• 대한의생명과학회지
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• 제16권3호
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• pp.201-206
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• 2010

In order to develop procedures for the rapid isolation of recombinant sugar transporter in functional form from away from the endogenous insect cell transporter, gene fusion techniques were exploited. Briefly, BamH1-digested human HepG2 type glucose transport protein cDNA was first cloned into a transfer vector pBlueBacHis, containing a tract of six histidine residues. Recombinant baculoviruses including the human cDNA were then generated by allelic exchange following transfection of insect cells with wild-type BaculoGold virus DNA and the recombinant transfer vector. Plaque assay was then performed to obtain and purify recombinant viruses expressing the human transport protein. All the cell samples that had been infected with viruses from the several blue plaques exhibited a positive reaction in the immnuassay, demonstrating expression of the glucose transport protein. In contrast, no color development in the immunoassay was observed for cells infected with the wild-type virus or no virus. Immunoblot analysis showed that a major immunoreactive band of apparent Mr 43,000~44,000 was evident in the lysate from cells infected with the recombinant baculovirus. Following expression of the recombinant fusion protein with the metal-binding domain and enterokinase cleavage site, the fusion protein was recovered by competition with imidizole using immobilized metal charged resin. The leader peptide was then removed from the fusion protein by cleavage with porcine enterokinase. Final separation of the recombinant protein of the interest was achieved by passage over $Ni^{2+}$-charged resin under binding conditions. The expressed transport protein bound cytochalasin B and demonstrated a functional similarity to its human counterpart.

• Journal of Microbiology and Biotechnology
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• 제26권10호
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• pp.1800-1807
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• 2016

To understand how human cytomegalovirus (HCMV) might change and evolve after reactivation, it is very important to understand how the nucleotide sequence of cultured HCMV changes after in vitro passaging in cell culture, and how these changes affect the genome of HCMV and the consequent variation in amino acid sequence. Strain JHC of HCMV was propagated in vitro for more than 40 passages and its biological and genetic changes were monitored. For each passage, real-time PCR was performed in order to determine the genome copy number, and a plaque assay was employed to get virus infection titers. The infectious virus titers gradually increased with passaging in cell culture, whereas the number of virus genome copies remained relatively unchanged. A linear correlation was observed between the passage number and the log₁₀ infectious virus titer per virus genome copy number. To understand the genetic basis underlying the increase in HCMV infectivity with increasing passage, the whole-genome DNA sequence of the high-passage strain was determined and compared with the genome sequence of the low-passage strain. Out of 100 mutations found in the high-passage strain, only two were located in an open reading frame. A G-T substitution in the RL13 gene resulted in a nonsense mutation and caused an early stop. A G-A substitution in the UL122 gene generated an S-F nonsynonymous mutation. The mutations in the RL13 and UL122 genes might be related to the increase in virus infectivity, although the role of the mutations found in noncoding regions could not be excluded.

• 대한수의학회지
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• 제38권2호
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• pp.304-313
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• 1998

Sixteen Korean field transmissible gastroenteritis viruses (TGEVs) were isolated using swine testicular cell (STC) and the genomic diversity of them was analyzed. All TGEV isolates produced a typical cytopathic effect in STC and were confirmed as TGEV by immunofluorescence assay using monoclonal antibody against TGEV and PCR using TGEV specific primers. RNAs from TGEV field isolates and vaccine TGEV were extracted and amplified by RT and PCR. The RT-PCR products were digested with selected restriction enzymes and analyzed RFLP patterns. The N-terminal end region of S gene and ORF 3 and 3-1 genes of TGEV amplified by TGEV specific primer pairs seemed to be conserved. Most specific variations were detected in S gene amplified by TGEV 4/6 primer pairs which includes antigenic sites A and D. When the PCR products were treated with Sau3AI and Ssp I, Bvac(vaccine strain), field isolates 133 and 347 were differentiated from Miller and Purdue types. In the case of D5 field isolates, it was classified into Purdue type by Sau 3AI but classified into independent TGEV by Ssp I. Two different TGEV strains from D2 sample were confirmed by plaque purification and RT-PCR-RFLP analysis. To investigate the change occurring in TGEV genome after serial passage, the TGEV P44 strain was passaged through STC. There were specific changes in S gene and a large deletion was observed in ORF 3 and 3-1 genes. These studies showed that a distinct difference in genome exists among TGEV field isolates.

• 약학회지
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• 제38권6호
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• pp.806-813
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• 1994

These experiments were conducted to investigate the effects of Cervi cornu extract on lymphocyte blastogenesis in spleen, thymus, lymph node, born marrow cells of Balb/c mouse, haemagglutination reaction against sheep red blood cell (SRBC), plaque forming cell (PFC) assay against SRBC and IL-2 production. Lymphocyte blastogenesis was determined by $[^3H]-thymidine$ incorporation. According to the lymphcoyte blastogenesis test on the immune cell. Ceriv cornu extrat was showed a potent mitogenic activity on the spleen and lymph node cells, but had mild mitogenic activity on the thymus and born marrow cells. Mitogenic active component of Crevi cornu extract was identified to be materials where molecular weights are higher than 5,000 by membrane filteration method. Cervi cornu extrat was shown to increase mitogenic effect on the lipopolysaccharide (LPS)-stimulated spleen cells significantly, but decrease mitogenic effect on the Con A stimulated spleen cell at the concentration 0.3%, 1% and 3%. Ceriv cornu extract didn't show to be haemagglutination reaction and showed to inhibit the Con A-induced haemagglutination reaction against SREC. Result of SRBC-PEC test. Ceriv cornu extract significantly increase the number of PEC at the concentration of 0.1% and 1%. When IL-2 or IL-4 production was determined by proliferation of CTLL-2 cells. Ceriv cornu extract was not shown to stimulate the production of IL-2. From the above results, it is shown that Ceriv cornu extract increased antibody production by B cells, but nor IL-2 production by helper T cells.

• Archives of Pharmacal Research
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• 제24권1호
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• pp.74-78
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• 2001

A preparation of water soluble components (EA) was made from carpophores of Elfvingia applanata (Pers.) Karst and its in vitro antiviral activity on vesicular stomatitis virus [(Indiana serotype, VSV(IND)] was investigated by plaque reduction assay. EA exhibited potent antiviral activity on VSV(IND) growth and negligible cytotoxicity on Vero cells, 50% effective concentration ($EC_{50}C$/) of 104$ug\textrm\/ml$ and 50% cytotoxic concentration ($CC_{50}C$) of 3,793$ug\textrm\/ml$, respectively. Selectivity index (Sl $CC_{50}C$/$EC_{50}C$) of EA on Vero cell and VSV(IND) was about 36.5. EA did not display either a direct virucidal effect on V5V(IND) or induction of antiviral substance by Vero cells upon its treatment. Thus, the mode of antiviral activity of EA was studied at steps of viral adsorption onto cell. When both EA and virus were added to cell monolayers, titer of cell-free virus in culture supernatant increased in ca. 30-40% compared with that of control group and titer of cell-associated virus was 60-100% higher than that of control group. These results suggested that antiviral activity of EA on VSV(IND) might be due to the hindrance of viral entry to cells at eITher endocytosis or loss of envelope.

• 한국식품위생안전성학회지
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• 제4권2호
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• pp.109-118
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• 1989

한국산 담자균류인 갓버섯 Lepiota procera의 균사를 액내 배양하여 항암성분인 단백성 다당체를 분리하였다. 이 성분은 DEAE-Sephadex A-50 이온교환수지와 Sepharose-4B gel Filtration을 이용하여 정제하여 Fraction C1을 얻었으며 이 Fr, Cr은 단백질과 다당체로 구성되어 있으며 항암 효과는 10mg/kg/day 투여군에서 64%의 저지유을 나타내었다. 이러한 항암작용의 기전을 밝히기 위한 연구의 일환으로 면역에 미치는 영향을 실험함 결과 이 단백다당체는 용혈반형성 세포수를 증가시켰으며, 저하된 지연성 과민반응을 회복시켰을 뿐만 아니라, carrageenan 투여에 의해 억제된 면역능을 다시 증강시켰음을 알 수 있었다. 이러한 결과들은 이 버섯의 항암작용이 세포독성에 의한 것이 아니라 종양에 대한 면역능을 강화시켜 발휘됨을 제시하고 있다.

• Journal of Periodontal and Implant Science
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• 제35권1호
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• pp.31-41
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• 2005

Fimbriae (fimA) of Porphyromonas gingivalis are filamentous components on the cell surface and are thought to play an important role in the colonization and invasion of periodontal tissue. P. gnigivalis fimA gene encoding fimbrillin, a subunit of fimbriae, has been classified into 5 genotypes (types I to V) based on the nucleotide sequences. In the present study, we examined the prevalence of these fimA genotypes in patients with dental implant and the relationship between prevalence of these genotypes and peri-implantitis. Dental plaque specimens obtained from 80 peri-implant sulci of 50 patients with dental implants were analyzed by 16S rRNA fimA gene-directed PCR assay. P. gingivalis were detected in 74.4% of the samples of the control group (healthy peri- implant sulci; probing depth<5mm) and in 92.0% of the samples of the test group (peri-implant sulci with peri-iimplantitis; probing $depth{\geqq}5mm$). Among the P. gingivalis-positive samples of the control group, the most prevalent fimA type was type I (29.3%), followed by type II (26.8%). In contrast, a majority among the P. gingivalis-positive samples of the test group was type II (56.S%), followed by type I (43.5%). TypeII fimA genotype organisms were detected more frequently in the test group and a significant difference in the occurrence of type II was observed between test and the control groups. A correlation between specific fimA types and peri-implant health status was found in type II (OR 3.545) and only a weak relationship was revealed in typeIV(OR 3.807). These findings indicate that P. gingivalis strains that possess type II fimA are predominant in peri-implant sulci with peri-implantitis and are closely associated with peri-implant health status. P. gingivalis with type II fimA may be involved in peri-implantitis.

• 대한바이러스학회지
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• 제30권1호
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• pp.11-18
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• 2000

Since $Hantavax^{TM}$, formalin inactivated Hantaan virus vaccine (10,240 ELISA units/ml), has been developed in 1990 to prevent against haemorrhagic fever with renal syndrome (HFRS) caused by Hantaan or Seoul virus, it has been commercially available in Korea. Twenty-one healthy people were booster shot once and twice after primary basic vaccination with $Hantavax^{TM}$. Seroconversion rates were measured by immunofluorescent antibody technique (IFAT), enzyme-linked immunosorbent assay (ELISA), high density composite particle agglutination (HDPA), and plaque reduction neutralization test (PRNT). Seroconversion rates of 21 vaccinees at one year after primary basic vaccination were 52.3%, 95.2%, 0.0%, 47.6%, and 28.6%, and 13 vaccinees at one month after 1st booster vaccination were 100%, 100%, 30.7%, 100% and 100% by IFAT, ELISA (IgG, IgM), HDPA and PRNT, respectively. Seroconversion rates declined slightly by twenty months, and they were 84.6%, 92.3%, 0.0%, 84.6% and 69.2% by IFAT, ELISA (IgG, IgM), HDPA and PRNT, respectively. Seroconversion rates of 9 vaccinees at three months after 2nd booster vaccination were 100%, 100%, 0.0%, 100%, and 88.9%, and 16 vaccinees at one year after the 2nd booster vaccination were 87.5%, 93.8%, 0.0%, 87.5% and 81.3% by IFAT, ELISA (IgG, IgM), HDPA and PRNT, respectively. Based on the above result $Hantavax^{TM}$ has proved a vigorous anamnestic response after the 1st and the 2nd booster vaccination and has persisted higher fluorescence, agglutination and neutralizing antibody titers in vaccinees.

• 대한바이러스학회지
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• 제30권2호
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• pp.125-130
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• 2000

Infection with human cytomegalovirus (HCMV) is of considerable clinical relevance after placental transmission and in immunosuppressed patients such as transplant recipients or patients with AIDS. The rapid detection method of HCMV has been required to overcome the time-consuming methods such as classical plaque assay or other immunological methods. This study was performed to establish the in situ ELISA, in which human lung fibroblasts infected with HCMV were fixed and used directly as antigen in 96 well culture plate. Expressed HCMV antigens were detected with HCMV-specific monoclonal antibodies. This method could detect HCMV dose-dependently upto $3{\times}10^2\;pfu/ml$. Antiviral activity of ganciclovir could be assayed within the known range of effective dose. This result showed that HCMV could be quantitated by in situ ELISA. The chemical, which was selected on the basis of component analysis in natural product, was tested to have the anti-HCMV activity by in situ ELISA, and three among five samples were found to have anti-HCMV activity with the dose-dependent manner. Conclusively in situ ELISA could be useful method for quantitation of HCMV and screening antiviral activity of samples to HCMV.

• Journal of Periodontal and Implant Science
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• 제36권1호
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• pp.265-272
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• 2006

Dental plaque, a biofilm consisting of more than 500 different bacterial species, is an etiological agent of human periodontal disease, It is therefore important to characterize interactions among periodontopathic microorganisms in order to understand the microbial pathogenesis of periodontal disease. Previous data have suggested a synergistic effect of tow major periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia in the periodontal lesion. In the present study, to better understand interaction between P. gingivalis and T. forsythia, the coaggregation activity between these bacteria was characterized. The coaggregation activity was observed by a direct visual assay by mixing equal amount (1 ${\times}$ $10^9$)of T. forsythia and P. gingivaJis cells. It was found that the first aggregates began to appear after 5-10 min, and that the large aggregates completely settled within 1 h. Electron and epifluorescence microscopic studies confirmed cell-cell contact between two bacteria. The heat treatment of P. gingivalis completely blocked the activity, suggesting an involvement of a heat-labile component of P. gingivalis in the interaction. On the other hand, heat treatment of T. forsythia significantly increased the coaggregation activity; the aggregates began to appear immediately. The coaggregation activity was inhibited by addition of protease, however carbohydrates did not inhibit the activity, suggesting that coaggregation is a protein-protein interaction. The results of this study suggest that coaggregation between P. gingivalis and T. forsythia is a result of cell-cell physical contact, and that coaggregation is mediated by a heat-labile component of P. gingivalis and T. forsythia component that can be activated on heat treatment.