Cloning and Expression of the Pasteurella haemolytica A1 leukotoxin Gene in Bacillus subtilis

유한상,Yoo, Han-sang,Maheswaran, Samuel K. The Korean Society of Veterinary Science 1996 大韓獸醫學會誌 Vol.36 No.3

Bovine Pneumonic Pasteurellosis는 수송열(輸送熱)로 일반적으로 알려져 있는 질병으로서, 여러가지 요인의 복합적(複合的)인 작용에 의해 발병하는 것으로 알려져 있으나, Pasteurella haemolytica A1이 가장 주요(主要)한 인자(因子)로 밝혀져 있다. P haemolytica A1은 leukotoxin(LKT), lipopolysaccharide(LPS), capsular polysaccharide 등 여러가지의 병원성인자(病原性因子)을 생성한다. 이들 인자중 LKT가 가장 중요한 병원성인자로 밝혀져 있다. 이에 본 실험은 P haemolytical A1의 LKT 유전자를 Bacillus subtilis에서 발현(發現)시킴으로서 LPS에 오염(汚染)되지 않은 LKT을 대량으로 생산할 목적으로 실시되었다. 실험의 첫 단계(段階)로서 pLKT52 plasmid을 Sau3 A1의 제한효소을 이용하여 부분소화(部分消化)시킨 후 이 부분 소화(消化)된 유전자들로부터 3~5kb 크기의 유전자들을 순수분리하여 pUC18와 결합시킨 후 E coli NM522에 형질전환(形質轉換)시켰다. 이때 형질전환된 균주들은 LKT에 대한 단크론 항체인 MAb601을 이용하여 colony blot 법에 의해서 LKT 유전자 보유 및 발현여부(發現與否)을 조사하였다. 이들 양성 clone들은 제한효소분석(制限酵素分析), 염기서열분석(鹽基序列分析) 및 Western blot 등에 의해서 재확인(再確認)하였다. 총 9개의 양성 clone중 위의 방법에 의해서 한 clone을 선택(選擇)하여 lktCA insert를 재분리하여 shuttle vector에 subcloning 하였다. Subcloning된 LKT 유전자들은 shuttle vector의 종류(種類)(pHPS9, p602/20, pHPS9-Sac)와 각기(各其) 다른 종류(種類)의 B subtilis(spoO12A, BR121, WB3O, Raj1105) 숙주내(宿主內)에서 발현정도를 Western blot 법에 의해서 비교(比較)하였다. 이때 최적발현조건(最適發現條件)은 p602/20와 pBL1의 dual plasmid system을 이용하여 B subtilis spoO12A에서 2시간동안 IPTG로 발현을 유도(誘導)하는 것이었다. B subtilis에서 발현된 LKT을 visual 법과 neutral red uptake 법을 이용하여 소 폐포(肺胞) 대식구(大食求)에 대한 biological activity를 확인하였다. 발현된 LKT에 대한 LPS 오염은 LKT을 SDS-PAGE 후 silver stain에 의해서 확인하였다. 본 실험을 통해서 볼 때에 lktCA 유전자를 보유(保有)하고 있는 p602/20는 B subtilis에서 매우 불안정(不安定)하였고, 발현된 LKT는 세균자체(細菌自體)에서 생성되는 protease들에 의해서 파괴(破壞)됨으로서 농도(濃度)가 매우 낮았다. 이러한 문제점들은 다음 단계(段階)의 실험에서 해결되어야할 문제들이다.

Escherichia coli의 pBR322 DNA 형질전환에 관여하는 인자에 관한 연구

유한상,마점술,Yoo, Han-sang,Mah, Jum-sool 대한수의학회 1984 大韓獸醫學會誌 Vol.24 No.1

To investigate the factors influencing the artifical transformation in Escherichia coli, E. coli C600 was transformed by pBR322 DNA with tetracycline and ampicillin resistant gene purified by CsCl-Etbr equilibrium density gradient centrifugation from E.coli HB 101. The influencing factors in the transformation such as concentration of calcium chloride, time of ice incubation, temperature and time of heat shock, time of gene expression, effects of plasmid DNA concentration and adding time were examined in these experiments. The results obtained were as follows; 1. The highest transformation frequency was observed in the treatments of 100 mM $CaCl_2$ before heat shock and the treatment of $CaCl_2$ was essential step in the process of E. coli transformation. 2. The highest transformation frequency was observed in the treatment of heat shock at $42^{\circ}C$ for 4 min. or $37^{\circ}C$ for 6 min., but the prolonged heat shock resulted a decreased transformation frequency. 3. Treatments of ice incubation at $0^{\circ}C$ for 45 min. before heat stocks or at $0^{\circ}C$ for 30min. after heat shock resulted an increased transformation frequency. 4. There was a linear relationship between DNA concentration and transformation frequency at the concentration of $8{\times}10^3$ recipient cells. The highest transformation frequency reached in carte of 7 mcg of donor DNA, but above 1 mcg of DNA concentration, transformation frequency was not remarkably increased. Addition of donor DNA just after the treatment of $CaCl_2$ was the best. 5. The best condition of gene expression at $37^{\circ}C$ were 40min. for TC-resistant gene and 100min. for AP-resistant gene. TC-resistant gene was higher in the transformation frequency and faster in the gene expression time than AP-resistant gene. In these results, the best conditions for the transformation of E. coli C 600 with pBR322 DNA were: treatment with 100mM $CaCl_2$, ice incubation at $0^{\circ}C$ for 45 min, heat shock at $42^{\circ}C$ for 4 min., 30 min. of ice incubation and incubation at $37^{\circ}C$ for 100min. for gene expression in that order.

병자호란 전후(1636-1638) 소의 역병(牛疫) 발생과 확산의 국제성

김동진(KIM Dong Jin),유한상(YOO Han Sang) 대한의사학회 2013 醫史學 Vol.22 No.1

This study reviewed the outbreak and prevalence of cattle plague around Byeongjahoran from the perspective of international war in East Asia. First of all, the epidemiological characteristics of cattle plague in Manchuria where the outbreak of cattle plague was first reported around Byeongjahoran were analyzed. From the study, it was found the military activities that Sarhu (Qing) had made during the growth into Empire promoted the exchanges of various germs which became naturalized in the regions in Northeast Asia, and that such extreme situation as war made various diseases taken place and dispersed. In particular, because of military activities of Sarhu (Qing), various contagious diseases including smallpox which was prevalent in Inner-Mongolia and Shanxi became prevalent in Manchuria. During the contacts with Chosun after Jeongmyohoran, pathogen occurring Rinderpest was introduced into Manchuria. Favorable conditions for the interactions with various pathogens were provided by frequent contacts with wild animals through hunting and various cultivation groups composed of Manchurians, Mongolians, Han-Chinese and Chosun people. Rinderpest breaking in Chosun around Byeongjahoran was originated in Shenyang in 1636. It was transmitted to cattle in the Korean Peninsula and expanded to Kansai Region. At that time Rinderpest occurred and rapidly expanded in a specific area due to the interactions of pathogens, hosts and environments, and suddenly disappeared because of the extinction and the separation of hosts. It is consistent with the symptoms of modern times ‘Rinderpest.’ In Chosun it occurred in Pyeongan-do 4 months before the outbreak of Byeongjahoran and gave great damage on the capital area and northern Gyeonggi region. Because of the large scale migration of militaries after Byeongjahoran, Rindpest was expanded to Hasamdo and was terminated in February to April leaving big damages. The damages of Byeongjahoran were very severe. From the statistical records, it was found that the mortality rate in Gyenggi-do was around 2/3, around 50% in Jeju area. The mortality rate of infected cattle was around 75%. In some records based on individual cases, 80-100% of mortality rate was addressed. It is comparable to 25% of mortality in 1627, and is near or less than the mortality rate of Rinderpest in the 19th and 20the century. When analyzing the expansion of Rinderpest from the perspective of place, the most damaged places were areas near the busy roads or the places with dense population. Therefore, the remote places far from busy roads or separated from the affected places right after the outbreak did not have much damage. Additionally, rich stock-feeders (豪民) had relatively small damages and poor households (小民) with 1 or 2 stokes were badly affected. The prevention and supply of medication by government made considerably positive effects on the prevention and treatment of Finderpest.

FSAE 2010 참관기

유한상(Han sang Yoo),이성욱(Seang wock Lee) 한국자동차공학회 2010 오토저널 Vol.32 No.4

Effective methods for the production of immunoglobulin Y using immunogens ofBordetella bronchiseptica, Pasteurella multocida and Actinobacillus pleuropneumoniae

Han Sang Yoo,InSooChoi,JongManKim,WonHur,유한상 대한수의학회 2002 Journal of Veterinary Science Vol.3 No.1

Swine respiratory diseases induce severe conomicloses in the swine industry worldwide. Severalmethods have ben developed and applied to controlthese diseases. However, there are stil problems ofdisease control in the swine industry. Recently, egyolk antibodies have ben found to offer several ad-vantages for disease control in animals and humans.In a previous tudy (24), antibodies to severaldeveloped. However, several problems remained,especialy in terms of reduced laying rates. Therefore,experimental vacines were reformulated with variousbacterial antigens of the swine respiratory diseases.After imunizing hens with the antigens, antibodyprofiles and other effects including laying rates wereinvestigated and compared to those of the previousstudy.Profiles of antibody titers were very similar withthose of the previous study. However, side fects,such as depression, weakness, reduction of layinglaying rates were increased in hens injected withcertain experimental vacines. In particular, layingrates of hens injected with vaccines against atrophicrhinitis were increased up to 84% by injecting avaccine composed of only the DNTs of B. bron-chiseptica and P. multocida D:4. Efficacies of thevaccines against swine pneumonic pasteurelosis andpleuropneumonia were very similar with those of theprevious study. These results uggest hat newvacines could be fective in the production of egyolk antibodies against the causative agents of swine*Adress: Department of Infectious Disease, Colege of VeterinaryMedicine and School of Agricultural Biotechnology, Seoul NationalUniversity, Suwon, 441-744 KoreaTel: +82-31-290-2737, Fax: +82-31-290-2737E.mail: yoohs@plaza.snu.ac.krrespiratory diseases.Key words ; IgY, Swine, Respiratory Diseases.

Session 2: 위장관 바이러스성 질환의 국내 역학 및 특성 : 돼지 Hepatitis E virus 특성 및 국내 발생상황

유한상 ( Han Sang Yoo ) 대한인수공통전염병학회 2013 창립총회 및 학술대회 초록집 Vol.2013 No.1

Hepatitis E virus (HEV), first recognized in the early 1980s, causes a significant health problem around the world with large outbreaks of acute hepatitis. HEV is a non-enveloped, single-strand, positive-sense RNA virus of approximately 7.2kb in length. HEV is classified in the family of Hepeviridae consisting of at least 4 mammalian genotypes and one avian genotype. There are four major genotypes of HEV that are recognized in the genus Hepevirus: genotype 1 (Burmese-like Asian strains), genotype 2(a Mexican strain and various African strains), genotype 3 (human strains from sporadic cases in industrialized countries, and animal strains from pigs, deer, and mongeese), and genotype 4 (Asian strains from sporadic cases, and swine strains in both developing and industrialized countries). Genotypes 1 and 2are infected in humans with large waterborne epidemics in developing countries with poor sanitation conditions, whereas genotypes 3 and 4 found both human and other animals with sporadic cases in both developing and developed countries. Especially, genotypes 3 and 4 HEV often show similar nucleotide identity between human and animal isolates, suggesting zoonotic infection. The mortality of HEV is generally low (1%) but is up to 25% in pregnant women. Moreover, the sporadic cases of HEV occasionally progress to chronic hepatitis causing liver cirrhosis in immunosuppressive patients. Recently, evidence is emerging for the occurrence of zoonotic infection in non-endemic areas, and chronic HEV is increasing in transplant recipients in industrialized countries. In addition, the number of animal species infected with HEV is increasing, and now includes pigs, chickens, and several wild species. Also, there are several evidences indicating zoonotic transmission in genotypes 3 and 4, thus particular attention on development of diagnostic methods and vaccine should be paid to prevent HEV infection. Swine HEV infection is widespread throughout the pig industry in both developing and industrialized countries. The infection rate in pigs generally the highest at 2-3 months of age, although seroprevalence appears to increase with age. The swine HEV is transmitted through fecal-oral contamination, as with human HEV, and feces containing large amounts of the viruses are the main source of transmission. The first report on swine HEV in 2003 indicated that the detection of swine HEV RNA-positive pigs and anti-HEV IgG-positive pigs were 2.3% and 14.8%, respectively. After the report, the prevalence was increasing by the year. At present, the seroprevalence of anti-HEV IgG was 39.5% and 80% in individual pigs and herd level prevalence, respectively. Also, anti-HEV antibody was detected in cats and zoo animals. Clinical signs of swine HEV-infected pigs, the infection are ubiquitous, as it exists in essentially all swine-producing regions worldwide. Pigs naturally infected with swine HEV are asymptomatic. In a prospective study of naturally infected piglets in the USA, gross pathogological lesions were not detected in pigs during early stage of swine HEV infection, although the piglets had microscopic evidence of hepatitis characterized by mild to moderate multifocal and periprotal lymphoplasmacytic hepatitis with mild focal hepatocellular necrosis. Similar observation was reported in the pigs experimentally infected with a genotype 3 strain of human HEV. HEV is encoded by 3 open reading frames (ORFs) in the genome. The ORF1 encodes nonstructural proteins including methyltransferases, papain-like cysteine proteases, helicase andRNA-dependent RNA polymerases. The ORF2and ORF3 encode highly immunogenic capsid proteins and cytoskeleton-associated phosphoprotein, respectively. The clinical symptoms of human hepatitis E could not be distinguished from other types of acute viral hepatitis, and thus accurate diagnosis of hepatitis E must rely on laboratory testing. HEV is a difficult virus to work with, as it cannot be efficiently propagated in cell cultures. Currently, the diagnosis of HEV infection is primarily based on PCR and ELISA. Therefore, a lot of RT PCR and ELISA methods were developed, but none has been approved by any international regulatory agency due to the absence of appropriate gold standards. The vaccine based on capsid protein showed good efficacy, but there is still limitations to prevent emerging zoonotic strains of HEV. Therefore, the study of accurate and well-validated diagnostic tests and development of vaccines against the animal strains for HEV may minimize the risks of zoonotic transmission. HEV is an important but still extremely understudied zoonotic pathogen. The evidences of interspecies infection between swine and human and recovery of viruses resembling swine HEV from human patients with acute hepatitis E raise a serious concern for zoonotic HEV infection. The recent discovery of avian HEV and other anti-HEV antibody positive animals suggest that there likely exist other animal reservoirs for HEV in addition to pigs. Therefore, HEV poses a potential public health risk for zoonosis and food safety. Also, development of an affordable vaccine against HEV will be needed by further research.

특집 : 산업동물에서 Zoonosis 연구동향 ; 소 결핵병의 국제 연구 동향

유한상 ( Han Sang Yoo ) 한국수의공중보건학회 2007 예방수의학회지 Vol.31 No.2

Field evaluation of a subunit vaccine against porcine pleuropneumoniae in Korea

유한상(Han Sang Yoo),박주연(Joo Young Park),석재필(Jae Phil Seok) 한국예방수의학회 1998 예방수의학회지 Vol.22 No.4

돼지 흉막폐렴은 임상증상에 따라서 다양한 질병의 경과와 높은 이환율 및 폐사율을 나타내는 돼지 주요한 전염성 호흡기 질병으로 전세계적으로 양돈업에 막대한 경제적인 피해를 주고 있다. 그러므로, 이 질병을 예방 및 근절을 위하여 다양한 노력이 시도되었으며, 이중 vaccine의 개발이 가장 활발 진행되어, 균체를 이용한 방법으로부터 시작하여 다양하게 개발되어 왔다. 특히, 이 질병은 원인체인 Actinobacillus pleuropneumoniae 혈청형 및 지역에 따라서 병원성에 많은 차이를 나타냄으로, 이 질병 예방을 위해 개발된 예방약의 전반적인 적용에 앞서서 새로운 지역적인 특성에서의 효능성에 대하여 사전실험이 요구되어왔다. 이에, 최근 개발되어 유럽 등에서 사용되고 있는 vaccine 중, A. pleuropneumoniae의 주요한 병원성인자인 ApxⅠ, ApxⅡ, ApxⅢ 및 42kDa 크기의 세포외막 단백질 (OMP)로 구성되어 있는 subunit vaccine을 국내에서 사용하기 전에, 국내 야외 농장에서의 안전성, 면역원성 및 야외 질병방어효능을 조사하였다. 안전성에서 기존량의 2배량을 접종하고 국소 또는 전신반응의 발생 유무를 관찰한 결과 어떠한 접종 반응도 나타내지 않았으며, ELISA 법을 이용하여 각각에 대한 항체가를 측정한 결과 접종후부터 각각에 대한 항체가의 상승이 확인되었으며, 접종 7주후에 ApxⅠ 및 ApxⅢ에 대한 항체가가 접종군과 대조군 사이에 뚜렷한 차이를 나타내었다(P<0.05). 또한 질병 발생율에서 백신군이 대조군에 비하여 뚜렷한 감소를 보였다. 이상의 결과를 바탕으로 볼 때에 이 subunit vaccine은 야외에서 안전성, 면역원성 및 질병 방어 효과가 인정됨으로 국내에서 돼지 흉막폐렴의 예방 및 근절에 많은 도움을 줄 것으로 사료된다.

광우병, AI 등 인수공통 전염병 방제기술 개발 : 국내, 외 광우병의 발생 현황과 대응 방안

유한상 ( Han Sang Yoo ) 한국환경농학회 2009 한국환경농학회 학술대회집 Vol.2009 No.2

The Molecular Response to Prion Infection in Mice

Han Sang Yoo(유한상) 한국실험동물학회 2009 한국실험동물학회 학술발표대회 논문집 Vol.2009 No.-