• Asian-Australasian Journal of Animal Sciences
• /
• 제31권8호
• /
• pp.1103-1109
• /
• 2018

Objective: This study aimed to screen and identify the target genes of miR-375 in pig Sertoli (ST) cells and to elucidate the effect of miR-375 on the proliferation of ST cells. Methods: In this study, bioinformatics software was used to predict and verify miR-375 target genes. Quantitative polymerase chain reaction (PCR) was used to detect the relationship between miR-375 and its target genes in ST cells. Enzyme-linked immunosorbent assay (ELISA) of rearranged L-myc fusion (RLF) and hypoxia-induced gene domain protein 1A (HIGD1A) was performed on porcine ST cells, which were transfected with a miR-375 mimics and inhibitor to verify the results. Dual luciferase reporter gene assays were performed to assess the interactions among miR-375, RLF, and HIGD1A. The effect of miR-375 on the proliferation of ST cells was analyzed by CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS). Results: Five possible target genes of miR-375, including RLF, HIGD1A, colorectal cancer associated 2, POU class 3 homeobox 1, and WW domain binding protein 1 like, were found. The results of quantitative PCR suggested that mRNA expression of RLF and HIGD1A had a negative correlation with miR-375, indicating that RLF and HIGD1A are likely the target genes of miR-375. The ELISA results revealed that RLF and HIGD1A were negatively correlated with the miR-375 protein level. The luminescence results for the miR-375 group cotransfected with wild-type RLF and HIGD1A vector were significantly lower than those of the miR-375 group co-transfected with the blank vector or mutant RLF and HIGD1A vectors. The present findings suggest that RLF and HIGD1A are target genes of miR-375 and that miR-375 inhibits ST cell proliferation according to MTS analysis. Conclusion: It was speculated that miR-375 affects cell proliferation through its target genes, which play an important role in the development of testicular tissue.

• Journal of Veterinary Science
• /
• 제23권4호
• /
• pp.55.1-55.12
• /
• 2022

Background: ASF was first reported in Kenya in 1910 in 1921. In China, ASF spread to 31 provinces including Henan and Jiangsu within six months after it was first reported on August 3, 2018. The epidemic almost affected the whole China, causing direct economic losses of tens of billions of yuan. Cause great loss to our pig industry. As ELISA is cheap and easy to operate, OIE regards it as the preferred serological method for ASF detection. P54 protein has good antigenicity and is an ideal antigen for detection. Objective: To identify a conservative site in the African swine fever virus (ASFV) p54 protein and perform a Cloth-enzyme-linked immunosorbent assay (ELISA) for detecting the ASFV antibody in order to reduce risks posed by using the live virus in diagnostic assays. Method: We used bioinformatics methods to predict the antigen epitope of the ASFV p54 protein in combination with the antigenic index and artificially synthesized the predicted antigen epitope peptides. Using ASFV-positive serum and specific monoclonal antibodies (mAbs), we performed indirect ELISA and blocking ELISA to verify the immunological properties of the predicted epitope polypeptide. Results: The results of our prediction revealed that the possible antigen epitope regions were A23-29, A36-45, A72-94, A114-120, A124-130, and A137-150. The indirect ELISA showed that the peptides A23-29, A36-45, A72-94, A114-120, and A137-150 have good antigenicity. Moreover, the A36-45 polypeptide can react specifically with the mAb secreted by hybridoma cells, and its binding site contains a minimum number of essential amino acids in the sequence 37DIQFINPY44. Conclusions: Our study confirmed a conservative antigenic site in the ASFV p54 protein and its amino acid sequence. A competitive ELISA method for detecting ASFV antibodies was established based on recombinant p54 and matching mAb. Moreover, testing the protein sequence alignment verified that the method can theoretically detect antibodies produced by pigs affected by nearly all ASFVs worldwide.

• Parasites, Hosts and Diseases
• /
• 제26권2호
• /
• pp.73-86
• /
• 1988

간흡충(Clonorchis sinensis)의 조항원(WWA), 정제항원(ABA)의 민감도와 특이도를 비교해 보는 한편, 발육단계별로 충란 항원(EGA)-피낭유충 항원(MEA)-성충 항원(WWA)의 단백질 구성물질의 차이를 SDS-polyacrylamide gel electrophoresis(SDS-PAGE)로 관찰하고 이들 항원의 유용성을 효소면역반응(enzyme-linked immunosorbent assay: ELISA)으로 비교하여 다음과 같은 결과를 얻었다. 간흡충의 조항원으로부터 정제된 항원(ABA)은 항체와 결합한 결합항원(affinity-purified antibody(IgG) binding antigen:ABA)으로서 Ouchterlony 반응으로 간흡충 감염 가토혈청과 4개의 침모대 (WWA는 7개)를 형성하였다. WWA의 major protein들은 분자량 16,300∼18,500 및 28,000∼29,000 dalton의 Polypeptide band였고, ABA는 분자량 18,000∼21,000 및 29,000∼31,000 dalton의 단백질로 다소의 차이를 보였다. ELISA시첩에서 ABA는 WWA에 비해 현저히 민감도가 낮았다. WWA와 ABA에 대한 폐흡충 감염 사람혈청의 ELISA 정사에서 WWA는 Ouchterlony 양성자 8예중 3예에서 교차반응을 나타내었으나 ABA는 교차반응을 나타내지 않았다. 간흡충의 발육단계별 항원 단백질의 분자량 범위는 WWA 11,000∼80,000, MEA 15,000∼100,000, EGA 15,000∼200,000 dalton이었다. 주류원 단백질의 분자량은 EGA는 각각 36,600 (band No. 22), 38,500(No. 20), 64,000(No. 9), 62,000(No. 10), 54,500(No. 11), 53,000(No. 12) dalton, MEA는 각각 65,600(No. 3), 44,700 (No.7), 43,900(No. 8) dalton, WWA는 각각 16,300∼18,500(No. 31-32), 28,000∼29,000(No. 25), 11,000w13,000 (No. 35) 및 31,000(No. 24) dalton이었다. 즉, MEA와 EGA가 WWA보다 고분자의 단백질로 구성되어 있었다. 각 발육단계별 항원의 항원성은 ELISA 반응으로 볼 때 WWA가 가장 높았고, MEA는 약한 항원성만을 나타내었으며 EGA는 음성이었다. WWA와 간흡충 감염 가토혈청은 감염 4∼6주에 OD>1.0, 12주 이후 항체가의 plateau를 나타내었으나 MEA는 Ouchterlony 반응에서 EGA와 함께 음성반응을 보였다. ELISA에서도 중감염군(12∼20주)에서만 OD>0.6으로 미약한 항원성이 인정되었으며 경감염군은 전감염기간중 OD<0.6를 나타내었다. 이상의 결과로 보아 성충 항원이 가장 강력한 함원성 물질을 포함하고 있음이 명백하다고 생각된다. 그러나 성충 조항원은 면역진단에 사용할 겹우 타흡충과의 빈번한 교차반응이 예상되므로 민감도 및 특이도가 높은 정제 항원물질을 분리하는 일이 중요하다고 생각된다.

• 대한한방내과학회지
• /
• 제27권4호
• /
• pp.864-878
• /
• 2006

목적 : 인삼은 다양한 생물학적 작용이 있다. 그 중 항염증작용과 관련하여 염증성 사이토카인 분비 및 저산소 유도인자-1${\alpha}$ 활성화 조절 효과를 살펴보고자 한다. 방법 : phorbol myristate acetate (PMA)+A23187에 유도된 세포에서 염증성 cytokines 분비의 변화와 인간의 mast cell인 HMC-1 cells에서 hypoxia-inducible factor-1 (HIF-1)${\alpha}$의 작용을 관찰하였다. 결과 : PMA+A23187은 대조군과 비교해서 interleukin $(IL)-1{\beta}$, IL-6와 tumor necrosis factor $(TNF)-{\alpha}$의 분비를 증가시킨다. 또한 증가된 cytokines IL-1, IL-6, $TNF-{\alpha}$가 인삼의 처리에 의해 두드러지게 억제하는 것을 확인하였다. 인삼(5 ${\mu}g/ml$)은 $IL-1{\beta}$, IL-6, $TNF-{\alpha}$의 분비를 약 105.1${\pm}$9.7%, 95${\pm}$9.4%, 29.7${\pm}$4.5%,(P<0.05)로 최대로 억제하였고, PMA+A23187에 유도된 vascular endothelial growth factor (VEGF)와 granulocyte macrophage-colony stimulating factor (GM-CSF)의 분비를 41.3%와 75.7%로 각각 억제하였다. 그리고 저자는 인삼이 PMA+A23187로 유도된 HIF-1${\alpha}$ 발현과 HIF-1에 대해 DNA binding activity를 억제하고 있음을 관찰하였다. 결론 : 인삼이 HIF-1에서 염증반응을 억제함을 나타내고, 이는 인삼이 염증성 질환을 치료하는데 유익한 효과가 있음을 의미한다.

• Journal of Nutrition and Health
• /
• 제48권1호
• /
• pp.9-18
• /
• 2015

본 연구에는 최근 항비만 소재로 연구되고 있는 건조, 미숙탱자의 물 추출물 (PF-W) 소재를 대상으로 폴리페놀 ($52.15{\pm}4.02mg/g$)과 플라보노이드 ($6.56{\pm}0.47mg/g$) 함량을 측정하고 항산화 활성과 세포독성을 시험한 후, 지방 흡수 제어 가능성을 확인하고자 lipoprotein lipase (LPL)의 억제효능을 배양배지와 세포 내의 LPL 함량, LPL mRNA 발현 그리고 LPL 효소활성측정을 통해 검토하였다. 그 결과 PF-W은 3T3-L1 adipocyte에서 LPL mRNA의 발현과 활성에는 영향이 없었으며, LPL의 분비를 억제하는 것을 알 수 있었다. PF-W의 LPL 분비억제기작을 확인하기 위해 다양한 단백질 이동 관련 유전자의 발현을 확인하였고, 그 결과 LPL의 이동과 분해에 관여하여 세포내 LPL의 활성을 조절하는 것으로 알려진 SorLA의 발현이 증가하는 것을 확인하였다. 이를 조절하는 transcription factor의 발현과 세포핵으로의 이동에 PF-W가 미치는 영향을 검토한 결과 PF-W를 처리함으로써 SorLA promoter 에 작용하는 $C/EBP{\beta}$의 단백질양이 세포핵에서 증가하는 것을 확인할 수 있었다. 본 연구를 통해 PF-W가 SorLA 유전자의 transcription factor인 $C/EBP{\beta}$의 단백질 발현을 세포핵에서 증가시킴으로써 SorLA의 발현이 증가되어 LPL의 분비억제가 가능함을 확인할 수 있었으며 이는 PF-W의 항비만 효과기전을 설명하는 기초자료를 제공하는 것이라 사료된다.

• IMMUNE NETWORK
• /
• 제4권1호
• /
• pp.23-30
• /
• 2004

Background: A human orthologue of mouse S100A6-binding protein (CacyBP), Siah-1-interacting protein (SIP) had been shown to be a component of novel ubiquitinylation pathway regulating $\beta$-catenin degradation. The role of the protein seems to be important in cell proliferation and cancer evolution but the expression pattern of SIP in actively dividing cancer tissues has not been known. For the elucidation of the role of SIP protein in carcinogenesis, it is essential to produce monoclonal antibodies specific to the protein. Methods: cDNA sequence coding for ORF region of human SIP gene was amplified and cloned into an expression vector to produce His-tag fusion protein. Recombinant SIP protein and monoclonal antibody to the protein were produced. The N-terminal specificity of anti-SIP monoclonal antibody was conformed by immunoblot analysis and enzyme linked immunosorbent assay (ELISA). To study the relation between SIP and colon carcinogenesis, the presence of SIP protein in colon carcinoma tissues was visualized by immunostaining using the monoclonal antibody produced in this study. Results: His-tag-SIP (NSIP) recombinant protein was produced and purified. A monoclonal antibody (Korea patent pending; #2003-45296) to the protein was produced and employed to analyze the expression pattern of SIP in colon carcinoma tissues. Conclusion: The data suggested that anti-SIP monoclonal antibody produced here was valuable for the diagnosis of colon carcinoma and elucidation of the mechanism of colon carcinogenesis.

• BMB Reports
• /
• 제29권2호
• /
• pp.105-110
• /
• 1996

Polyclonal antiserum R101 against aflatoxin $B_1$ ($AFB_1$) was raised in New Zealand white rabbits after injection of bovine serum albumin-$AFB_1$ conjugate. Competitive ELISA (enzyme linked immuno-sorbent assay) demonstrated that antiserum R101 has the highest binding for $AFB_1$ (50% inhibition at 170 fmol) and aflatoxicol II (50% inhibition at 112 fmol). It also reacts with other aflatoxins such as $AFB_2$, $AFG_1$, $AFG_2$, and aflatoxin metabolites ($AFM_1$, $AFM_2$, $AFP_1$, and $AFQ_1$), but it does not cross-react with $AFG_2a$. Using this antiserum, aflatoxins were quantitated in 100 urine samples of undergraduate students at the College of Pharmacy, Sung Kyun Kwan University, Republic of Korea. By ELISA, $AFB_1$ and its metabolites were detected in human urine samples (N=100, male=89, female=11, ages=20~31 yrs) with a range of 1.4~200.6 ng/kg/day (mean$\pm$SD=$18.11{\pm}33.01\;ng\;AFB_1/kg/day$ in males, $3.82{\pm}2.65\;ng/kg/day$ in females). Assuming that urinary excretion is about 7.6% of $AFB_1$ intake (Groopman et al., 1992), we estimated that Koreans were daily exposed to a total dietary $AFB_1$ of $240.20{\pm}438.67\;ng/kg/day$ in males and $50.35{\pm}29.88\;ng/kg/day$ in females, respectively. When the human monitoring data was applied to a linear regression model of Y=21.67X-10.04 {Y=liver cancer incidence per 100,000, X=Log $AFB_1$ intake (ng/kg/day), r=0.99} developed from previously reported epidemiological data, calculated liver cancer incidences attributed to $AFB_1$ exposure were 41.56/100,000 in males and 26.84/100,000 in females. The incidences were similarly correlated with liver cancer mortality rates of 43.43/100,000 in males and 11.23/100,000 in females in Korea. These results suggest that aflatoxin exposure may be an important risk factor for the high incidence of liver cancer in Korea.

• Journal of Microbiology and Biotechnology
• /
• 제19권5호
• /
• pp.502-510
• /
• 2009

Although the Escherichia coli heat-labile enterotoxin B subunit (LTB) has already been expressed in several different systems, including prokaryotic and eukaryotic organisms, studies regarding the synthesis of LTB into oligomeric structures of pentameric size in the budding yeast Saccharomyces cerevisiae have been limited. Therefore, this study used a functional signal peptide of the amylase 1A protein from rice to direct the yeast-expressed LTB towards the endoplasmci reticulum to oligomerize with the expected pentameric size. The expression and assembly of the recombinant LTB were confirmed in both the cell-free extract and culture media of the recombinant strain using a Western blot analysis. The binding of the LTB pentamers to intestinal epithelial cell membrane glycolipid receptors was further verified using a GM1-ganglioside enzyme-linked inmmunosorbent assay (GM1-ELISA). On the basis of the GM1-ELISA results, pentameric LTB proteins comprised approximately 0.5-2.0% of the total soluble proteins, and the maximum quantity of secreted LTB was estimated to be 3 mg/l after a 3-day cultivation period. Consequently, the synthesis of LTB monomers and their assembly into biologically active aligomers in a recombinant S. cerevisiae strain demonstrated the feasibility of using a GRAS microorganism-based adjuvant, as well as the development of carriers against mucosal disease.

• Asian Pacific Journal of Cancer Prevention
• /
• 제15권6호
• /
• pp.2613-2618
• /
• 2014

Aims: Dysfunction of the host immune system in cancer patients can be due to a number of factors, including lymphocyte apoptosis. Several studies showed that $Foxp3^+T$ cells take part in inducing this process by expressing FasL in tumor patients. However, the relationship between apoptosis, $CD8^+T$ cells and $Foxp3^+T$ cells in HCC patients is still unclear. The present study was designed to investigate the correlation between apoptosis levels and Fas/FasL expression in $CD8^+T$ lymphocytes and $Foxp3^+T$ cells in patients with HCC. Methods: $CD8^+T$ cells and $CD3^+Foxp3^+T$ cells were tested from peripheral blood of HCC patients and normal controls and subjected to multicolor flow cytometry. The expression of an apoptosis marker (annexin V) and the death receptor Fas in $CD8^+T$ cells and FasL in $CD3^+Foxp3^+T$ cells were evaluated. Serum TGF-${\beta}1$ levels in patients with HCC were measured by enzyme-linked immunosorbent assay. The relationship between apoptosis and Fas expression, as well as FasL expression in $CD3^+Foxp3^+T$ cells was then evaluated. Results: The frequency of $CD8^+T$ cells binding annexin V and Fas expression in $CD8^+T$ cells, were all higher in HCC patients than normal controls and the proportion of apoptotic $CD8^+T$ cells correlated with their Fas expression. Serum TGF-${\beta}1$ levels correlated inversely with $CD3^+Foxp3^+T$ cells. Conclusions: Fas/FasL interactions might lead to excessive turnover of $CD8^+T$ cells and reduce anti-tumor immune responses in patients with HCC. Further investigations of apoptosis induction in $Fas^+CD8^+T$ cells in vitro are required.

• 대한약침학회지
• /
• 제19권1호
• /
• pp.37-44
• /
• 2016

Objectives: This study examined the relative efficacies of a derivative of betulinic acid (dBA) and its poly (lactide-co-glycolide) (PLGA) nano-encapsulated form in A549 lung cancer cells in vivo and in co-mutagen [sodium arsenite (SA) + benzo[a]pyrene (BaP)]-induced lung cancer in mice in vivo. Methods: dBA was loaded with PLGA nanoparticles by using the standard solvent displacement method. The sizes and morphologies of nano-dBA (NdBA) were determined by using transmission electron microscopy (TEM), and their intracellular localization was verified by using confocal microscopy. The binding and interaction of NdBA with calf thymus deoxyribonucleic acid (CT-DNA) as a target were analyzed by using conventional circular dichroism (CD) and melting temperature (Tm) profile data. Apoptotic signalling cascades in vitro and in vivo were studied by using an enzyme-linked immunosorbent assay (ELISA); the ability of NdBA to cross the blood-brain barrier (BBB) was also examined. The stage of cell cycle arrest was confirmed by using a fluorescence-activated cell-sorting (FACS) data analysis. Results: The average size of the nanoparticles was ~ 110 nm. Confocal microscopy images confirmed the presence of NdBA in the cellular cytoplasm. The bio-physical properties of dBA and NdBA ascertained from the CD and the Tm profiles revealed that NdBA had greater interaction with the target DNA than dBA did. Both dBA and NdBA arrested cell proliferation at G0/G1, NdBA showing the greater effect. NdBA also induced a greater degree of cytotoxicity in A549 cells, but it had an insignificant cytotoxic effect in normal L6 cells. The results of flow cytometric, cytogenetial and histopathological studies in mice revealed that NdBA caused less nuclear condensation and DNA damage than dBA did. TEM images showed the presence of NdBA in brain samples of NdBA fed mice, indicating its ability to cross the BBB. Conclusion: Thus, compared to dBA, NdBA appears to have greater chemoprotective potential against lung cancer.