose pyrophosphorylase genes and characterization of their expressed subunits

The ADP‐glucose pyrophosphorylase small (S) and large (L) subunit genes from the unicellular algae Ostreococcus tauri were synthesized by PCR, subcloned, and the recombinant expression products characterized. This enzyme regulates the biosynthesis of starch in photosynthetic eukaryotes and has a reversible reaction in vitro. In higher plants, the L subunit makes the S subunit more sensitive to the activator. To understand the evolution of the subunit roles, we partially purified the S+L heterotetramer and S homotetramer from O. tauri, the simplest and smallest photosynthetic eukaryote. On the other hand, the L subunit could not be expressed alone with activity. We found that S+L is more sensitive to 3‐phosphoglycerate (activator) and phosphate (inhibitor) (A0.5 1.35 mM, I0.5 0.21 mM, forward direction, A0.5 0.80 mM, I0.5 0.51 mM reverse) than the homotetramer S (A0.5 2.20 mM, I0.5 1.40 mM forward, A0.5 2.80 mM, I0.5 1.20 mM reverse) with an activation of 33.5‐ and 7.6‐fold in the forward and 22.0‐ and 9.9‐fold in the reverse direction. The difference in regulation between the homotetramer (S) and heterotetramer (S+L) is not as pronounced as found in other plant ADP‐glucose pyrophosphorylases. These subunits in unicellular algae may not have evolved to play distinct roles as in higher plants. This research was supported by NSF grant no. MCB 0615982.

Expression of pseudorabies virus gE epitopes in Pichia pastoris and its utilization in an indirect PRV gE-ELISA

Pseudorabies virus glycoprotein E (PRV gE) has been recognized as a suitable diagnostic antigen for pseudorabies. In order to produce gE antigen in large quantities and at low cost, a gene fragment encoding PRV gE epitopes was expressed in Pichia pastoris expression system. SDS-PAGE and Western blotting revealed that the expression product was two recombinant proteins, approximately 38 and 32 kDa, in the culture supernatant of P. pastoris integrant 72 h after induction. Protein concentration assay showed the expression product amounted to 106.7 mg/l, accounting for 66.67% of total culture supernatant proteins. An indirect PRV gE-ELISA was then established by using the recombinant expression product as a coating antigen. Cross-reactivity assay showed that this antigen was PRV specific. Reproducibility experiment displayed good consistency. Comparison of detection results of 348 field serum samples between PRV gE-ELISA and a commercially available PRV diagnostic kit showed there was no significant difference between these two methods ( P>0.05).

Intact Protein Analysis for Site-Directed Mutagenesis Overexpression Products: Plasmid-Encoded R67 Dihydrofolate Reductase

Mass spectrometry is currently the method of choice for the analysis of recombinant protein expression products. By combining proteolytic digestion with peptide mapping and tandem mass spectrometry techniques, verification of site-directed mutagenesis products can be obtained. The proteolytic digestion step converts a purified recombinant protein into a mixture that must be reseparated, thus greatly increasing the analysis time associated with the confirmation of site-directed mutagenesis products. Ion/ion reaction chemistry combined with quadrupole ion trap mass spectrometry provides a fast and efficient way to analyze intact proteins for the correct site-directed mutagenesis products, without heavy reliance on the proteolytic digestion step. Analysis of a series of protein variants (I68M, I68Q, Y69F, and Q67Y) from plasmid-encoded R67 dihydrofolate reductase using ion/ion reaction chemistry confirmed the presence of the correct site-directed mutagenesis products. For the I68M mutant, ion/ion separations detected the presence of extensive degradation from the N-terminal end of the protein. In the case of the Q67Y mutant, a mixture of Q67Y and Q67C species was detected by employing tandem mass spectrometry combined with ion/ion reactions. The ion/ion reaction technique was also performed on a partially purified lysate of the Q67Y/C mixture and successfully screened for the presence of both components in a complex mixture. The ion/ion reaction approach achieved the same results as the proteolytic-digestion-based methodology in a much shorter analysis time.

Monitoring the expression and purification of recombinant proteins by MALDI-TOF mass spectrometry

1Samples coming from biologic sources usually contain several contaminants that interfere seriously with Mass Spectrometry (MS) measurements. In this paper we report the application of MALDI-TOF MS to monitor recombinant protein expression and purification. The technique is based on the use of a C18 resin to clean and concentrate proteins in batch. The utility of this method is demonstrated for samples coming from different bacterial cultures expressing secreted and intracellular proteins ranging from 4 to 53 kDa. MALDI-TOF MS of peptide and proteins can be accomplished directly from complex bacterial cultures or from any purification step in a few minutes using the conventional stainless steel sample targets, allowing for a nearly instantaneous monitoring of the nature and integrity of recombinant expression products.

Complementary DNA cloning and expression of a newly recognized high molecular mass allergen phl p 13 from timothy grass pollen (Phleum pratense).

Grass pollen extracts contain a range of different allergenic components that can be classified as having low, middle or high molecular mass. Almost 75% of patients allergic to grass pollen display immunoglobulin (Ig) E-reactivity to allergens in the high molecular mass range of 55-60 kDa. These proteins have not yet been fully characterized on the protein and DNA level. The aim of this study was to identify and characterize an allergen of the high molecular mass fraction of Phleum pratense pollen by N-terminal protein sequencing and molecular cloning. A previously uncharacterized allergen which migrates as a double band with a molecular mass of 55-60 kDa was biochemically purified and investigated by N-terminal sequencing. Subsequently, a DNA primer was designed to amplify the corresponding cDNA using PCR. The cloned cDNA and deduced amino acid sequence were compared with sequence data bases. Immunoblots carrying the recombinant expression product were developed with monoclonal antibodies and sera derived from allergic subjects. The IgE-binding capacity of natural and recombinant allergen was determined using EAST. The nucleic acid sequence as well as the deduced amino acid sequence consisting of 394 amino acids indicated homology with pollen specific polygalacturonases. Four potential sites for glycosylation and 16 cysteine residues were found. The recombinant expression product exhibited the same molecular size as the natural allergen and was clearly IgE-reactive. The newly characterized allergen Phl p 13, which shows homology with polygalacturonases, is clearly different from the allergen designated as Phl p 4 and therefore the high molecular mass fraction is composed of at least two different allergens. A possible reason why this important allergen has not been detected until now is that Phl p 13 and Phl p 4 are hardly separable by one dimensional SDS-PAGE.

Cloning, expression, purification, and characterization of the murine lysosomal acid α-mannosidase

Catabolism of α-linked mannose residues on eukaryotic glycoproteins is accomplished by a broad specificity lysosomal α-mannosidase (EC 3.2.1.24). Based on regions of protein sequence conservation between the lysosomal α-mannosidase from Dictyostelium discoideum and the murine Golgi glycoprotein processing α1,3/1,6-mannosidase, α-mannosidase II, we have cloned a cDNA encoding the murine lysosomal α-mannosidase. The longest of the clones was 3.1 kb in length and encoded a polypeptide of 992 amino acids containing a putative NH 2-terminal signal sequence and 11 potential N-glycosylation sites. The deduced amino acid sequence was 76.5% identical to the human lysosomal α-mannosidase and 38.1% identical to the lysosomal α-mannosidase from D. discoideum. Expression of the cDNA in Pichia pastoris resulted in the secretion of an α-mannosidase activity into the culture medium. This recombinant expression product was purified and was shown to have enzymatic characteristics highly similar to the enzyme purified from mammalian sources and to the human lysosomal α-mannosidase cDNA expressed in Pichia. These characteristics include a similar pH optimum, K m, V max, inhibition by swainsonine, and activity toward natural substrates. Northern blots identified a major 3.5 kb RNA transcript in all murine tissues tested. A minor transcript of 5.4 kb was also detected in some murine tissues similar to the alternatively spliced transcripts that have been previously identified in human tissues.

Expression of recombinant microfilarial chitinase and analysis of domain function

A family of chitinase isozymes was previously characterized from the microfilariae of Brugia malayi and Brugia pahangi. The expression of these enzymes correlates with the onset of microfilarial infectivity for the mosquito vector. To study the role of chitinase activity in filarial transmission, the p70 chitinase from Brugia malayi was cloned and expressed in two forms: a full-length product of ∼ 62 kDa and a truncated product of 43 kDa containing only the N-terminal catalytic domain. Two epitopes defined by monoclonal antibodies were preserved only in the full-length recombinant enzyme. It was found that deletion of the cysteine-rich C-terminal domain increased the yield of the recombinant expression product, and did not affect the Km for di- or trisaccharide substrates. However, affinity for high molecular weight chitin was specific to the full-length molecule, and is apparently mediated by the cysteine-rich domain, suggesting a role for this part of the protein in targeting the secreted enzyme to its substrate.

Eimeria refractile body proteins contain two potentially functional characteristics: transhydrogenase and carbohydrate transport.

cDNA encoding an immunogenic protein from partially sporulated oocysts of Eimeria acervulina was cloned and used to search for the homologous counterpart in Eimeria tenella. Monospecific antibodies were raised against the recombinant expression product. Using these antibodies, the parasite proteins were found to be localised in the refractile bodies. The derived amino-acid sequences were compared by computer using the SWISSPROT protein database. In addition to high homology between the Eimeria species, extensive similarity was found with pyridine nucleotide transhydrogenase from Escherichia coli. Comparison with the sugar signature database also resulted in a possible sugar binding domain present only in the Eimeria proteins. It is possible that the corresponding parasite proteins play a role in the recently discovered mannitol cycle of Eimeria.

Monoclonal antibodies to Escherichia coli beta-galactosidase and their use for detection and purification of recombinant expression products.

A panel of seven mouse monoclonal antibodies (BG-01-BG-07) was prepared against beta-galactosidase derived from E. coli. The antibodies are beta-galactosidase specific, show no cross-reactivity with other E. coli proteins and can be used for identification and characterization of beta-galactosidase fusion proteins expressed in lambda expression vectors. One of the antibodies allows a simple, one-step isolation of the fusion proteins directly from the crude bacterial lysates using immunoaffinity chromatography.

Mutation in the Escherichia coli htpR locus results in stabilization of recombinant expression products that are susceptible to proteolytic degradation

We compared the expression and degradation of three cloned malarial proteins in a pair of isogeneic strains of Escherichia coli that differed at the htpR locus. The htpR locus encodes an alternate σ factor necessary for the transcription of heat shock promoters. Plasmodium sequences were cloned from polymerase chain reaction-amplified DNA initiated by oligonucleotide primers that were specific for the gene coding regions to be expressed. The amplified DNA was cloned and expressed in a vector that encodes a strong T7 promoter and translation-initiation signal. The total cell yield of two of the expressed proteins was found to be increased when synthesis occurred in a E. coli htpR mutant. Pulse-chase experiments showed that the increased protein yield correlated with a decrease in the degradation of the protein in the htpR strain. A two- to seven-fold increase in the half-life of the malaria proteins was observed in the E. coli htpR − background as compared to htpR +. We found no difference in survival of the E. coli K165 htpR mutant and isogeneic parent during thermal induction. Since the synthesis of the heat shock σ factor did not significantly influence survival of E. coli and htpR expression results in increased degradation of foreign proteins, the E. coli htpR mutant was a valuable host strain for production of foreign proteins.

Demonstration of alternative splicing of a pre-mRNA expressed in the blood stage form of Plasmodium falciparum.

By screening of a lambda gt11 library from Plasmodium falciparum genomic DNA with an antiserum raised against a 41-kDa protein band, which was shown to confer protective immunity to monkeys, the phage clone 41-3 was identified. The entire 41-3 gene was isolated, and its coding regions were determined by amplification and sequencing of 41-3 specific mRNA fragments. The 41-3 gene has a complex structure consisting of nine exons, encoding 375 amino acids in total with a calculated molecular weight of 43,400. Provided that the N-terminal hydrophobic residues function as signal sequence which is cleaved off, the molecular weight of the 41-3 protein decreases to 41,200 and could therefore be considered to be a component of the protective Mr = 41,000 protein band. Indeed, a 41-kDa protein could be detected by Western blot analysis using antisera raised against different recombinant expression products of the 41-3 gene. We furthermore demonstrate an alternative splice process for the mRNA precursor transcribed from the 41-3 gene to yield at least three distinct mRNAs. The major splice product carries all exons E1 to E9, whereas at least two minor 41-3 mRNA species can be identified which show deletions in the region between exons E5 and E7. The possible role of this differential splice process for the parasite is discussed.

4826957 Immunogenic recombinant yeast expression product and method for purifying it: Victor Nussenzweig, Philip J Barr assigned to New York University; Chiron Corporati

4826957 Immunogenic recombinant yeast expression product and method for purifying it: Victor Nussenzweig, Philip J Barr assigned to New York University; Chiron Corporati

Defined monoclonal antibodies to Escherichia coli β-galactosidase as a tool for characterisation of recombinant expression products

Mouse monoclonal antibodies were prepared against β-galactosidase (EC 3.2.1.23) of Escherichia coli. The binding sites of these monoclonal antibodies within the β-galactosidase molecule were estimated by immunoblot analyses to various defined peptide regions of β-galactosidase, encoded by expression plasmids. Monoclonal antibodies, were characterised, which either bind to the amino-terminal or to the carboxy-terminal region or to an internal section of β-galactosidase. These defined monoclonal antibodies were shown to be a useful tool for characterisation of β-galactosidase fusion proteins expressed in Escherichia coli. Galactosidase, β-; Monoclonal antibody; Recombinant expression product; Epitope mapping; ( E. coli)

Induction of protective immunity against Schistosoma mansoni by vaccination with schistosome paramyosin (Sm97), a nonsurface parasite antigen.

Paramyosin (Sm97), a 97-kDa myofibrillar protein identified by the unusually monospecific antibody response induced by intradermal vaccination of mice with a complex soluble worm antigen preparation (SWAP) of adult Schistosoma mansoni administered with bacillus Calmette-Guérin (BCG), was purified and tested for its capacity to protect mice against challenge infection. When administered intradermally with BCG at total doses of only 4-40 micrograms per mouse, both the native molecule and a recombinant expression product containing approximately 50% of the whole protein were found to confer significant resistance (26-33%) against challenge infection, while 2 mg of unfractionated SWAP was required to induce similar levels of protection. In addition, paramyosin was shown to stimulate T lymphocytes from vaccinated mice to produce lymphokines [e.g., gamma interferon (IFN-gamma)] that activate macrophages to kill schistosomula. Neither schistosome myosin nor a heterologous paramyosin from a different invertebrate genus were protective, indicating a requirement for specific epitopes in the immunization. That the protection induced by paramyosin involves a T-cell-mediated mechanism was supported by the failure of anti-paramyosin antibodies to passively transfer significant resistance to infection to recipient mice. Lymphocytes from mice vaccinated with paramyosin were found to produce IFN-gamma in response to living schistosomula, suggesting that during challenge infection of vaccinated hosts, paramyosin (a nonsurface antigen) may elicit a protective T-cell response as a consequence of its release from migrating parasite larvae. Paramyosin-depleted SWAP was also found to be protective as well as stimulatory for T lymphocytes from SWAP-vaccinated mice, indicating that other antigens in this preparation may have immunoprophylactic potential. In summary, these results (i) suggest that the induction of T-cell-dependent cell-mediated immunity against soluble nonsurface antigens may be an effective strategy for immunization against multicellular parasites and (ii) in the case of schistosomes, identify paramyosin as a candidate vaccine immunogen in this category.