Producing novel antibodies by expression of recombinant immunoglobulin genes.
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Animals↗
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Mapping of the complement regulatory domains in the human factor H-like protein 1 and in factor H1.
The human factor H-like protein 1 (FHL-1) is composed of seven repetitive domains (short consensus repeats; SCRs) that are identical in sequence to the seven NH2-terminal SCRs of the complement regulatory protein factor H. We have identified the native FHL-1 protein as a 42-kDa human plasma protein by immunoblotting and by comparing the mobility to that of a recombinant FHL-1 protein. Here, we demonstrate the existence of two distinct co-migrating human plasma proteins that represent the 42-kDa FHL-1 protein and the previously identified 43-kDa factor H-related 1 beta protein. Similar to factor H, the recombinant FHL-1 protein displays cofactor activity in factor I-mediated cleavage of C3b. To identify relevant SCRs of factor H and FHL-1, we recombinantly expressed the domains shared between the two proteins in the baculovirus expression system. Recombinant FHL-1 and all truncated forms that include SCRs 1 to 4 displayed cofactor activity. All four NH2-terminal SCRs are essential, as deletion mutants composed of SCR 1 and 4 only; of SCRs 1, 2, and 4 only, or of SCRs 1, 3, and 4 only were functionally inactive. Similarly, the distance between these individually folding domains is critical for function, as a recombinant protein that had two and four amino acids inserted between SCRs 1 and 2 or between SCRs 3 and 4, respectively, had no activity. These results demonstrate that all four NH2-terminal SCRs of FHL-1 (and of factor H) are required for cofactor activity in factor I-mediated cleavage of C3b, and that the distance between these SCRs is essential.
Base Sequence↗
[The prospects for using the genetically engineered surface antigen of the hepatitis B virus (HBsAg) expressed by recombinant poxvirus strains as the basis for vaccines against hepatitis B].
The use of a recombinant poxvirus (RPV) strain, expressing HBsAg in the process of reproduction in different bioreactor systems under stationary and bioreactor conditions of cultivation, made it possible to obtain highly purified HBsAg. The identity and purity of HBsAg was confirmed by the analysis of its amino acid composition, SDS electrophoresis in polyacrylamide gel, electron microscopy and high-performance liquid chromatography. Good prospects of the use of RPV-expressed gene engineering HBsAg as the basis vaccines against hepatitis B was demonstrated in 10 experimental batches of vaccine. All batches of the preparation had pronounced immunogenicity and were safe and nontoxic in animal experiments. The ID50 of experimental batches did not exceed 211 ng/ml, which, according to the data of comparative experiments, was lower than, or equal to, corresponding values of analogous foreign commercial preparations, based on plasma or yeast HBsAg.
Animals↗
Induction of recombinant gene expression in stably transfected cell lines using attenuated vaccinia virus MVA expressing T7 RNA polymerase with a nuclear localisation signal.
There are major drawbacks using vaccinia virus (VV) expressing T7 polymerase for eukaryotic expression. VV is infectious for humans and due to cytosolic replication of Poxviridae, transient transfection of T7 promoter containing plasmids is necessary, which varies in efficiency. Several improvements have been introduced to this system to enhance expression of herpes viral glycoproteins. Stably transfected cell lines were generated with an EBV-based episomal plasmid vector which can be pushed to increasing copy numbers under selective pressure. The avirulent vaccine MVA strain was adopted to generate a safe laboratory vector for inserting the bacteriophage T7 RNA polymerase gene with (+) or without (-) a nuclear localisation signal. Constructs were designed for recombination into the VV haemagglutinin gene as recombinants could not be isolated successfully when inserting into the MVA thymidine kinase locus. Both T7 MVA recombinants induced foreign protein expression in transiently transfected cells but only the T7-/+ MVA induced target protein expression in stably transfected cells. The level of protein expression by this induction mechanism was comparable to, or superior to levels obtained with VV recombinants expressing the gene under control of the VV 11 k IE promoter. The results suggests that the T7+ MVA virus can be used to induce gene expression in stable recombinant cell lines and offers an attractive and safe alternative to other inducible eucaryotic expression systems.
Animals↗
Oral immunization of swine with attenuated Salmonella typhimurium aroA SL3261 expressing a recombinant antigen of Mycoplasma hyopneumoniae (NrdF) primes the immune system for a NrdF specific secretory IgA response in the lungs.
Salmonella typhimurium SL3261 (aroA mutant) expressing a recombinant Mycoplasma hyopneumoniae antigen was used to orally immunize swine against porcine enzootic pneumonia. This construct, designated S. typhimurium aro A SL3261 (pKF1), expressed a recombinant protein containing the carboxy-terminal 11 kDa of a 42 kDa M. hyopneumoniae NrdF ribonucleotide reductase R2 subunit protein. Here we demonstrate that this antigen is present in all seven geographically diverse strains of M. hyopneumoniae tested, and is recognized by the swine immune system after experimental infection with the virulent M. hyopneumoniae Beaufort strain. The immune response of swine orally immunized twice with S. typhimurium SL3261 (pKF1) on day 0 and day 14 was evaluated. Oral immunization with S. typhimurium SL3261 (pKF1) primed the immune system to elicit a significant (P<0.05) secretory IgA response against the 15 kDa NrdF antigen in the respiratory tract of swine, post-challenge, compared to control groups. Blood lymphocytes from swine immunized with S. typhimurium SL3261 (pKF1) proliferated significantly (P<0.05) following stimulation with M. hyopneumoniae whole-cell extracts compared to control groups 14 days post-vaccination. Following challenge with virulent M. hyopneumoniae, swine immunized with S. typhimurium SL3261 (pKF1) showed higher average daily weight gains and reduced lung pathology compared to control groups.
Administration, Oral↗
[Recombinant eukaryotic expression plasmid of bcr-abl gene fragment induces specific immune response in mice].
OBJECTIVE: To study the specific immune response induced by a recombinant eukaryotic expression plasmid encoding bcr-abl fusion gene fragment so as to explore new immunotherapy in mouse. METHODS: A recombinant eukaryotic vector pVbcr-abl expression cDNA fragment of bcr-abl fusion gene was constructed and used to immunize BALB/c mice. Serum level of bcr-abl specific antibody was detected by enzyme-linked immunosorbent assay (ELISA). Twenty days later the immunized mice were subcutaneously inoculated SP2/0/bcr-abl cells. The survival time, tumor growth time and lymphocytic infiltration were observed. T cells infiltration into tumor tissue was analyzed by immunohistochemistry. Changes of T cell subset in the spleen of mice was analyzed by fluorescent-activated cell sorting (FACS) and the cytotoxicity T lymphocyte (CTL) activity in spleen by lactate dehydrogenase (LDH)-release assay. RESULTS: The eukaryotic expression vector pVbcr-abl was constructed successfully, and highly expressed the cDNA fragment of bcr-abl fusion gene. The BALB/c mice immunized with the vector could generate the specific antibody and CTL, resulting in a specific immunoprotection. There were dramatic differences in the tumor-forming time, tumor ulcer appearing time and tumor-growing speed between the immunized and the control groups. The mice had longer survival time in the immunized group than in the control group. There were a large amount of CD3(+) T cells infiltration in tumor tissue of the immunized mice. The spleen cells from the immunized mice had higher CTL activity with a alteration of T cell subset, the CD4(+)/CD8(+) ratio being 1.54 +/- 0.29, higher than that of control group (1.18 +/- 0.30). CONCLUSION: The recombinant eukaryotic expression plasmid pVbcr-abl can induce in vivo not only the generation of specific antibody, but also high level of specific CTL activity, resulting in killing the SP2/0/bcr-abl tumor cells directly and inhibiting the tumor growth.
Animals↗
Baculovirus-based expression of mammalian caveolin in Sf21 insect cells. A model system for the biochemical and morphological study of caveolae biogenesis.
Caveolae were originally defined morphologically as 50-100 nm noncoated vesicular organelles located at or near the plasma membrane. Caveolin, a vesicular integral membrane protein of 21 kDa, is a principal protein component of caveolae membranes in vivo. Caveolin interacts with itself to form high molecular mass oligomers, suggesting that it might play a structural role in the formation of caveolae membranes. However, it remains controversial whether recombinant expression of caveolin is necessary or sufficient to generate caveolae membranes in vivo. To directly address this issue, we have taken a different experimental approach by exploiting a heterologous expression system. Here, we have recombinantly expressed mammalian caveolin in Sf21 insect cells using baculovirus-based vectors. Two isoforms of caveolin have been identified that differ at their extreme N terminus; alpha-caveolin contains residues 1-178, and beta-caveolin contains residues 32-178. After recombinant expression in Sf21 insect cells, both alpha- and beta-caveolin formed SDS-resistant high molecular mass oligomers of the same size as native caveolin. Morphologically, expression of either caveolin isoform resulted in the intracellular accumulation of a homogeneous population of caveolae-sized vesicles with a diameter between 50 and 120 nm (80.3 +/- 14.8 nm). This indicates that each caveolin isoform can independently generate these structures and that caveolin residues 1-31 are not required for this process. Using caveolin as a marker protein and a detergent-free procedure to purify caveolae from mammalian cells, we purified these recombinant caveolin-induced vesicles from insect cells. These purified recombinant vesicles: (i) have the same buoyant density as mammalian caveolae; (ii) appear as approximately 50-100 nm membranous structures by whole-mount electron microscopy; and (iii) contain approximately 95% of the recombinantly expressed caveolin protein by Western blotting. Immuno-labeling of these structures with anti-caveolin IgG confirmed that they contain caveolin. Thus, ectopic overexpression of caveolin in this heterologous system is sufficient to drive the formation of caveolae-like vesicles. Further functional analysis demonstrated that caveolin was capable of interacting with a known caveolin-interacting protein, Ha-Ras, when coexpressed in insect cells by co-infection with two recombinant baculoviruses. Taken together, our results demonstrate that baculovirus-based expression of caveolin in insect cells provides an attractive experimental system for studying the biogenesis of caveolae.
Animals↗
In vivo expression of recombinant vascular endothelial growth factor in rabbit carotid artery increases production of superoxide anion.
OBJECTIVE: Vascular endothelial growth factor (VEGF) is one of the most important pro-angiogenic cytokines. Ability of VEGF to stimulate formation of superoxide anion in vivo has not been studied. We hypothesized that in vivo expression of recombinant VEGF in the rabbit carotid artery increases production of superoxide anion. METHODS AND RESULTS: Plaque-forming units (10(9)) of adenovirus-encoding human VEGF165 (AdVEGF) or beta-galactosidase (AdLacZ) were delivered into the lumen of rabbit carotid arteries. Three days after gene delivery, expression of recombinant proteins was detected in endothelium and smooth muscle cells. Endothelium-dependent relaxations to acetylcholine were impaired in AdVEGF-transduced arteries (P<0.01; n=5). Treatment with superoxide dismutase mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride (10(-5) mol/L), improved relaxations to acetylcholine (P<0.01; n=5). Western blot analysis demonstrated increased expression of p47(phox) in AdVEGF-transduced arteries (P<0.05; n=8). Lucigenin chemiluminescence showed significantly higher production of superoxide anion in AdVEGF-transduced arteries (P<0.05; n=5 to 10). CONCLUSIONS: Our results suggest that in vivo expression of recombinant VEGF in the vascular endothelium increases local production of superoxide anion. Superoxide anion appears to be an important mediator of vascular effects of VEGF in vivo.
Animals↗
Expression of recombinant anti-E-selectin single-chain Fv antibody fragments in stably transfected insect cell lines.
We have investigated the possibility of improving the yield of properly folded recombinant single chain Fv fragments (sFv) of an antibody by expressing the protein in stably transfected Drosophila melanogaster SC-2 cells. The DNA encoding the variable regions of the 1.2B6 anti-E-selectin antibody were used to generate a recombinant sFv. This construct was cloned into the pHEN1 vector for expression in Escherichia coli and the pRmHa-3 vector to generate stably transfected Drosophila SC-2 cell lines. Following expression in the bacterial system, and using standard refolding protocols to obtain active material, it was shown that the majority of the sFv formed non-covalent aggregates. In addition SDS-PAGE analysis indicated that even the monomeric material was heterogeneous. In contrast, expression of sFv in Drosophila SC-2 cell lines allowed purification of active sFv directly from the culture supernatant. Only a small proportion of the sFv formed aggregates, and the purified material was homogeneous as determined by SDS-PAGE. Thus the use of stably transfected insect cells has a number of potential advantages in expressing recombinant antibody fragments.
Animals↗
[Expression of truncated Toxoplasma gondii GRA8 in the prokaryotic expression plasmids].
OBJECTIVE: To construct the prokaryotic recombinant expression plasmids of Toxoplasma gondii GRA8 and analyze their expression in E. coli containing the prokaryotic recombinant plasmids. METHODS: The full gene and its truncated fragment of GRA8 were amplified by PCR from T. gondii genomic DNA, and cloned into pMD18-T vector. The right genes in positive clones sequenced with ABI PRISMTM 377XL DNA Sequencer were digested with restrictive endonucleases and subcloned into pGEX-4T-2. The recombinant plasmids were transformed into E. coli JM109. The recombinant clones were characterized by PCR and digested with restriction endonucleases. Positive clones were induced with IPTG to express target protein and characterized by SDS-PAGE. The recombinant protein was purified from E. coli lysate by affinity chromatography and SDS-PAGE. The immunological activity of this protein was analyzed by Western blotting. RESULTS: The gene fragments encoding GRA8 were amplified by PCR from T. gondii genomic DNA. The inserts of GRA8 in positive clones were coincident with the original sequence of GRA8 gene from GenBank. The recombinant expression plasmids were constructed through subcloning the right inserts of GRA8 into pGEX-4T-2. The expression level of GRA8 from the recombinant expression plasmids in E. coli was low, and there was almost no full-length GRA8 expressed in E. coli. The purified protein reacted with the sera from rabbits infected with T. gondii RH strain. CONCLUSION: The expression level of GRA8 from the recombinant expression plasmids in E. coli is low and the purified truncated GRA8 shows certain antigenicity.
Animals↗
High-level expression of recombinant protein A in Escherichia coli.
A high-level expression plasmid pPA-3 was constructed, which yielded up to 20% of soluble cell proteins as recombinant Protein A in E. coli DH5 alpha strain by heat shock induction. The recombinant Protein A contained only the five domains of native Protein A that bind with the Fc part of IgG. The molecular weights of the recombinant Protein A expressed in E. coli were determined to be 33kDa, 32.2kDa, 29.5kDa and 28.6kDa by SDS-PAGE and Western-blotting. The diversity of the molecular weights may be due to proteolysis, and a possible cleavage site is proposed. Some of the protein was purified with coupled human IgG by one-step affinity chromatography. The reactivity of the protein was compared with that of native Protein A and found that this protein could bind with more IgG than native Protein A in equal quantities of proteins.
Amino Acid Sequence↗
Enzymic, spectroscopic and calorimetric studies of a recombinant dextranase expressed in Pichia pastoris.
Conformational stability and structural characterization of an rDex (recombinant dextranase) expressed in Pichia pastoris were studied by enzymic assays, fluorescence, CD and DSC (differential scanning calorimetry). We also identified two disulphide bridges (Cys9-Cys14, Cys484-Cys488) and two free Cys residues (Cys336, Cys415) that are not conserved between bacterial and fungal dextranases of GH-49 (glycoside hydrolase family 49) by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS. Enzymic and fluorescence studies revealed that rDex is biological and conformationally stable at acidic pH, with maximum activity at pH 4.5-5.0, while CD spectra indicated a secondary structure basically composed of beta-sheets. rDex loses biological activity at neutral pH without total disruption of its conformation. In addition, rDex preserves its conformation close to 60 degrees C, but it is thermally denatured with appreciable aggregation at temperatures above 75 degrees C. DSC studies always displayed irreversible transitions and a strong dependence on the scan rate. Our combined analysis suggested that the denaturation process of rDex is under kinetic control, which is described reasonably well by the two-state kinetic scheme.
Amino Acid Sequence↗
[High level expression of recombinant human sCD40L-IZ and identification of its biological activation].
AIM: To clone and express the recombinant human soluble CD40 ligand(CD40L) with biological activation. METHODS: The isoleucine zipper (IZ) gene was fused at N-terminal of CD40L gene coding E107-L261 contributing to form trimer. The gene coding hexahistidine was fused at the N-terminal of IZ because the sCD40L-IZ fusion protein could be purified by affinity chromatography. Then the fusion gene was amplified and cloned into expression plasmid pET30a. RESULTS: The protein sCD40L-IZ with His-tag at the N-terminal was effectively expressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was performed to acquire soluble sCD40L-IZ. The fusion protein sCD40L-IZ was conveniently purified using HiTrap affinity column with above 95% purity. The gel filtration chromatography and non-reduced SDS-PAGE identified the trimeric structure of the recombinant protein. The microscope analysis showed the sCD40L-IZ interacted with the membrane CD40 on XG2, a multiple myeloma cell line. CONCLUSION: The recombinant human trimeric CD40L-IZ was expressed in prokaryotic cells successfully, which has provided a basis for further study of the relationship between CD40L and apoptosis, CD40L and pathogenesis of illness. It is also useful to clinical treatment.
Animals↗
Adenovirus vectors for cytokine gene expression.
Recombinant Adenovirus type 5 constructs containing IL-6 cDNA can be used to infect cells in vitro and obtain a high level of IL-6 expression and secretion into culture media. Furthermore, Ad5-IL-6 viruses can also be used to infect Balb/c mice or Sprague-Dawley rats and obtain a high level of IL-6 expression that is sustained over a period of 3-5 days. Intratracheal infection was accompanied by dramatic increases in virus-encoded IL-6 mRNA levels in rat lung tissue, raised levels of IL-6 detected in bronchoalveolar lavage fluids and in serum, and IL-6-dependent sequelae such as liver acute phase responses. This occurs in a tissue-specific manner, depending on routes of infection by the virus. Rat lungs showed a prominent expansion (10 fold in numbers) of all classes of lymphocytes, including B cells, T helper cells (CD4+) and CTL (CD8+) at day 7 after infection which resolved significantly by day 12. Thus the associated biological effects of viral vector mediated IL-6 over-expression was also transient in nature. Other tissues can be infected with Ad5 and thus can also be induced to express selected genes in a transient fashion. We are currently examining the potential for Ad recombinant cytokine vectors in therapy for cancer and for bone marrow reconstitution after transplantation. Thus the use of recombinant Ad5 vectors may have a broad application in the study of cytokine function and possibly in future therapy as a transient gene transfer approach.
Adenoviruses, Human↗
Immunoselection of recombinant baculoviruses expressing high levels of biologically active herpes simplex virus type 1 glycoprotein D.
The DNA sequence encoding the complete herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) was inserted into a baculovirus transfer vector under control of the polyhedrin gene promoter of the baculovirus Autographa california nuclear polyhedrosis virus (AcNPV). After co-transfection of Spodoptera frugiperda (Sf9) insect cells with wild-type AcNPV DNA and the recombinant transfer vector DNA, polyhedrin-negative recombinants that expressed high levels of HSV-1 gD were isolated using immunoaffinity selection with antibody coated magnetic particles followed by plaque purification. These recombinant baculoviruses expressed a protein that was slightly smaller than virion HSV-1 gD made in Vero cells. This recombinant protein was expressed at high levels. The expressed protein was glycosylated, was found on the membrane of Sf9 cells, and reacted with gD specific antibodies. Antibodies raised in mice to the recombinant gD neutralized HSV-1 as measured by plaque reduction assays. Mice inoculated with the recombinant baculovirus were completely protected from lethal challenge with HSV-1.
Animals↗
In utero delivery of adeno-associated viral vectors: intraperitoneal gene transfer produces long-term expression.
Recombinant adeno-associated viruses (rAAV) are promising gene transfer vectors that produce long-term expression without toxicity. To investigate future approaches for in utero gene delivery, the efficacy and safety of prenatal administration of rAAV were determined. Using luciferase as a reporter, expression was assessed by whole-body imaging and by analysis of luciferase activity in tissue extracts, at the time of birth and monthly thereafter. Transgene expression was detected in all injected animals. Highest levels of luciferase activity were detected at birth in the peritoneum and liver, while the heart, brain, and lung demonstrated low-level expression. In vivo luciferase imaging revealed persistent peritoneal expression for 18 months after in utero injection and provided a sensitive whole-body assay, useful in identifying tissues for subsequent analyses. There was no detectable hepatocellular injury. Antibodies that reacted with either luciferase or rAAV were not found. AAV sequences were not detected in germ-line tissues of injected animals or in tissues of their progeny. In utero AAV-mediated gene transfer in this animal model demonstrates that novel therapeutic vectors and strategies can be rapidly tested in vivo and that rAAV may be developed to ameliorate genetic diseases with perinatal morbidity and mortality.
Animals↗
Immunogenicity of nonreplicating recombinant vaccinia expressing HLA-A201 targeted or complete MART-1/Melan-A antigen.
The effect on immunogenicity of different tumor T cell epitope formulations was evaluated in vitro using nonreplicating recombinant vaccinia vector expressing two forms of the melanoma-associated MART-1/Melan-A antigen. The first recombinant virus expressed a minigene encoding a fusion product between an endoplasmic reticulum (ER)-targeting signal and the HLA-A201 binding 27-35 peptide. The second viral construct encoded the complete MART-1/Melan-A protein. The capacity of HLA-A201 cells infected with either viral construct to generate and to stimulate MART-1/Melan-A 27-35 specific cytotoxic T-lymphocytes (CTL), was comparatively characterized. The results obtained here with a tumor antigen confirmed the capacity of vaccinia virus-encoded ER-minigene to generate a very strong antigenic signal. In cytotoxicity assays, recognition of target cells infected with high amounts of both recombinant viruses with activated specific CTL clones, resulted in similar lytic activity. With regard to calcium mobilization, TCR down-regulation, IFN-gamma release, and T cell proliferation assays, the targeted epitope elicited 10- to 1000-fold stronger responses. Remarkably, the immunogenic difference between the two formulations, in their respective capacity to generate CTL from naive HLA-A2 peripheral blood mononuclear cells in vitro as measured by tetramer detection, was lower (2- to 3-fold). Recombinant vectors expressing complete antigens have demonstrated their capacity to generate specific responses and such vaccines might take advantage of a broader potential of presentation. However, as demonstrated here for the HLA-A201-restricted MART-1/Melan-A immunodominant epitope, nonreplicative vaccinia virus expressing ER-targeted minigenes appear to represent a significantly more immunogenic epitope vaccine formulation.
Antigen Presentation↗
Reduced fimbria-associated activities of Porphyromonas gingivalis induced by recombinant fimbrial expression.
The adhesion properties of the recombinant fimbriae (r-fimbriae) recovered from a YH522 transformant of Porphyromonas gingivalis which harbors a chimeric plasmid, pYHF2, containing the fimA gene of strain 381 were compared with those of the endogenous fimA fimbriae of strain 33277. The adhesion level of the r-fimbriae to Actinomyces viscosus was clearly lower than that of the endogenous fimbriae. In addition, the r-fimbriae were shown to lack some minor components detectable in the endogenous fimbriae. The plasmid pYHF2 prepared from the YH522 transformant was then transformed into six different P. gingivalis strains and the resultant pYHF2-containing strains were examined for their fimbrial expression. In spite of the presence of a considerable diversity in the expression level of the r-fimbriae among these transformants, it was evident that the strains expressing higher levels of the r-fimbriae exhibited a greater decrease in adhesion activity to other bacteria and to oral epithelial cells, as well as in self-aggregation.
Actinomyces viscosus↗