Baculovirus Vector System에 의해 발현된 재조합 Pseudorabies Virus Major Capsid Protein의 면역원성

전무형,안동준,장경수,조용성,박종현,송재영,현방훈,안수환 충남대학교 생물공학연구소 1997 생물공학연구지 Vol.5 No.-

The recombinant pseudorabies virus major capsid protein (rMCP) was produced by expression of the MCP gene in Sf-9 cell using baculovirus transfer vector system. Following evaluation of the immunochemical properties of the rMCP, the immunogenicity of the recombinant subunit protiens were investigated in guinea pig and swine to obtain the preliminary guide line for the subunit vaccine using rMCP and gP50. It was proved that ultrasonication and 30% ammonium sulfate was most efficient to concentrate and purify the protein. The rMCP was safe in mice, guinea pigs and piglets. In guinea pigs, rMCP mixed with various adjuvants induced substantial degree of serum neutralizing antibody titers, but revealed incomplete protecivity against challenge. In swine, the combination of rMCP and gP50 showed the higher serum neutralizing antibody titers and cellular immune responses than rMCP alone. However, the protectivity was lower in comparison with the commercial gI-deleted inactivated vaccine. We expect these results to contribute to characterization of MCP gene of Korean isolate of PRV and to ultilize as preliminary information for prodution and evaluation of PRV recombinant subunit vaccines.

국내에서 분리된 Aujeszky's Disease Virus의 면역원성 : Ⅰ. Aluminum Hydroxide겔 흡착 불활화 Aujeszky's Disease Virus항원의 면역원성 Ⅰ. Immunogenicity of the Inactivated Aujeszky's Disease Virus with Aluminum Hydroxide Gel Adjuvant

전무형,이헌준,박정우,안수환 충남대학교부설 생명공학연구소 1992 생물공학연구지 Vol.2 No.-

Aujeszky's disease virus(ADV), NYJ-1 strain, isolated from the diseased piglets in Korea was inactivated with binary ethyleneimine or formalin and absorbed in aluminum hydroxide gel. Safety and humoral and cellular immunogenicity of the antigens were investigated in mouse, guinea pig and piglets. In mouse inoculation test, the antigens showed 100% of safety and induced a significant level of ADV antibody. The protective potency following intracerebral challenge with the virulent ADV at 10^7.5TCID_50/0.2㎖ were 60% to 64.3%. In guinea pig inoculation test, it was found that the antigens were quite safe, inducing 63.2 to 124.9 of virus neutralization(VN) titers at the 5th week post inoculation(pi). The protectivity of the antigens against the challenge of the virulent ADV were 62.5% to 75.0%. In piglet inoculation test, the antigens were quite safe after the primary and booster injections, and induced 28.7 to 39.1 of VN titers at the 5th week pi. The high levels of ADV antibodies were also recognized by agar gel precipitation test, latex agglutination test and immunoenzyme assays. Leucocyte adherence inhibition(LAI) test for the piglets revealed 22.3±3.8% to 32.8±6.9% of LAI index at the 4th week pi, and the evident reactions of delayed type hypersensitivity was observed in subcutaneous inoculation of the antigen to the immunized piglets. Following the challenge, the immunized piglets showed none of specific clinical signs of AD and revealed the higher gain of body weight, and the lower secretion of ADV from the nasal cavity as compared with the control group.

소 傳染性鼻氣管炎 바이러스에 대한 monoclonal antibody 生産과 診斷法 開發 : Ⅱ. Monoclonal antibody를 이용한 소 傳染性鼻氣管炎의 診斷 Ⅱ. Diagnosis of infectious bovine rhinotracheitis by using monoclonal antibody

全茂炯,金德煥,安壽煥,李重馥,閔元其 충남대학교부설 생명공학연구소 1991 생물공학연구지 Vol.1 No.-

To develop more specific and sensitive diagnostic methods for infectious bovine rhinotracheitis, 7-C-2 monoclonal antibody specific to polypeptides of infectious bovine rhinotracheitis virus (IBRV) was applied in indirect immunofluorescence antibody assay(IFA), indirect immunoperoxidase assay(IPA) and radial immunodiffusion enzyme assay (RIDEA). It was found that IBRV infected in MDBK cells could be detected as early as 8 hours post infection by IFA, and that IFA was more rapid and specific to identify IBRV antigen than IPA. The diagnostic efficacy of RIDEA and SN test was studied with 88 bovine sera. It was evident that RIDEA could eliminate the false positive reaction encountered in serum neutralization(SN) test, being more rapid and sensitive than the latter. Highly significant correlation coefficiency(r=0.76, p<0.01) was evaluated between the titers of sera and the diameters of RIDEA. Tracheal membranes and sera collected from 96 slaughtered cattle with lesions in respiratory organs were examined to detect IBRV antigen and antibody by IFA, RIDEA and SN test. It was presented that positive rates were 32.3% in IFA, 20.8% in RIDEA and 21.9% in SN test, and that coincidence rate between RIDEA and SN test were 100% in positive sera and 98.7% in negative sera. In conclusion, it was assumed that application of monoclonal antibody could improve the diagnostic efficacy of IBR by enhancing sensitivity and specificity of IPA, IFA and RIDEA.

Ciprofloxacin의 돼지 호흡기 감염증 및 설사증에 대한 치료효과

전무형,김지영,채미경,서상희,김명철,박창식,김태용,윤효인 충남대학교 수의과대학 동물의과학연구소 2003 動物醫科學硏究誌 Vol.11 No.-

To evaluate the clinical efficacy of ciprofloxacin (CFX) on treatment of the pigs with respiratory diseases or diarrhea, CFX at various dosages were administered intramuscularly to the diseased pigs selected from the pig farms located in Chungnam province. One hundreds and fourteen heads of pigs with respiratory diseases were divided into five groups and CFX at 0.25, 0.5, 1.0 ( = 2.5mg CFX/Kg B.W.) and 2.0 doses were administered i.m. for 5 days. The recovery rates were found 31.8%, 73.9%, 91.3% and 92.0%, respectively, being higher than that of the untreated control group (19.0%). The mortality of the treated groups were 0%, that is lower than that of the control (9.5%). Seven kinds of bacteria were isolated from the respiratory organs of 86 pigs, and 34 (39.5%) of 86 pigs showed the complicated infection. In analyzing by the pigs infected with a single pathogen as P. multocida, A. pleuropneumonia, Staphylclcoccus spp. or Mycoplasma spp., the recovery rates were 0∼66.6% at 0.25 dose. 66.6∼100% at 0.5 dose and 100% in all of 4 pathogens at 1.0 to 2.0 doses. At 0.25 dosage the pigs infected with A. pleuropneumonia showed the lowest recovery rate. One hundreds and twenty two heads of pigs with diarrhea were divided into five groups and CFX at 0.25, 0.5, 1.0 ( = 2.5mg CFX/Kg B.W.) and 2.0 doses were administered i.m. for 5 days. The recovery rates were 29.1%, 40.0%, 80.0% and 88.0%, respectively. The mortality of the treated groups were 0-8.3%, being much lower than that of the control. Four kinds of bacteria were isolated from the fecal specimens of 94 pigs, and 84 (89.0%) of 94 pigs showed the complicated infection.

Ciprofloxacin의 돼지 호흡기 감염증 및 설사증에 대한 치료효과

전무형,김지영,채미경,서상희,김명철,박창식,김태용,윤효인 충남대학교 형질전환복제돼지연구센터 2004 논문집 Vol. No.8

To evaluate the clinical efficacy of ciprofloxacin (CFX) on treatment of the pigs with respiratory diseases or diarrhea. CFX at various dosages were administered intramuscularly to the diseased pigs selected from the pig farms located in Chungnam province. One hundreds and fourteen heads of pigs with respiratory diseases were divided into five groups and CFX at 0.25, 0.5, 1.0 (= 2.5mg CFX/Kg B.W.) and 2.0 doses were administered i.m. for 5 days. The recovery rates were found 31.8%, 73.9%, 91.3% and 92.0%, respectively, being higher than that of the untreated control group (19.0%). The mortality of the treated groups were 0%, that is lower than that of the control (9.5%). Seven kinds of bacteria were isolated from the respiratory organs of 86 pigs, and 34 (39.5%) of 86 pigs showed the complicated infection. In analyzing by the pigs infected with a single pathogen as P. multocida; A. pleuropneumonia. Staphyloccus spp. or Mymplasma spp., the recovery rates were 0∼66.6% at 0.25 dose, 66.6∼100% at 0.5 dose and 100% in all of 4 pathogens at 1.0 to 2.0 doses. At 0.25 dosage the pigs infected with A. pleuropneumonia showed the lowest recovery rate. One hundreds and twenty two heads of pigs with diarrhea were divided into five groups and CFX at 0.25, 0.5, 1.0 (= 2.5mg CFX/Kg B.W.) and 2.0 doses were administered i.m. for 5 days. The recovery rates were 29.1 %, 40.0%, 80.0% and 88.0%, respectively. The mortality of the treated groups were 0∼8.3%, being much lower than that of the control. Four kinds of bacteria were isolated from the fecal specimens of 94 pigs, and 84 (89.0%) of 94 pigs showed the complicated infection.

Mirizzi 증후군의 변형된 분류와 치료

김형철,강길호,채만규,김성용,백무준,이문수,박상흠,이문호,김창호,송옥평,조무식,박희주 순천향의학연구소;Soonchunhyang Medical Research Institute 2000 Journal of Soonchunhyang Medical Science Vol.6 No.1

Purpose : The Mirizzi syndrome is relatively rare and preoperative diagnosis of this disease is difficult. In 1978, Morelli suggested the subclassification of the Mirizzi syndrome into acute or chronic form. We experienced 5 cases of acute form. We analysed clinical features, preoperative radiologic findings and operative findings of 18 cases including acute forms which were diagnosed as Mirizzi syndrome and should suggest the modified classification of Mirizzi syndrome for choice of appropriate treatment. Method : From January 1995 to December 1998, 18 cases, of which 8 cases were diagnosed at Soonchunhyang University Chunan Hospital, and 10 cases were reported in the Korean Journal were retrospectively analysed with regard to clinical features, preoperative radiologic findings and operative findings. According to the clinical features, whole cases were divided into type Ⅰ(acute form) and type Ⅱ(chronic form) and then each type of cases were subclassified according to preoperative radiologic findings and operative findings. Results : Of 18 cases there were 5 cases in type Ⅰ(27.8%), 13 cases in type Ⅱ(72.2%). Type Ⅱb was most common. Type Ⅰa cases were treated only with cholecystectomy. We applied cholecystectomy, T-tube choledochostomy and patch technique in type Ⅰb and thpe Ⅰc cases. Cholectystectomies including removal of gallstones and internal drainage procedures were done in type Ⅱ chronic forms. Conclusion : The acute form(Type Ⅰ) of Mirizzi syndrome was suggested by Morelli might be subclassified into typeⅠa,Ⅰb and Ⅰc following the presence of the necrotic defect in common hepatic duct. Through the modified classification of Mirizzi syndrome based on clinical feature, preoperative radiologic findings and operative findings, we can choice appropriate treatment.

Pseudorabies Virus의 Major Capsid Protein 유전자의 클론닝과 Baculovirus Vector System에 의한 발현

안동준,전무형,송재영,박종현,현방훈,장경수,안수환 충남대학교 생물공학연구소 1997 생물공학연구지 Vol.5 No.-

Pseudorabies is caused by Pseudorabies virus (PRV: Aujeszky's disease virus) of Herpesviridae that is characterized by 100 to 150nm in size with a linear double-stranded DNA molecule with of approximately 90 × 10 exp (6)Da. This disease affects most of domestic animals such as swine, cattle, dog, sheep, cat, chicken, etc. causing high mortality and economic losses. In swine, young piglets show high mortality and pregnant sows, reproductive failures. However the adult swine reveals no clinical signs in general. But they become a carrier state and play an importnat role for propagation of the disease. In this study, the nucleotide sequence of major casid protein gene of PRV, Yangsan strain isolated from the diseased swine in Korea was analyzed, and the recombinant MCP was produced by expression of the MCP gene in Sf-9 cell using baculovirus transfer vector system. As result, in BamHI digestion, MCP gene locus of PRV YS strain showed different from that of Indiana S strain. The patterns of enzyme mapping were also found to be unidentical each other. The sequence of the MCP gene partially analyzed showed 98.09% identity to Indiana S strain. The expression of MCP in Sf-9 cell cotransfected by pVLMCP-44 baculovirus expression vector was characterized by Southern blot hybridization, immunofluoresent and immunocytochemical tests, SDS-PAGE and Western blotting. The rMCP with M.W. 142kDa was most effectively expressed in Sf-9 cells at the 3-4th days post inoculation of the recombinant baculovirus by 2 moi.

젖소 足皮膚炎에 대한 오존연고의 治療 效果

이수진,전무형,조성환,김덕환,박창식,김명철 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10

Thirty dairy cows with pododermatitis were selected and treatment effect of ozone ointment for bovine pododermatitis was investigated. In addition, bactericidal effect of ozone ointment on etiological agent of bovine pododermatitis was evaluated. The pathohistological examination for the pododermatitis, according to application with ozone ointment was investigated. Thirty dairy cows were divided two groups: control group(vaseline group: 15 cows), treatment group(ozone ointment group: 15 cows). Various parameters were evaluated in terms of the lameness score, swelling score, lesion score, WBC, neutrophil, pathohistological finding, and antimicrobial action. As compared with vaseline group, ozone ointment group revealed significant decrease of lameness (p<O.05), swelling (p<O.O1) and lesion score (p<O.05) were shown in hoof lesions on 14 days after application. In hematological findings, WBC count revealed slightly high values within normal range before treatment, however, this was I Corresponding author improved on 14 days after application of ozone ointment. The number of neutrophils was slightly higher than that of normal, however. this was improved on 14 days after application of ozone ointment. In pathohistological findings, normal dermal tissue was found in tissues with pododermatitis on 14 days after application of ozone ointment. In antimicobial action, marked decrease rate of bacteria was observed in feet of all cases treated with ozone ointment. The decreasing rate of bacteria in anaerobic culture was higher than that in aerobic culture.

꿩에서 분리된 Newcastle Diesase Virus 내열성주 (CBP)의 Fusion(F) 유전자 클론닝과 염기서열 분석

장경수,전무형,송희종,김귀현,박종현 충남대학교 생물공학연구소 1999 생물공학연구지 Vol.7 No.-

The gene encoding F protein of CBP-1 strain, a heat-stable Newcastle disease virus (NDV) isolated from the diseased pheasants in Korea, was characterized by reverse transcriptionpolymerase chain reaction (RT-PCR), nucleotide and amino acid sequences. Virus RNA was prepared from the chorioallatoic fluid infected with NDV CBP-1 virus and cDNA was amplified by RT-PCR, cloned and sequenced to analyze. The PCR was sensitive as to detect the virus titer above 2^5 hemagglutination unit. 1.7kb (1,707bp) size of the cDNA was amplified and cloned into BamHI site of pVL1393 Baculo transfer vector. The uncleotide sequences for F protein were determined by dye terminator cyclic sequencing using four pairs of primers, and 553 amino acid sequences were predicted. In comparison of the nucleotide sequence of F gene of CBP-1 with those of other NDV strains, the homology revealed 88.8%, 98.5% and 98.7% with Kyojungwon (KJW), Texas GB and Beaudette C strains, respectively. As the deduced 553 amino acid sequences of F protein of CBP-1 were compared with those of other NDV strains, the homology appeared 89.9%, 98.7% and 98.9% with KJW, Texas GB and Beaudette C strains, respectively. The putative protease cleavage site (112-116) was R-R-Q-K-R, indicating that CBP-1 strain is velogenic type. The amino acid sequences include 6 sites of N-asparagine-linked glycosylation and 13 cysteine residues. These data indicate that the genotype of CBP-1 strain is more closely associated with the strains of Texas GB and Beaudette C than KJW strain.

국내에서 분리된 Infectious Bovine Rhinotracheitis Virus DNA의 제한효소분석

민원기,전무형,이중복,김병한,안수환 충남대학교부설 생명공학연구소 1991 생물공학연구지 Vol.1 No.-

Nine strains of infectious bovine rhinotracheitis virus(IBRV) isolated in Korea since 1970 were investigated to elucidate the pathogenicity on various cell lines and the reactivity in cross serum neutralization test. The genomes of the viruses were also studied by restriction endonuclease analysis to examine the genetical patterns and the origin of the viruses. Results obtained by experiment were as follows ; 1. In pathogenicity test on various cell lines by cytopathology and immunofluorescence antibody assay, MDBK cells were found to be very susceptible to all of the domestic isolates, Colorado and Oxford strains and RK-13 cells, mildly reactive to all of viruses. However, CV-1 and BHK cells revealed negligible reactions against all of viruses tested. 2. In cross serum neutralization(SN) test with the positive serum from the cattle immunized with PQ7 strain of IBRV, PQ7, SQ, VS, A14, TSV and S′74-7 strains showed 64 of SN titer, and IQ, QW, A37, Colorado and Oxford strains, 128 of SN titer. It was assumed that there is no difference in major antigenic determinants among the viruses. 3. By analysis of restriction endonuclease Hind Ⅲ, it was found that 8 domestic isolates except A37 and Colorado strain revealed the identical banding patterns. However, Oxford strain showed quite different patterns, displaying H band at 6.9×10 exp (6) daltons and K′band at 4.5×10 exp (6) daltons. A37 strain exclusively contained the longer bands of A′ and A″. 4. When the viral DNA were analyzed by restriction endonuclease EcoRI, all of the domestic isolates and Colorado strain showed very similar banding patterns, whereas Oxford strain revealed a considerable discrepancy, displaying B′ band at 12.5×10 exp (6) daltons. 5. By analysis of restriction endonuclease BamHI, it was found that the domestic isolates and Colorado strain revealed similar patterns displaying 8 fragments. However, Oxford strain was cleaved into 9 fragments displaying G′and G″ bands.