• 한국식품영양과학회지
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• 제33권7호
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• pp.1169-1174
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• 2004

본 연구에서는 감마선 및 전자선종이 ovalbumin(OVA)의 구조 변화에 미치는 영향을 비교 평가하였다. 표준항원 OVA을 3,5,7 및 10 kGy의 흡수선량으로 감마선 및 전자선 조사하였으며 그에 따른 구조적인 변화는 SDS-PAGE, GPC-HPLC 및 단클론 항체를 사용한 Ci-ELISA법으로 측정하였다. Native OVA분자는 감마선 및 전자선 조사에 의해 소편화 및 응집화 되었으며, 조사 선량이 증가할수록 OVA의 분자량이 감소하였다. 또한 OVA의 면역화학적 인 구조는 방사선 조사에 의해 항원-항체간의 결합능이 감소하는 것으로 나타났으나 감마선 및 전자선종에 따른 차이는 없었다. 이상의 결과들은 식품 알레르겐의 제거 및 면역원의 개발에 대한 전자선 조사의 기초자료로 이용될 수 있을 것으로 사료된다.

• Biomolecules & Therapeutics
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• 제11권3호
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• pp.157-162
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• 2003

The generation of secretory IgA antibodies(Abs) for specific immune protection of mucosal surfaces depends on stimulation of the mucosal immune system, but this is not effectively achieved by parenteral or even oral administration of most soluble antigens. Thus, to produce a possible vaccine antigen against urinary tract infections, the uropathogenic E. coli (UPEC) adhesin was genetically coupled to the ctxa2b gene and cloned into a pMAL-p2E expression vector. The chimeric construction of pMALfimHIctxa2b was then transformed into E. coli K-12 TB1 and its nucleotide sequence was verified. The chimeric protein was then purified by applying the affinity chromatography. The purified chimeric protein was confirmed by SDS-PAGE and western blotting using antibodies to the maltose binding protein (MBP) or the cholera toxin subunit B (CTXB), plus the N-terminal amino acid sequence was analyzed. The orderly-assembled chimeric protein was confirmed by a modified $G_{M1}$-ganglioside ELISA using antibodies to adhesin. The results indicate that the purified chimeric protein was an Adhesin/CTXA2B protein containing UPEC adhesin and the $G_{M1}$-ganglioside binding activity of CTXB. This study also demonstrate that peroral administration of this chimeric immunogen in mice elicited high level of secretory IgA and serum IgG Abs to the UPEC adhesin. The results suggest that the genetically linked CTXA2B acts as a useful mucosal adjuvant, and that the adhesin/CTXA2B chimeric protein might be a potential antigen for oral immunization against UPEC.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.1093-1098
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• 2001

The gene coding for a Lepidoptera-specific insecticidal crystalline (or control) protein (ICP), recognized as cryIAc, from Bacillus thuringiensis subsp. kurstaki HD-73, was cloned into the vector pBluscript ll SK-, and then transformed in Escherichia coli $DH5{\alpha}$. The clone was named EBtIAc and the chimeric phagemid, as pEBtIAc. Hyperexpression of CryIAc protoxin was observed in the extract of the culture of E. coli harboring pEBtIAc. Crystalline protoxin was purified by differential solubility. It was dissolved in alkaline pH, and exposed to trypsin to be activated. The molecular weights of the pro- and activated toxins on SDS-PAGE were estimated to be ca. 130 kDa and 60 kDa, respectively. The toxicity was tested by force-feeding larvae of gypsi moth (Lymantria diapar) with trypsinized protoxin. Using the batch of biologically active form of the toxin as an immunogen, anti-CryIAc antiserum was raised in a New Zealand white rabbit. Immunoglobulin G was fractionated from the seam by Protein-A sepharose affinity chromatography. Immunoreactivity of the antibody was examined by dot and Westerns blottings. It has been found that the anti- CryIAc antibody recognized the purified toxin at a level below a nanogram in terms of quantity. Using the antibody some of Bt-corns were able to be differentiated from tons of corn kernels which were imported from America as forage crops.

• 한국가금학회지
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• 제39권4호
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• pp.273-278
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• 2012

본 연구는 식중독은 물론, 아토피성 피부염의 원인물질로 알려져 있는 포도상구균 장내독소 B(Staphylococcal enterotoxin B; SEB)에 대한 특이 난황항체를 개발하고자 실험을 실시하였다. 우선, SEB 유전자를 클로닝한 다음, 대장균 발현시스템을 이용하여 약 30 kDa 정도의 재조합 SEB 단백질을 생산하였다. 재조합 SEB 단백질을 산란계에 2주 간격으로 3회 면역접종을 실시하고, 혈청 및 난황 내 항체가를 측정한 결과, 면역 후 4주경에 항체가가 최고치에 달하였으며, 산란계로부터 획득한 난황항체를 이용한 Western blot 결과, 재조합 SEB 단백질은 물론, 상용화 SEB 단백질과도 특이적으로 반응한다는 것을 규명하였다. 결론적으로, 식중독과 아토피성 피부염 등의 원인물질로 알려진 SEB에 특이적인 난황항체를 생산에 성공하였으며, 이러한 특이적인 난황항체는 식중독 및 아토피성 피부염의 예방 및 치료에 활용 가능할 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제15권4호
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• pp.570-575
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• 2002

Effects of active immunization against native 14-mer somatostatin (SRIF, somatotropin releasing inhibiting factor) and its two 14-mer-somatostatin analogues on the milk production in rat mammary cells were studied. Native SRIF, Tyr11-somatostatin (Tyr11-SRIF), and D-Trp8, D-Cys14-somatostatin (Trp8Cys14-SRIF) were conjugated to bovine serum albumin (BSA) for immunogen preparation. Twenty-four female Sprague-Dawley rats were divided into four groups and immunized against saline (Control), SRIF, Tyr11-SRIF, and Trp8Cys14-SRIF at five weeks of age. Booster immunizations were performed at 7, 9, and 11 weeks of age. SRIFimmunized rats were mated at 10 weeks of age. The blood and mammary glands were collected at day 15 post-pregnancy and -lactation. To measure the amount of milk protein synthesis in the mammary gland, mammary cells isolated from the pregnant and the lactating rats, were cultured in the presence of $^3H$-lysine. No significant differences in growth performance, concentration of growth hormone in the circulation, and the amount of milk protein synthesis were observed among the groups. Inductive levels of serum anti-SRIF antibody in the SRIF and Tyr11-SRIF groups but not in the Trp8Cys14-SRIF group, were significantly higher than that of the control group during the pregnancy and lactation periods. The result suggests that active immunization against native 14-mer SRIF and Tyr11-SRIF was able to induce anti-SRIF antibodies, but did not affect the milk protein synthesis.

• Journal of Microbiology and Biotechnology
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• 제18권1호
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• pp.95-103
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• 2008

Mushrooms are regarded as one of the well-known foods and biopharmaceutical materials with a great deal of interest. Polysaccharide ${\beta}$-glucan is the major component of mushrooms that displays various biological activities such as antidiabetic, anticancer, and antihyperlipidemic effects. In this study, we compared the immunostimulatory potency of polysaccharide fractions, prepared from liquid culture of pine-mushroom Tricholoma matsutake, with a potent immunogen lipopolysaccharide (LPS), and their molecular mechanisms on the functional activation of macrophages. We found that fraction II (TMF-II) was able to comparably upregulate or highly enhance the phenotypic functions of macrophages such NO production and cytokine (IL-$1{\beta}$, IL-6, IL-12, and TNF-${\alpha}$) expression, to LPS. TMF-II triggered the phosphorylation of $I{\kappa}B{\alpha}$, a critical step for NF-${\kappa}B$ activation and translocation. Of the upstream signaling enzymes tested, Src and Akt were thought to be the responsible upstream signaling components in induction of NO production, although TMF-II strongly upregulated the phosphorylation of all MAPK pathways. Therefore, our data suggest that T. matsutake-derived ${\beta}$-glucan may exert its immunostimulating activities with similar potency to LPS via activation of multiple signaling pathways linked to NF-${\kappa}B$ activation.

• 대한수의학회지
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• 제41권1호
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• pp.43-49
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• 2001

As compared with reaction of antibody for sonicated antigen of Brucella abortus strain RB51 and 1119-3 by Western blot analysis, Brucella field positive sera was detected strong reaction at 40~80 kDa LPS of strain 1119-3, but detected very weak reaction at strain RB51 partly. Otherwise, as we analyzed major immunogen of RB51 by antisera bled periodically during 6 months after RB51 vaccination. we detected strong immunological reaction at 17, 18 and 8 kDa antigen of RB51. Especially, reaction of 8 kDa antigen by Western blot coincided with reaction of dot-blot assay in RB51-antibody detection method. We also compared with reaction of field sera by STAT(standard tube agglutination test), dot-blot assay and Western blot (reaction of 8 kDa antigen of strain RB51). 16 sera of 4~5 months after RB51 vaccination are all negative by STAT, and 12 field brucellosis positive serum are all positive, and also 12 of 16 sera vaccinated RB51 are positive by dot-blot assay and reaction of 8kDa antigen by Western blot. but 1 of 15 Brucellosis negative sera reacted nonspecifically dot-blot assay.

• 미생물학회지
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• 제38권1호
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• pp.38-44
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• 2002

국내 분리 침습성 균주중선별된 S. pneumoniae KNIH1156 (type 19F)으로부터 페렴구균의 병원성 인자이며 항원학적으로 다양한 표면단백항원인 pneumococcal surface protein A (PspA)를 분리${\cdot}$정제하였다. 폐렴구균을 CDM-ET배지에서 배양하게 되면 배지내로 PspA가 방출된다는 점과 PspA가 인간의 lactoferrin에 특이적으로 결합한다는 사실을 이용하여 CDM-ET 배지에서 S. pneumoniae KNIH1156 을 배양한 후 배양액을 농축하여 lactoferrinaffinity chromatography에 통과시켜 PspA를 분리, 정제하였다. 정제 후 anti-PspA antiserum으로 PspA를 확인하여 순수분리, 정제되었음을 확인하였으며 또한 인간의 lactoferrin과의 결합능력을 유지하고 있음을 확인하였다. 순수하게 분리하여 정제된 PspA의 면역원성을 확인하기 위하여 ICR mice에 욕강주사하였을 때 $LD_{50}$ 가 $1{\times}10^{7.5}$ CFU/ml께서 $1{\times}10^{10}$ CFU/ml로 약 500배 중가함을 관찰하였다. 따라서 본 실험에서S. pneumoniae KNIH1156 으로부터 분리${\cdot}$ 정제한 PspA가 면역원성과 방어능을 가지고 있음을 확인할 수 있었다.

• 한국축산식품학회지
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• 제33권2호
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• pp.198-204
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• 2013

This study aimed to apply the Parallux system to detect (fluoro)quinone antibiotics residues in raw bovine milk. The immunogen enabled the generation of a specific antiserum with a titer of 1/40,000. The $Parallax^{TM}$ kit using the antibody displayed $IC_{50}$ value of 10 to 150 ppb for (fluoro)quinolone antibiotics. $Parallax^{TM}$ kit was also sensitive for the detection of incurred (fluoro)quinolone at Korean Maximum Residual Levels in raw bovine milk as the result of dose response test. Cross reactivities of the antibody with the common (fluoro)quinolones were determined to be norfloxacin, 100%; enrofloxacin, 100%; ciprofloxacin, 100%; danofloxacin, 100%; nalidixic acid, 40%. Lower detection limit (LOD) values of the $Parallax^{TM}$ kit in raw bovine milk were determined to be norfloxacin, 4 ppb; enrofloxacin, 5 ppb; danofloxacin, 5 ppb; ciprofloxacin, 5 ppb and nalidixic acid, 10 ppb. The $Parallax^{TM}$ kit was run 8 times with five different concentrations of norfloxacin to determine the coefficient of variation (CV, %) of intra-assay, which was between 2.7% and 11.8%. To confirm the precision among kit batches for the inter-assay, five different batch kits were tested with 2 different concentration of norfloxacin. The CVs of the inter assay were 4.2% at 50 ppb, and 7.2% at 10 ppb norfloxacin, respectively.

• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.474-481
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• 1996

Since the commercially available rabbit anti-cyclin D3, generated from c-terminal 16 amino acid residues which are common to human and murine cyclin D3, is highly cross-reactive with many other cellular proteins of mouse, a new rabbit polyclonal anti-cyclin D3 has been raised by using murine cyclin D3 protein expressed at a high level in Escherichia coli as the immunogen. To express murine cyclin D3 protein in E. coli, the cyclin D3 cDNA fragment encoding c-terminal 236 amino acid residues obtained by polymerase chain reaction (PCR) was inserted into the NcoI/BamHI site of protein expression vector, pET 3d. Molecular mass of the cyclin D3 overexpressed in the presence of IPTG (Isopropyl $\beta$-D-thiogalactopyranoside) was approximately 26 kDa as calculated from the reading frame on the DNA sequence, and the protein was insoluble and mainly localized in the inclusion bodies that could be easily purified from the other cellular soluble proteins. When renaturation was performed following denaturation of the insoluble cyclin D3 protein in the inclusion bodies using guanidine hydrochloride, 4.4 mg of soluble form of cyclin D3 protein was produced from the transformant cultured in 100ml of LB media under the optimum conditions. Four-hundred micrograms of the soluble form of cyclin D3 protein was used for each immunization of a rabbit. When the antiserum obtained 2 weeks after tertiary immunization was applied to Western blot analysis, it was able to detect 33 kDa cyclin D3 protein in both murine lymphoma cell line BW5147.G.1.4 and human Jurkat T cells at 3,000-fold dilution with higher specificity to murine cyclin D3, demonstrating that the new rabbit polyclonal anti-murine cyclin D3 generated against c-terminal 236 amino acid residues more specifically recognizes murine cyclin D3 protein than does the commercially available rabbit polyclonal antibody raised against c-terminal 16 amino acids residues.