Baculovirus를 이용한 Aujeszky's Disease Virus gⅢ 단백질 발현

송재영,이중복,현방훈,박종현,김병한,권창희,전무형,안수환 충남대학교 생물공학연구소 1993 생물공학연구지 Vol.3 No.-

The g Ⅲ gene located in U_L region of Yangsan strain, a field isolate of Aujeszky's disease virus (ADV) in Korea, was cloned into pTZ18R and sequenced. The gⅢ gene consisting of 1,437 nucleotides showed 98% sequence homology with that of Becker strain, a reference strain of ADV. The gene encoding gⅢ of Yangsan strain was placed under the control of Autographa californica nucleopolyhedrosis virus (AcNPV) polyhedrin promoter, and expressed by the derived recombinant baculovirus using Spodoptera frugiperda 9 (Sf9) cells. The expressed gⅢ was a protein with molecular weight of 72kd determined by immunoprecipitation and Western blotting assay using anti-ADV polyclonal antibodies and anti-gⅢ monoclonal antibody. The partially purified gⅢ protein was utilized as antigen in the radial immunodiffusion enzyme assay (RIDEA) to detect to specific antibody against ADV in pig sera. The results indicated that the sensitivity of RIDEA with the recombinant gⅢ protein antigen (98%) was as high as that with the conventional glycoprotein antigen extracted from the ADV infected cells. In addition, the false positive and false nagative reactions in gⅢ RIDEA were significantly reduced than the conventional glycoprotein RIDEA as judged from the results of standard serum neutralization test.

Baculovirus를 利用한 Canine Parvovirus VP2蛋自質의 發現

朴鍾賢,宋載永,玄芳動,安動濬,姜永源,全茂炯,安壽煥 충남대학교 생물공학연구소 1998 생물공학연구지 Vol.6 No.-

국내에서 分離된 개파보바이러스주(V20주)의 VP2 遺傳子를 baculovirus system을 이용하여 발현시켜 다음의 結果를 얻었다. 1. 개파보바이러스의 VP2 유전자를 PCR에 의해 增幅하여 1755bp의 VP2遺傳子를 pUC19에 클로닝하여, 클로닝된 遺傳子를 polyhedrin promoter를 가지는 baculovirus expression vector인 pVL1393에 옮겨 VP2 발현벡터인 pVL1393-VP2를 얻을 수 있었다. 2. pVL1393-VP2 plasmid와 baculovirus DNA와의 homologous recombination에 의해 재조합바이러스인 VP2-BV를 얻을 수 있었으며, 그 발현효율은 2.000-5.000 HAU/0.05 ml이었다. 3. 免疫沈澱法에 의해 발현된 단백질은 개파보바이러스의 VP2단백질과 유사한 64 kb에 달하는 것이었으며, 血球凝集能을 지니고 있었다 4. 血球凝集能을 지닌 蛋白質이 여러 陽性血淸에 있어서 抗體수준을 測定할 수 있는지 개파보바이러스항원과의 相關性을 比較한 바 0.94 (n=125. p<0.01)의 相關係數를 보였다 5. 발현 VP2白은 virus-like particles를 形成하였으며, 그 크기로는 개파보바이러스와 비슷한 25 ㎚의 크기를 갖았다. Canine parvovirus(CPV) is a member of autonomous replicating parvoviruses and is aetiologically associated with enteritis and mycoarditis in puppies. The capsids of CPV are composed of three structural proteins: VP1, VP2 and VP3. The VP2 protein is the major component of capsid. The VP2 gene of a canine parvovirus. V20 strain isolated in Korea was cloned into baculovirus expression vector, and subsequently the VP2 protein was expressed by a recombinant baculovirus under the control of polyhedrin promoter. The recombinant VP2 protein expressed in Spodoptera frugiperda 9(Sf9) cells was detected by haemagglutination(HA) test and immunofluorescent antibody assay. Molecular weight of the recombinant VP2 protein expressed was estimated as 64Kd when tested by immunoprecipitation test using anti-CPV monoclonal antibody. In haemagglutination inhibition(Ⅲ) test. 8 HA units of the recombinant VP2 protein antigen was successfully utilized to determine a level of antibody against CPV in various positive sera. The recombinant VP2 protein showed also the capability to form virus like particles similar in size and appearance to the CPV virions.

미세혈관 협심증 환자의 임상상 및 지표에 관한 연구

박상호,온영근,김현정,이태훈,길효욱,김현건,박수진,송해정,한대희,방덕원,신원용,이내희,김성구,권영주 순천향의학연구소 2003 Journal of Soonchunhyang Medical Science Vol.9 No.1

Background : Microvascular angina or syndrom X is heterogenous and encompasses different pathogenetic entities. Several studies suggest that microvascular angina have good prognosis and occurs predominantly in postmenopausal women. Lipoprotein (a), total cholesterol, LDL-cholesterol, HDL-cholesterol, triglyceride, homocystein, CRP and fibrinogen are the independent risk factor of acute coronary syndrome and are the chief underlying cause of atherosclerosis. We investigated clinical characteristics and markers in patients with microvascular angina. Methods : We studies 23 patients diagnosed with microvascular angina and 16 control group with negative treadmill exercise test and no significant coronary angiogram. We investigated clinical characteristics in patients group and evaluated symptom during a follow-up period of 4.2± 2.5 month. Exercise time in treadmill exercise test was compared between patients and control group. We assessed the level of markers, such as lipoprotein (a), total cholesterol, LDL-cholesterol, HDL-cholesterol, triglyceride, homocystein, CRP and ESR in two groups. Results : There is no difference in age between two groups. Patients group had a tendency to increase in female, hypertension, DM, and smoking history, but there was no statistical significance difference. Exercise duration time was significantly decreased in patients with microvascular angina (p<0,05). Serum lipoprotein (a), total cholesterol, LDL-cholesterol, HDL-cholestrol, Triglyceride, ESR, Fibrinogen and Homocystein value were no significant difference between microvascular angina and control group. Only CRP was significantly high in patients with microvascular angina (p<0.05). Conclusion : There was no statistical significance difference in age, sex, hypertension, DM and smoking history between two groups but patients group had a tendency to increase the prevalence in hypertension, DM and smoking history, female. Also, Symptom improvement and disappearance in follow-up was presented in 11 patients of 19(57.9%). Exercise tolerance was worse in patients group than control group. CRP value was significantly higher in patients group than control group. There was no statistical significance difference in lipoprotein (a), total cholesterol, LDL-cholesterol, HDL-cholesterol, triglyceride, homocystein, ESR and Fibrinogen between two groups.

국내에서 분리된 canine parvovirus의 구조유전자 cloning과 염기서열 분석

박종현,송재영,이중복,현방훈,안수환,전무형 충남대학교 생물공학연구소 1993 생물공학연구지 Vol.3 No.-

In this study gene encoding structural proteins of a CPV isolate was cloned and saquenced to elucidate the molecular genetical properties of the canine parvoviruses isolated from the field. Six recombinant plasmids of pEP3, p1471, p2070, pEP069, pEP338 and p14711p were constructed from the map positions 22 to 98 of RF DNA to clone the VP1 and VP2 genes of CPV-V20. Sequentialy the gene comprising 3780 nucleotides were sequenced by dideoxy chain termination method. When nucleotide sequence of gene encoding the structural proteins of CPV-V20 was compared with those of other strains, CPV-N, CPV-d and CPV-780929 published previously. DNA homologies to CPV-V20 were 99.87% with CPV-NM, 99.73% with CPV-d, 96.85% with CPV-780929 AND 98.4% with FPLV-Carl, respectively. The DNA sequence data of CPV-V20 showed seven point mutations and also deletion of 135 nucleotides from the nucleotide position 4745 to 4879 located in the 3-noncoding region of CPV-N.

Pocine Adenovirus-3의 E1B Region의 鹽基序列 分析

朴鍾賢,宋載永,李重馥,玄芳勳,安東濬,車相昊,裵用泰,姜永源,Reddy, P S,全茂炯,安壽煥 충남대학교 생물공학연구소 1999 생물공학연구지 Vol.7 No.-

돼지 아데노바이러스(PAV-3). 6618주의 EIB region이 包含되어 있는 map unit 4.0에서 9.7까지의 유전자에 대한 1,984 bp의 염기서열을 決定하였으며, 이 結果를 알려진 여러 아데노바이러스 유전자와 비교하여 다음과 같은 결과를 얻었다. 1. PAV-3의 EIB유전자는 10개의 ORF로 구성되어 있으며, 그 중 아데노바이러스의 단백질과 유사성이 있는 것은 ORF1, ORF2 및 ORF3이었다. ORF1은 Ad41의 19kd 와 BAV-2에서의 EIB ORF2에서의 아미노산의 一致率은 각각 32%와 31%이었다. 2. ORF2는 Ad2 55kd protein과 tupaia adenovirus 44kd protein가 각각 34%로 아미노산 一致率이 가장 높았으며, Ad41의 52kd protein. BAV-3의 EIB ORF3에서도 33%의 一致率을 보였다. 3. ORF1은 61-666 uncleotide (606 bp), ORF 2에서는 429-1,850 uncleotide (1,422 bp)의 부위로 각각 202, 474 a.a로 構成되었으며, 예상되는 분자량은 20 kd와 52 kd이었다. 4. ORF3는 hexon-associated pIX유전자로 추정되며 내부에 1개의 polyadenylation signal(ATAAA)이 1938-1942 uncleotide에 위치하였으며, 이 부위는 TATA box (1937-1942 uncleotide)와 중복되어 존재하였다. Porcine adenovirus type 3 (PAV-3) does not cause severe infection in pigs. Adenovirus has been suggestive of live vaccine vector carrying foreign gene. One of insertion regions is delayed early (EIB) region. However, EIB region of PAV-3 has not been molecularly characterized to date. Nucleotide sequence of EIB of PAV-3 was determined. The EIB region was composed of 1,984 bp and located between 4.0 and 9.7 map units. Three potential open reading frames(ORFs) with low level of homology to other adenoviruses and a polyadenylation signal were identified in the rightward direction of genome. The nucleotide and the predicted amino acid sequences of EIB were compared to those of human and animal adenoviruses. One of the three potential ORFs. ORF1 encoded a polypeptide homologous to bovine adenovirus type 2(BAV-2) ORF2 and human adenovirus type 41(Ad41) 19 kd protein. ORF2 encoded a polypeptide homologous to human adenovirus type 2(Ad2) 55 kd protein, bovine adenovirus type 3(BAV-3) ORF3 and porcine adenovirus type 4(PAV-4) ORF2. The predicted protein of ORF1 had homology to those of Ad41 and BAV-2 with 32 and 31% respectively, whereas the deduced protein of ORF2 had homology to those of Ad2. BAV-3 and PAV-4 with 34, 33 and 29%, respectively.

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.

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.

구리 지역에서 조사한 고혈압의 인지도, 치료률 및 관리률

홍철호,김순길,홍택원,이제,신진호,이재웅,김경수,김정현,임헌길,이방헌 대한간질학회 2003 대한간질학회지 Vol.9 No.1

Epidemiology and Molecular Biological Characterization of Rabies in Korea

Bang-Hun Hyun(현방훈) 한국보건교육건강증진학회 2005 한국보건교육·건강증진학회 학술대회 발표논문집 Vol.- No.-

Development of the in vivo Bioaerosol Experiment System for Virus Filtration Efficiency (VFE) Test

Bang Hun Hyun,Jie Yeun Park,Hyun Mi Pyo,Hyung Kwan Jang,Soo Dong Cho,Dong Gun Yang,Jaejo Kim,Seong In Lim,Jae young Song,Chun Gui Park 대한수의학회 2009 대한수의학회 학술대회발표집 Vol.2009 No.-