Plaque assay방법을 이용한 수두 바이러스의 역가 검정

우규진,황규계,전복환,박송용,문홍모 대한바이러스학회 1994 Journal of Bacteriology and Virology Vol.24 No.2

Use of Nested Polymerase Chain Reaction for Identification of Rickettsia tsutsugamushi Serotype Cultured in Human Embryonic Lung Cells

안창남,우규진,김태연,신광순,김철중,백락주,An, Chang-Nam,Woo, Gyu-Jin,Kim, Tae-Yeon,Shin, Kwang-Soon,Kim, Chul-Joong,Baek, Luck-Ju 대한미생물학회 1996 Journal of Bacteriology and Virology Vol.26 No.2

Rickettsia tsutsugamushi의 원형균주인 Karp주와 Gilliam주를 초대 배양된 사람 정상 2배체 폐세포(LuMA cell)를 이용하여 증식과 세포병변들의 속도를 비교할 수 있었고, 배양된 균주는 네스티드 프라이머를 사용하여 혈청형을 동정할 수 있었다. R. tsutsugamushi의 세포벽 외막에 존재하며 혈청형을 결정하는 주요항원은 54-56Kd 단백인 것으로 밝혀지고 있는데, 이 단백 유전자의 DNA 염기서열을 분석하여 Karp주와 Gilliam주의 공통서열로 첫번째 프라이머쌍을 만들었고 첫번째 프라이머쌍의 안쪽에 위치한 혈청형 사이에 차이가 있는 서열로 두번째 프라이머쌍을 만들었다. 네스티드 뉴클레오티드 프라이머는 중합효소 연쇄반응의 특이성을 증가시킬 수 있는데 이 실험 결과로 이 PCR 방법은 scrub typhus의 진단과 혈청형의 동정에 적용될 수 있을 것으로 보여진다. We selected the adequate cell line to be used for propagation and plaquing of R. tsutsugamushi in laboratory and identified R. tsutsugamushi serotype cultured in LuMA cells by nested PCR. As in this study, we concluded that. 1. LuMA cell was suitable for the study of the biology of rickettsiae-host cell interaction. 2. The plaque-forming unit (PFU) per ml of R. tsutsugamushi Karp strain propagated in embryonated egg yolk sacs was $10^{8.8}$ and the PFU/ml of Gilliam strain was $10^{7.1}$. 3. The rate and extent of cytopathic changes depended on the PFU titer of R. tsutsugamushi. 4. PCR with nested primer pairs was useful for identification of R. tsutsugamushi serotype cultured in human embryonic lung cells.

Siderophore A3에 의한 Pseudomonas synxantha A3의 철(Fe) 흡수와 그 Receptor Protein에 관한 연구

조영배,우규진,전홍기 부산대학교 유전공학연구소 1994 분자생물학 연구보 Vol.10 No.-

P. synxantha A3로 부터 siderophore A3의 생성이 결핍된 변이주(fluroescent negative;Flu^- )와 siderophore A3에 의한 철의 흡수가 결손된 변이주(iron uptake negative;Iu )를 얻었다. P. synxantha A3의 생육은 EDDA농도가 증가할수록 감소 하였으며, siderophore A3의 생성도 균의 생육과 함께 감소하였다. 철(Fe)이 고농도로 존재하는 상태(100μM)에서의 생육정도는 P. synxantha A3와 Flu^- 변이주의 경우 비슷한 생육도를 보였으며, 철이 저농도로 존재하는 상태(0μM)에서는 P. synxantha A3의 성장속도는 약간 느리나 Flu^- 변이주보다 약간 높은 생육도를 보였다. 여과멸균한 Fe-siderophore A3 complex를 100μM/ml 되게 최소배지에 첨가한 상태에서의 생육도는 P. synxantha A3가 가장 양호하였으며, Flu^-, Iu^-변이주의 순으로 생육정도를 나타내었다. Citrate농도가 높아질수록 균의 생육은 증가하였으나, siderophore A3 생성은 저하되었다. P. synxantha A3의 경우 복합배지에서 자란 균체보다 siderophore A3생성용 죄소배지에서 자란 균체에서 약 150,000 dalton정도의 2개의 protein band가 더 존재하였으며, Iu^- 변이주의 경우 M.W.30,000과 45,000사이에 뚜렷한 2개의 protein band가 형성되었다. In an iron-limited enviornmemt, pesudomonas synxantha A3 produces a yellow-green fluorescent siderophore called sideropable of iron uptake(Iu^-)from Fe-siderophore A3 complex and a nonfluorescent mutamt(Flu^-)unable to synthesize siderophore a# were obtained, and their outer membrane protein(OMP) extracts were analyzed on SDS-PAGE. When increase the concentration of ethylendiamine-N,N'-diacetic acid(EDDA) in medium, the growth and production of siderophore A3 of P. synxantha A3 was gradually decreased and rapidly reduced ovwe 1.0㎎/ml of EDDA. Responding to the increased concentration of Fe(Ⅲ)and Fe-siderophore A3 complex in the iron limitted siderophore A3-production medium, the growth of P. synxantha A3, Flu mutant and Iu^- mutant were increased, but the that of Iu-mutant was much lower. Depending on the increase the concentrations of citrate, the grpwth of the test strains was increased but the production of siderophore A3 was inhibited. When P. synxantha A3 was cultured in siderophore A3 production medium, two more bands around the zone of M.W. 150,000 were identified. Qnd two distinct protein bands between the zone of M.W.30,000and 45,000 were also identified in OMP from Iu^- strain compared to that of mother strain.

Microcarrier 배양에서 세포부착과 바이러스 역가변화에 미치는 바이러스 감염 비율의 영향

전복환,박호선,우규진,황규계,박송용,문홍모 대한바이러스학회 1995 Journal of Bacteriology and Virology Vol.25 No.2

We had previously evaluated the possibility of using microcarrier culture system to produce attenuated cell-associated and cell-free varicella-zoster virus (VZV). We have investigated the attachment characteristics of both normal human embryonic lung (LuMA) cells and virus-infected LuMA cells on cell-grown microcarriers with various multiplicities of infection. In cell at tachment period for virus infection, a portion of the unattached normal cells in culture medium was increased with the decrease of unattached VZV-infected cells regardless of infection ratio. VZV yield was enhanced with increase of initial virus-infected cells onto cell-laden microcarriers. By understanding relationship between the attachment kinetics of cells and virus titers and by determining virus infection ratio, it might be possible to propagate the VZV efficiently using mi- crocarrier culture system.

수두 바이러스 증식에 미치는 혈청의 영향

전복환(Bok-Hwna Chun),우규진(Gyu-Jin Woo),황규계(Kyu-Kye Hwang),박호선(Ho-Sun Park),박송용(Song-Yong Park),문홍모(Hong-Mo Moon) 한국생물공학회 1994 KSBB Journal Vol.9 No.3

무혈청배지에서 수두 바이러스의 증식

전복환,박호선,최윤,황규계,우규진,정현용,박증석,박송용 대한바이러스학회 1995 Journal of Bacteriology and Virology Vol.25 No.1

A human embryonic lung fibroblast (LuMA) cell line has been established for production of a live attenuated varicella vaccine. We have investigated the possibility of using serum-free medium for growth of LuMA cells and propagation of both cell-associated and cell-free varicella- zoster virus (VZV). The medium was supplemented with serum-free supplements (HITE: human serum albumin, insulin, transferrin, and monoethanolamine) in a mixture of basal serum-free medium (a mixture of DME and Ham's F12 medium, 1: 1). We have also investigated the effects of lipid supplementation and low serum addition for promotion of virus propagation in serum-free medium supplemented with HITE. Although the serum-free medium was not adequate for LuMA cell growth, the medium supplemented with concentrated lipid emulsion (CLE) was comparable to DME medium supplemented with 2% fetal bovine serum (FBS) in respect of VZV propagation.

Evaluation of the Efficacy of a Pre-pandemic H5N1 Vaccine (MG1109) in Mouse and Ferret Models

송민석,문호진,권혁일,Philippe Noriel Q. Pascua,이준한,백윤희,우규진,최주희,이상호,유현승,오인경,윤엽,노종복,성문희,홍승표,김철중,최영기 한국미생물학회 2012 The journal of microbiology Vol.50 No.3

The threat of a highly pathogenic avian influenza (HPAI) H5N1 virus causing the next pandemic remains a major concern. In this study, we evaluated the immunogenicity and efficacy of an inactivated whole-virus H5N1 pre-pandemic vaccine (MG1109) formulated by Green Cross Co., Ltd containing the hemagglutinin (HA) and neuraminidase (NA) genes of the clade 1 A/Vietnam/1194/04 virus in the backbone of A/Puerto Rico/8/34 (RgVietNam/04xPR8/34). Administration of the MG1109 vaccine (2-doses) in mice and ferrets elicited high HI and SN titers in a dose-dependent manner against the homologous (RgVietNam/04xPR8/34) and various heterologous H5N1 strai(RgKor/W149/06xPR8/34,RgCambodia/ 04xPR8/34,RgGuangxi/05xPR8/34), including a heterosubtypic H5N2 (A/Aquatic bird/orea/W81/05) virus. However, efficient cross-reactivity was not observed against heterosubtypic H9N2 (A/Ck/Korea/H0802/08) and H1N1 (PR/8/34) viruses. Mice immunized with 1.9 μg HA/dose of MG1109 were completely protected from lethal challenge with heterologous wild-type HPAI H5N1 A/EM/Korea/W149/06 (clade 2.2) and mouse-adapted H5N2 viruses. Furthermore, ferrets administered at least 3.8 μg HA/dose efficiently suppressed virus growth in the upper respiratory tract and lungs. Vaccinated mice and ferrets also demonstrated attenuation of clinical disease signs and limited virus spread to other organs. Thus, this vaccine provided immunogenic responses in mouse and ferret models even against challenge with heterologous HPAI H5N1 and H5N2 viruses. Since the specific strain of HPAI H5N1 virus that would potentially cause the next outbreak is unknown, pre-pandemic vaccine preparation that could provide crossprotection against various H5 strains could be a useful approach in the selection of promising candidate vaccines in the future.