• Parasites, Hosts and Diseases
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• v.61 no.4
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• pp.439-448
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• 2023

Tick infestation causes a significant threat to human and animal health, requiring effective immunological control methods. This study aimed to investigate the potential of recombinant Haemaphysalis longicornis enolase protein for tick vaccine development. The exact mechanism of the recently identified enolase protein from the H. longicornis Jeju strain remains poorly understood. Enolase plays a crucial role in glycolysis, the metabolic process that converts glucose into energy, and is essential for the motility, adhesion, invasion, growth, and differentiation of ticks. In this study, mice were immunized with recombinant enolase, and polyclonal antibodies were generated. Western blot analysis confirmed the specific recognition of enolase by the antiserum. The effects of immunization on tick feeding and attachment were assessed. Adult ticks attached to the recombinant enolase-immunized mice demonstrated longer attachment time, increased bloodsucking abilities, and lower engorgement weight than the controls. The nymphs and larvae had a reduced attachment rate and low engorgement rate compared to the controls. Mice immunized with recombinant enolase expressed in Escherichia coli displayed 90% efficacy in preventing tick infestation. The glycolytic nature of enolase and its involvement in crucial physiological processes makes it an attractive target for disrupting tick survival and disease transmission. Polyclonal antibodies recognize enolase and significantly reduce attachment rates, tick feeding, and engorgement. Our findings indicate that recombinant enolase may be a valuable vaccine candidate for H. longicornis infection in experimental murine model.

• Reproductive and Developmental Biology
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• v.33 no.2
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• pp.77-83
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• 2009

Erythropoietin (EPO), a glycoprotein hormone produced from primarily cells of the peritubular capillary endothelium of the kidney, is responsible for the regulation of red blood cell production. We have been investigating the roles of glycosylation site added in the biosynthesis and function of recombinant protein. We constructed three EPO mutants ($\Delta$69, $\Delta$105 and $\Delta$69,105), containing an additional oligosaccharide chains. EPOWT and EPO$\Delta$69 were effectively expressed in transient and stably transfected CHO-K1 cell lines. But, it wasn't detected any protein in the culture medium of EPO$\Delta$105 and EPO$\Delta$69,105 mutants. The growth and differentiation of EPO-dependent human leukemic cell line (F36E) were used to measure the cytokine dependency and in vitro bioactivity of rec-hEPO. MTT assay values were increased by survival of F36E cells at 24h. To analysis biological activity in vivo, two groups of ICR-mice (7 weeks old) were injected subcutaneously with 10 IU per mice of rec-hEPO molecules on days 0 and 2. Red blood cell and hematocrit values were measured on 6 days after the first injection. The hematocrit values were remarkably increased in all treatment groups. The pharmacokinetic analysis was also affected in the mice injected with rec-hEPO molecules 2.5 IU by tail intravenous. Protein samples were detected by Western blotting. An EPO$\Delta$69 protein migrated as a broad band with an average apparent molecular and detected slightly high band. Enzymatic N-deglycosylation resulted in narrow band and was the same molecular size. The biological activity of EPO$\Delta$69 was enhanced to compare with wt-hEPO. The half-life was longer than wt-hEPO. The results suggest that hyperglycosyalted recombinant human erythropoietin (EPO$\Delta$69) may have important biological and therapeutic good points.

• KSBB Journal
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• v.15 no.2
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• pp.181-187
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• 2000

The production of heterologous protein using GAL promoter in conventional S. cerevisiae has several problems to s이ve for c commercialization. In this research, S. cerevisiae mutant(reg1-501, gaI1), which cannot use galactose and has alleviated g glucose repression level, is used as host for optimizing induction of GAL promoter. In this experiment, the effects of specific g growth rate on specific recombinant protein expression rate were tested in both cases and optimum fed batch fermentation m method was obtained in both cases. Through these experiments, optimum condition of recombinant protein production by G GAL promoter using S. cerevisiae mutant (reg1-501, gal1) were found.

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2003.10a
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• pp.118-119
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• 2003

Baculovirus occlusion bodies have been recently engineered to incorporate foreign protein such as the Bacillus thuringiensis (Bt) CrylAc protein for improvement of insecticidal activity. In this study, polyhedrin, cylAc, egfp and crylCa genes were fused to produce occlusion bodies that contain novel recombinant crystal protein by homologous recombination between cylAc and crylCa genes in insect cells. (omitted)

• KSBB Journal
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• v.25 no.3
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• pp.257-264
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• 2010

Bone morphogenetic protein-7 (BMP-7) is one of important growth factors for skeletal development and bone growth. In this work, BMP-7 was efficiently expressed in recombinant Bacillus subtilis. The mature BMP-7 protein indicated molecular weight of 15.4 kDa by Western blot assay and was secreted into culture medium with 0.35 ng/mL. The extracellular and intracellular rhBMP-7 proteins were purified by using a FPLC system with an ion exchange column and a gel filtration column. The extracellular and intracellular rhBMP-7 proteins had finally a 57.1% purity and a 36.2% purity, respectively. The purified rhBMP-7 proteins showed an intact biological activity which stimulated alkaline phophatase (ALP) activity in MC3T3-E1 cells.

• Microbiology and Biotechnology Letters
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• v.23 no.6
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• pp.784-788
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• 1995

The lambda Integrase (Int) carries out site-specific recombination between the two partner DNA sequences, attachment P (attP) and attachment B (attB). In order to study the recombination mechanism, a large quantity of pure integrase is required. Then, we constructed an int gene inserted recombinant plasmid (pNYL3) by using the pQE31 HIS-Tag vector, and produced the fusion protein, 6xHIS-Int from the E. coli TG1 strain carrying the pNYL3 plasmid. The recombinant protein produced was purified by phosphocellulose and Ni$^{++}$-NTA affinity column chromatographies. The result of the in vitro recombination assay using the standard reaction mixture containing 6xHIS-Int and partially purified integration host factor (IHF) showed that the 6xHIS-Int tagged recombination Integrase had the full recombination activity.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.711-714
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• 2001

The production of the recombinant Plasmodium vivax merozoite surface protein (PvMSP) has been investigated in the recombinant E.coli system. Experimental optimization of the culture conditions, such as the effect of initial pH, and operating temperature has been tried on the growth of recombinant E.coli and on the overproduction of the target foreign protein.

• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.783-788
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• 2004

Marker proteins of genetically-modified organisms (GMO) and their antibodies were prepared and characterized as major components of an analytical system. We selected two GMO markers, neomycin phosphotransferase II and 5- enolpyruvylshikimate-3-phosphate synthase, and produced them from E. coli employing genetic recombination technology. After purification, their structural conformation and binding affinities to the respective antibodies were characterized. The results showed that the recombinant proteins were identical with commercially obtained reference proteins. We further used them as immunogens to raise polyclonal antibodies capable of discriminating GMO containing protein from non-GMO. Well-characterized marker proteins and antibodies will be valuable as immunoreagents in constructing analytical systems such as biosensors and biochips to measure quantities of GMO.

• Macromolecular Research
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• v.17 no.7
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• pp.464-468
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• 2009

Gelatin nanoparticles derived from bovine or porcine have been developed as various types of drug delivery system, and they need to be cross-linked to maintain their physicochemical properties in aqueous environments. Although gelatin is a widely used material in pharmaceutical industries, the safety issue of animal-origin gelatins, such as transmissible mad cow disease and anaphylaxis, remains to be solved. The purpose of this study was to prepare type A gelatin (GA) nanoparticles by modified, two-step, desolvation method and compare the toxicity of the resulting GA nanoparticles with recombinant human gelatin (rHG) nanoparticles. The GA nanoparticles were characterized, and drug loading and release pattern were measured. FITC-BSA, a model protein, was efficiently loaded in the nanoparticles and then released in a biphasic and sustained release pattern without an initial burst. In particular, the cell viability of the GA nanoparticles was less than that of the rHG nanoparticles. This finding suggests that rHG nanoparticles should be considered as an alternative to animal-origin gelatin nanoparticles in order to minimize the safety problems.

• IMMUNE NETWORK
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• v.4 no.3
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• pp.184-189
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• 2004

Background: Hu syndrome, a neurological disorder, is characterized by the remote effect of small cell lung cancer on the neural degeneration. The suspicious effectors for this disease are anti-Hu autoantibodies or Hu-related CD8+ T lymphocytes. Interestingly, the same effectors have been suggested to act against tumor growth and this phenomenon may represent natural tumor immunity. For these diagnostic and therapeutic reasons, the demand for antibodies against Hu protein is rapidly growing. Methods: Polyclonal and monoclonal antibodies were generated using recombinant HuR protein. Western blot analyses were performed to check the specificity of generated antibodies using various recombinant proteins and cell lysates. Extracellular stimuli for HuR expression had been searched and HuR-associated proteins were isolated from polysome lysates and then separated in a 2-dimensional gel. Results: Polyclonal and monoclonal antibodies against HuR protein were generated and these antibodies showed HuR specificity. Antibodies were also useful to detect and immunoprecipitate endogenous HuR protein in Jurkat and BJAB. This report also revealed that TNF-${\alpha}$ treatment in BJAB up-regulated HuR expression. Lastly, protein profile in HuR-associated mRNAprotein complexes was mapped by 2-dimensional gel electrophoresis. Conclusion: This study reported that new antibodies against HuR protein were successfully generated. Currently, project to develop a diagnostic kit is in process. Also, this report showed that TNF-${\alpha}$ up-regulated HuR expression in BJAB and protein profile associated with HuR protein was mapped.