• Journal of Microbiology and Biotechnology
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• 제26권9호
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• pp.1613-1619
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• 2016

Francisella tularensis is a highly virulent pathogen of humans and other mammals. Moreover, F. tularensis has been designated a category A biothreat agent, and there is growing interest in the development of a protective vaccine. In the present study, we determine the in vitro and in vivo immune responses of a subunit vaccine composed of recombinant peptides Tul4 and FopA from epitopes of the F. tularensis outer membrane proteins. The recombinant peptides with adjuvant CpG induced robust immunophenotypic change of dendritic cell (DC) maturation and secretion of inflammatory cytokines (IL-6, IL-12). In addition, the matured DCs enabled ex vivo proliferation of naive splenocytes in a mixed lymphocyte reaction. Lastly, we determined the in vivo immune response by assessment of antibody production in C57BL/6 mice. Total IgG levels were produced after immunization and peaked in 6 weeks, and moreover, Tul4-specific IgG was confirmed in the mice receiving peptides with or without CpG. Based on these results, we concluded that the recombinant peptides Tul4 and FopA have immunogenicity and could be a safe subunit vaccine candidate approach against F. tularensis.

• Journal of Microbiology and Biotechnology
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• 제21권5호
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• pp.529-535
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• 2011

Single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulin, connected with a short linker peptide of 10 to about 20 amino acids. In this study, the scFv of a monoclonal antibody against the third domain of human CD4 was cloned from OKT4 hybridoma cells using the phage display technique and produced in E. coli. The expression, production, and purification of anti-CD4 scFv were tested using SDS-PAGE and Western blot, and the specificity of anti-CD4 scFv was examined using ELISA. A 31 kDa recombinant anti-CD4 scFv was expressed and produced in bacteria, which was confirmed by SDS-PAGE and Western blot assays. Sequence analysis proved the ScFv structure of the construct. It was able to bind to CD4 in quality ELISA assay. The canonical structure of anti-CD4 scFv antibody was obtained using the SWISS_MODEL bioinformatics tool for comparing with the scFv general structure. To the best of our knowledge, this is the first report for generating scFv against human CD4 antigen. Engineered anti-CD4 scFv could be used in immunological studies, including fluorochrome conjugation, bispecific antibody production, bifunctional protein synthesis, and other genetic engineering manipulations. Since the binding site of our product is domain 3 (D3) of the CD4 molecule and different from the CD4 immunological main domain, including D1 and D2, further studies are needed to evaluate the anti-CD4 scFv potential for diagnostic and therapeutic applications.

• 생명과학회지
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• 제13권3호
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• pp.308-313
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• 2003

식중독 병원균인 장염비브리오균 (V. parahaemolyticus)의 세포외 분비 효소 중 콜라겐분해효소를 발현벡터인 pET-29b에 클로닝시키고 대장균에서 발현시킨 다음, 부분정제하여 그 특성을 조사하였다. V. parahaemolyticus collagenase는 $(NH_4)_2So_4$침전, affinity adsorption, 그리고 Sephacryl S-100 gel filtration 과정을 통하여 부분정제 되었다. 이 collagenase는 73%의 회수율과 43.7의 정제도를 나타내었으며, 전기영동시 분자량은 약 35 kDa로 나타났다. 이 효소의 최적 pH 및 온도는 6~12와 $35^{\circ}C$이었고, 온도안정성 조사에서 $55^{\circ}C$까지는 90% 잔존 찬성을 유지하였으나 $60^{\circ}C$이상에서는 급격하게 효소활성이 실활되었다. 기질특이성조사에서 type I collagen과 콜라겐분해효소의 합성기질로 알려진 Z-GPGGPA에서 gelatin과 casein에 비해 높은 활성을 보이는 것으로 보아 이 효소가 진정한 콜라겐분해효소라는 것을 알 수 있었다.

• Tuberculosis and Respiratory Diseases
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• 제61권3호
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• pp.239-247
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• 2006

연구배경: Mycobacteria 배양액 여과 단백질은 결핵에 대한 세포성 면역반응 및 진단 연구에 중요한 역할을 하는 것으로 알려져 있다. 새로운 결핵균 특이 유전자를 클로닝하여 약 14-kDa의 결핵균 재조합 단백질(TB-14)을 분리 정제 한 뒤 전혈 배양(whole blood culture)을 통해 재조합 단백질 항원의 자극에 따른 IFN-${\gamma}$ 분비 유도를 PPD와 비교 측정하여 TB-14 단백질이 결핵균에 대한 숙주의 세포성 면역반응 유도에 어떤 역할을 하는 지를 알아보고자 하였다. 방 법: M. avium 배양 여과액에서 항혈청과 강하게 반응하는 하나의 M. avium 특이 단백질을 확인하여 아미노산 서열을 규명한 뒤 이 단백질과 높은 상동성을 나타내는 M. tuberculosis 유전자를 클로닝하여 다량의 결핵균 재조합 단백질(TB-14)을 분리 정제하였다. 재조합 단백질 TB-14에 대한 세포성 면역 반응 유도를 알아보기 위해 결핵 환자(9명), PPD 양성 정상인(7명) 및 PPD 음성 정상인(7명)들로부터 얻은 전혈(whole blood)를 RPMI로 희석한 뒤 $10{\mu}g$의 PPD와 TB-14 단백질로 48 시간 동안 자극하여 상층액에 분비된 IFN-${\gamma}$ 농도를 ELISA로 측정하였다. 결 과: 1. M. avium LR114F 균주의 배양 여과액 내에서 M. intracellulare 항혈청에 강하게 반응하는 새로운 M. avium 특이 단백질을 규명하여 아미노산 서열을 확인하였다. 2. M. avium 특이 단백질과 상동성을 보이는 M. tuberculosis 특이 재조합 단백질인 TB-14은 148개의 아미노산으로 구성되어 있고 30개의 아미노산으로 이루어진 signal peptides를 가지며 M. avium과 78%의 상동성을 보였다. 3. PPD 양성인의 전혈 배양에서 TB-14 단백질 항원으로 자극된 군에서 PPD로 자극된 군보다 월등히 높은 IFN-${\gamma}$ 분비를 나타내었다. 반면 결핵환자에서는 질환의 양상이나 치료 정도에 따라 IFN-${\gamma}$의 분비 양상이 일정하지 않았다. 결 론: 새로운 결핵균 특이 재조합 단백질 TB-14는 결핵에 대한 인체 내 세포성 면역반응 유도에 중요한 역할을 할 수 있는 단백질이라 생각되며 특히 PPD 양성자에서 높은 IFN-${\gamma}$의 분비 양상을 보여 정상인과 결핵 감염자의 감별진단에도 활용될 수 있으리라 생각된다. 또한 TB-14 단백질에 대한 항혈청을 제작하여 환자의 가검물에 분비되는 결핵균 특이 단백질을 탐색하는 결핵의 진단 연구에도 활용될 수 있을 것으로 사료된다.

• Journal of Microbiology
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• 제44권5호
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• pp.548-555
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• 2006

FaeG is the key factor in the infection process of K88ad enterotoxigenic Escherichia coli (ETEC) fimbrial adhesin. In an attempt to determine the possibility of expressing recombinant FaeG with immunogenicity for a new safe and high-production vaccine in E. coli, we constructed the recombinant strain, BL21 (DE3+K88), which harbors an expression vector with a DNA fragment of faeG, without a signal peptide. Results of 15% SDS-polyacrylamide slab gel analysis showed that FaeG can be stably over-expressed in BL21 (DE3+K88) as inclusion bodies without FaeE. Immunoglobulin G (IgG) and M (IgM) responses in pregnant pigs, with boost injections of the purified recombinant FaeG, were detected 4 weeks later in the sera and colostrum. An in vitro villius-adhesion assay verified that the elicited antibodies in the sera of vaccinated pigs were capable of preventing the adhesion of K88ad ETEC to porcine intestinal receptors. The protective effect on the mortality rates of suckling piglets born to vaccinated mothers was also observed one week after oral challenge with the virulent ETEC strain, $C_{83907}$ (K88ad, $CT^+,\;ST^+$). The results of this study proved that the adhesin of proteinaceous bacterial fimbriae or pili could be overexpressed in engineered E. coli strains, with protective immune responses to the pathogen.

• 한국수산과학회지
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• 제50권6호
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• pp.770-776
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• 2017

This study investigated the effects of recombinant eel gonadotropin hormone (rJeGTH) produced in silkworms, with and without a carboxyl-terminal peptide from equine chorionic gonadotropin (eCG), on the induction of sexual maturation in female eels Anguilla japonica. Experiments were conducted both in vivo and in vitro. In in vitro trials, germinal vesicle breakdown (GVBD) induction did not significantly differ between rJeFSH and $rJeFSH{\cdot}eCG$ treatments and the control group. However, previous studies did find that rJeLH and $rJeLH{\cdot}eCG$ treatments induced GVBD in female eels. Our in vitro exploration of $estradiol-17{\beta}$ ($E_2$) levels in immature ovarian tissues revealed significantly higher $E_2$ levels in the group treated with $rJeFSH{\cdot}eCG$ $1{\mu}g/mL$ than in the control group. In contrast, the in vivo experiments showed no effect of recombinant hormones on the sexual maturation of feminized eels. Previous studies and our own in vitro results have clearly shown that rJeGTH and $rJeGTH{\cdot}eCG$ have a positive effect on the sexual maturation of feminized eels. To develop the activity of rJeGTH in vivo, further studies should confirm circulation time and activity of these hormones in eels' bloodstream, modify the structure of the recombinant gene, and implement additional glycosylation.

• Biotechnology and Bioprocess Engineering:BBE
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• 제6권4호
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• pp.212-230
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• 2001

Recent advances in recombinant DNA technology have resulted in development of many new protein drugs. Due to the unique properties of protein druges, they have to be delivered by parenteral injection Although delivery of protein drugs by other routes, such as pulmonary and nasal routes, has shown some promises, to date most protein drugs are administered by par-enteral routs. For long-term delivery of protein drugs by parenteral administration, they have been formulated into biodegradable microspheres. A number of microencapsulation methods have been developed, and the currently used microencapsulation methods are reviewed here, The microen-capsulation methods have been divided based on the method used. They are: solvent evapora-tion/extraction; phase separation (coacervation);spray drying; ionotropic gelation/polyelectrolyte complexation; interfacial polyumerization and supercritical fluid precipitation. Each method is de-scribed fro its applications, advantages, and limitations.

• BMB Reports
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• 제45권1호
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• pp.14-19
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• 2012

A feruloyl esterase encoding gene (designated fae6), derived from a leachate metagenomic library, was cloned and the nucleotide sequence of the insert DNA determined. Translational analysis revealed that fae6 consists of a 515 amino acid poly-peptide, encoding a 55 kDa pre-protein. The Fae6 primary structure contained the G-E-S-A-G sequence, which corresponds well with a typical catalytic serine sequence motif (G-x-S-x-G). The fae6 gene was successfully over-expressed in E. coli and the recombinant protein was purified to 8.4 fold enrichment with 17% recovery. The $K_M$ data showed Fae6 has a high affinity to methyl sinapate while thermostability data indicated that fae6 was thermolabile with a half life ($T_{1/2}$) < 30 min at $50^{\circ}C$. High affinity for Fae6 against methyl sinapate, methyl ferulate and ethyl ferulate suggest that the enzyme can be useful in hydrolyzing ferulated polysaccharides in a biorefinery process.

• 생명과학회지
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• 제11권2호
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• pp.190-195
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• 2001

The cuclodextrin glucanotransferase (CGTase) gene of Bacillus macerans was subcloned at the downstream of yeast ADH1 promoter, and then the resulting plasmid pVT-CGTM(9.15 kb) was introduced into the yeast host strain, Saccharomyces cerevisias 2805. The transformed yeast, S. cerevisiae 2805/pVT-CGTM, showed the starch-hydrolyzing activity on the starch-azure plate. The optimal conditions for the CGTase expression were found to be 2% dextrose, initial pH5.5, 3$0^{\circ}C$, and 48hr cultivation. Under this condition, the extracellular CGTase activity reached at 0.53 U/mL, whereas the intracellular activity was about 0.03U/mL. This result indicates that the signal peptide of Bacillus CGTase functioned well in S. cerevisiae.

• 미생물학회지
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• 제30권1호
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• pp.60-64
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• 1992

The secretion vector with promoter and signal sequence region of neutral protease gene (npr) from Bacillus amyloliquefaciens was constructed by the technique of polymerase chain reaction (PCR). A unique restriction iste was introduced into the 3' of the signal coding region by the synthesis of PCR primer. To demonstrate the function of cloned promoter and signal sequence, we used the E. coli .betha.-lactamase structural gene as a foreign gene. The signal sequence of .betha.-lactamase gene was deleted by Bal31 exonuclease and only mature region was introduced into the secretion vector. Bacillus subtilis cells transformed by the recombinant vector synthesized the fusion protein and were also capable of removing the signal peptide from the original fusion protein, as judged by the assay of .betha.-lactamase activity and secretion into the growth medium by western blotting.