hwp

Size: px

Start display at page:

Download "hwp"

세윤 상
6 years ago
Views:

1 유전자재조합단백질 Adenylate Kinase, Nucleoside Diphosphate Kinase 와 Heat-Shock Protein 7 의결핵균에대한방어면역효능분석 대한결핵협회결핵연구원, 연세대학교의과대학미생물학교실이승헌, 이은계, 김수연, 조상래, 박영길, 배길한 Protective Efficacy of Recombinant Proteins Adenylate Kinase, Nucleoside Diphosphate Kinase, and Heat-Shock Protein 7 against Mycobacterium tuberculosis Infection in Mice Seung-Heon Lee, Eun-Gae Lee, Su-Yeon Kim, Sang-Nae Cho, Young-Kil Park, Gill-Han Bai Department of Molecular Biology, Korean Institute of Tuberculosis, Seoul, Korea, Department of Microbiology, Yonsei University College of Medicine, Seoul, Korea Background : Priming and boosting vaccination strategy has been widely explored for new vaccine development against tuberculosis. As an effort to identify other vaccine candidates, this study was initiated to evaluate protective efficacy of adenylate kinase (AK), nucleoside diphosphate kinase (NdK), and heat shock protein 7 (Hsp7) of Mycobacterium tuberculosis. Method : M. tuberculosis genes encoding AK, NdK, and Hsp7 proteins were amplified by PCR and cloned into E. coli expression vector, pqe3. Recombinant AK, NdK, and Hsp7 was purified through Ni-NTA resin. To evaluate immune responses, we performed enzyme-linked immunosorbent assay (ELISA) for IgG isotype and IFN-γ after mice were immunized subcutaneously with recombinant proteins delivered in dimethyl dioctadecylammonium bromide (DDA). Immunized- and control groups were challenged by aerosol with M. tuberculosis. The spleens and lungs of mice were removed aseptically and cultured for CFU of M. tuberculosis. Result : Vaccination with recombinant proteins AK, NdK, and Hsp7 delivered in DDA elicited significant level of antibody and IFN-γ responses to corresponding antigens but no protective immunity comparable to that achieved with Mycobacterium bovis. Conclusion : Recombinant proteins AK, NdK, and Hsp7 do not effectively control growth of M. tuberculosis in mice when immunized with DDA as an adjuvant. (Tuberc Respir Dis 5; 58:4-5) Key words : Adenylate kinase, Nucleoside diphosphate kinase, Heat shock protein7, Mycobacterium tuberculosis, Protective efficacy 서 세계적으로한해 9 만명이상의사망자가발생하는결핵은여전히주요보건문제의하나로남아있으며, HIV/AIDS의확산과다제내성 (multidrug-res istant) 결핵의출현은결핵문제를더욱심각하게하고있다. 현재전세계적으로신생아들에게접종되고 본연구는보건복지부보건의료기술연구개발사업의지원에의하여이루어진것임 (3-PJ-PG-4-). Address for correspondence : Gill-Han Bai Ph.D. Korean Institute of Tuberculosis 4 Woomyundong, Sochogu, Seoul, 37-4, Korea Phone : Fax : gbai@hotmail.com Received : Dec.. 4 Accepted : Jan 론 있는결핵예방백신은우형결핵균의독성을약하게만든 (Bacille Calmette-Guérin) 이지만결핵에대한예방효과는 ~8% 로보고되고있어보다개선된효능의백신이필요하다. 근래에결핵균 (Mycobacterium tuberculosis) 에대한유전자의염기서열이분석됨으로써결핵균에대한 T세포항원을이용한새로운백신개발이여러차례시도되고있다 3. 이새로운 T 세포항원중에 adenylate kinase (AK) 는 ATP와 AMP사이에서가역적고에너지인산화작용을촉매하여 ADP를생성하는효소 (Mg. ATP + AMP Mg. ADP) 로서에너지물질대사와 nucleotide 합성에관여한다. Markaryan 등은 ATP가존재한상태에서 Pseudomonas aeruginosa의 AK가대식세포 (macrophage) 의사멸을유도하는독성인자임을증명하였고 4, Lehmann 등은결핵균의 AK가짧은단편으 4

2 Tuberculosis and Respiratory Diseases Vol. 58. No., Feb, 5 로구성된세균성 AK의새로운아과 (subfamily) 에포함된다고보고하였다 5. ATP를기질로이용하는효소로서 nucleoside di phosphate kinase (NdK) 는 nucleoside (NTP) 혹은 deoxynucleoside triphosphate (dntp) 들의 γ-인산기 (phosphate) 를 nucleoside (NDP) 혹은 deoxynucleoside diphosphate (dndp) 들에전이함으로써 NTP와 dntp 들을생성하는중요한효소로서성장, 신호전달계와병원성에중요한역할을한다 6. 결핵균의 NdK는 X-ray 구조상짧은 polypeptide chain 으로구성된.6A 의매우안전한육량체로서 7, ATP에의한마우스대식세포의사멸을유도한다는보고가있다 8. 항원 7kDa는 M. leprae 와 M. bovis 의배양추출물에서처음동정되었으며, 결핵균의항원 7kDa 과교차반응이일어나는것으로보고되었다 9,. 이들항원들은사람의 T 세포면역원으로알려져있으며, 이에대한근거로 의 7kDa 단백질이 를면역주사한사람과 tuberculoid leprosy 환자에서 T 세포반응을일으킨다는보고가있다,. 또한이들단백질들은염기서열분석을통하여 7kDa Heat shock protein (Hsp7) 종류의단백질로서인식되어오고있으며, D- 전기영동을이용하여동정된결핵균의단백질중에서 Hsp7은진단및백신개발에이용할결핵균항원후보물질로서제안되어왔다 3. 본실험에서는독성이약하고세포성면역반응 (cellmediated immunity) 을유도하는 dimethyl dioctade cylammonium bromide (DDA) 면역보강제를이용하여결핵균 AK, NdK와 Hsp7 가백신후보물질로서가능한지를알아보고자하였다. 대상및방법. 마우스특이적병원성이없는 6-7주된 C57BL/6 암컷마우스를구입하였으며, 연세대학교임상의학센터 BL-3 biohazard 동물실에유치하였다.. 균주 Mycobacterium tuberculosis H37Rv 와 Mycobacterium bovis Pasteur 73P 는 Saution 배지를이용하여 37 에서정체상태로배양하였다. 3. 재조합단백질들의클로닝및정제 ak (rv733), ndk (rv445c) 와 hsp7 (rv35) 유전자를포함한 M. tuberculosis H37Rv 게놈을주형으로클로닝에이용할제한효소염기서열이삽입된 primer 들을이용하여중합효소연쇄반응하였다. 중합효소연쇄반응산물을 pdrive T-vector (QIAGEN, Hilden, German) 에클로닝한후, 염기서열분석법을통하여중합효소연쇄반응산물 (pdrive:ak, ndk, hsp7) 을확인하였다. 확인된이들클론들에서중합효소연쇄반응산물을각각의제한효소를이용하여분리하고, 동일한제한효소로절단된 E.coli 발현벡터 pqe3 (QIAGEN, Hilden, German) 에삽입하였다. 이재조합플라스미드를 E.coli M5에형질전환하였고, 재조합단백질들을 QIAGEN 실험방법을이용하여 Ni-NTA resin을이용한친화성크로마토그래피 (affinity chromatography) 법으로분리하였다. 4. 면역법실험방법은 Andersen 4 의방법을기초로변형하여실시하였다. 생리식염수 μl에 DDA (5 μg/dose; Sigma, St. Louis, MO, USA) 와재조합단백질 (4 μ g/dose) 들을혼합하여 주간격으로세번에걸쳐마우스 C57BL/6의등에피하주사하였다. 양성대조군으로는 Pasteur 73P 균주 ( x 6 CFU) 를동일한위치에한번주사하였다. 5. 체액성면역반응 (humoral immune response) 마지막면역주사후, 4, 8, 주째에마우스로부터혈청을분리하여효소면역측정법 (enzyme-linked im munosorbent assay, ELISA) 을이용하여항체생성을통한체액성면역반응을알아보았다..5 M bicarbonate 완충액 (ph 9.6) 에각각의재조합단백질혹은결핵균 CFP (culture filtrate protein; 43

3 SH Lee, et al.: Protective efficacy of recombinant proteins AK, NdK, and Hsp7 against Mycobacterium tuberculosis infection in mice μg/ml) 를첨가하여 4 에서 6시간방치함으로써 96-well microtiter 판 (Costar, Cambridge, MA) 에코팅 (coating) 하였다..5 % Tween 이포함된 PBS (PBST) 용액을이용하여 microtiter 판을세척한후 % fatal bovine serum이포함된 PBS 용액 ( % FBS-PBS) 을첨가하고 37 에서 시간방치하였다. 다시 PBST 용액으로세척한 microtiter 판에 % FBS-PBS 용액에 / 희석한혈청을첨가하고 37 에서 시간혹은 4 에서 6시간방치한뒤다시 PBST 용액으로세척하였다. 여기에 biotinated antimouse IgG (Gibco BRL, Rockville, MD, USA) 를첨가하여 37 에서 시간반응시키고, PBST 용액으로세척한후 anti-biotin-horseradish peroxidase (HRP; Sigma, St. Louis, MO, USA) 를첨가하고 37 에서 시간반응시켰다. PBST 용액으로 microtiter 판을여러차례세척하고 ortho-phenylene-diamine (.4 mg/ml) 과 3 % H O (.4 μl/ml) 가혼합된.5 M citrate butter (ph 5.) 를첨가하여 37 에서 5분간암실에서반응시킨뒤.5 N H SO 4 를첨가하여반응을종결시켰다. 반응결과는 49 nm optical density에서 ELISA 판독기를이용하여측정하였다. 위와동일한방법으로 biotinated anti-mouse IgG 과 IgGa (Serotec, Raleigh, NC, USA) 를이용하여항체 isotype 생성능도실시하였다. 위실험들은마지막면역주사후, 4, 8, 주째에각그룹당 3마리마우스에서분리한혈청을이용하여 회실시하였다. 6. 비장세포분리및 IFN-γ 생성능측정마지막면역주사후, 8, 주째에마우스를사멸하고비장을무균적으로분리하였다. 3마리마우스에서분리한세포를모으고, 5 μm -mercaptoethanol, % penicillin-streptomycin, mm glutamate와 %(v/v) fetal calf serum이포함된 RPMI 64배지에 well당 x 6 량의비장세포와재조합단백질 ( μ g/ml) 혹은결핵균 CFP ( μg/ml) 을첨가하고 37, CO 에서배양하였다. 배양 6일후배양상층액을채취하여 mouse IFN-γ OptEIATM set (Pharmingen, San Diego, CA, USA) 를이용하여 IFN-γ 생성능을측정하였다. 위실험들은마지막면역주사후, 8, 주째에각그룹당 3마리마우스에서분리한비장세포를이용하여 회실시하였다. 7. 결핵균의공기감염 (aerosol challenge) 및방어면역효능 (protective efficacy) 마지막면역주사후, 주째에 M. tuberculosis H37Rv 5 x 6 cell/5 ml을각그룹당마우스 마리씩공기감염시켰다. 이는마우스의폐당 4 결핵균수를감염시킬수있는양이었다. 공기감염후 6 주, 8주째에각그룹당 5마리마우스의폐와비장을무균적으로분리하여동질분쇄하였다. 결핵균수를확인하기위하여동질분쇄한조직액을희석하고 7H 배지 (Difco, Sparks, MD, USA) 에분주하여 37 에서 3주간배양하였다. 각그룹간의결핵균수의차이는 Student's t-test 에의해신뢰수준 95% 범위에서의유의성 (P<.5) 을통계학적으로분석하였다. 결과. 항체생성능마지막면역주사후, 4, 8, 주째에마우스로부터혈청을분리하고효소면역측정법을이용하여항체생성을통한체액성면역반응을알아보았다. 재조합단백질을면역주사한마우스에서각단백질에대한 IgG 생성능은높은항체생성능을나타내면서조금씩감소하였다. 특히 를면역주사한마우스에서는 4주째의반응에서 AK와 NdK에대한항체생성능이높게나타났으나 8주째에급격히감소하였다. IgG과 IgGa 에대한생성능에서는 IgGa 에대한생성능에비하여 IgG 항체생성능이매우높게나타났다 (Fig. -3). 결핵균 CFP에대한항체반응에서는 Hsp7 을면역주사한마우스에서만 IgG와 IgG 생성능이높게 44

4 Tuberculosis and Respiratory Diseases Vol. 58. No., Feb, 5 나타났다 (Fig.4).. IFN-γ 생성능 AK 에대한 IFN-γ 생성능에서는, 마지막면역주사 후 8주째에 를면역주사한마우스에서음성대조군인생리식염수와 DDA를면역주사한마우스와비교하여통계적으로 IFN-γ 의생성이증가되었고 (P<.), 주째에서는증가된양상을나타내지못하였다. 재조합단백질 AK를면역주사한마우스에서는 A A.5.5 O.D 49nm.5 O.D 49nm.5 saline DDA Hsp7 saline DDA AK B B.5.5 O.D 49nm.5 O.D 49nm.5 Hsp7 AK C C.5.5 O.D 49nm.5 O.D 49nm.5 Hsp7 AK Figure. IgG isotype responses to Hsp7 induced by experimental vaccine. Mice were immunized three times with recombinant Hsp7 in dimethyl dioctadecyl ammounium bromide(dda) or received a vacci nation. Four, eight and twelve weeks after the last immunization, serum from three mice in each group was pooled, specific A) IgG, B) IgG, and C) IgGa levels against Hsp7 were determined. Each bar repr esents the mean of triplicate values ± standard error of the mean. :4wk, :8wk, and :wk after the last immunization Figure. IgG isotype responses to AK induced by experimental vaccine. Mice were immunized three times with recombinant AK in dimethyl dioctadecyl ammounium bromide(dda) or received a vacci nation. Four, eight and twelve weeks after the last immunization, serum from three mice in each group was pooled, specific A) IgG, B) IgG, and C) IgGa levels against AK were determined. Each bar repre sents the mean of triplicate values ± standard error of the mean. :4wk, :8wk, and :wk after the last immunization 45

5 SH Lee, et al.: Protective efficacy of recombinant proteins AK, NdK, and Hsp7 against Mycobacterium tuberculosis infection in mice 생리식염수를면역주사한마우스와비교하여면역주사후 8주째 (P<.5) 와 주째 (P<.) 에서 IFN-γ 의생성이증가되었고, DDA를면역주사한마우스와비교에서는 주째에증가되었다 (P<.5). 와재조합단백질 AK를면역주사한마우스간의비교에서는 A.5 면역주사후 8주째에는 를면역주사한마우스에서 IFN-γ 의생성이증가되었고 (P<.), 주째에는재조합단백질 AK를면역주사한마우스에서 IFN-γ 의생성이증가되었다 (P<.5). NdK에대한 IFN-γ 생성능에서는, 마지막면역주사후 8주째에 를면역주사한마우스에서생리식염수와 DDA를면역주사한마우스와비교하여통계적으로 IFN-γ 의생성이증가되었고 (P<.), 주 O.D 49nm A..5 saline DDA NdK O.D 49nm.8.4 B saline DDA AK NdK Hsp7.5 B O.D 49nm..5 NdK O.D 49nm.8.4 C AK N dk Hsp7.5 C O.D 49nm.5 O.D 49nm..8.4 NdK AK NdK Hsp7 Figure 3. IgG isotype responses to NdK induced by experimental vaccine. Mice were immunized three times with recombinant NdK in dimethyl dioctadecyl ammounium bromide(dda) or received a vaccin ation. Four, eight and twelve weeks after the last immunization, serum from three mice in each group was pooled, specific A) IgG, B) IgG, and C) IgGa levels against NdK were determined. Each bar repre sents the mean of triplicate values ± standard error of the mean. :4wk, :8wk, and :wk after the last immunization Figure 4. IgG isotype responses to CFP induced by experimental vaccine. Mice were immunized three times with recombinant proteins in dimethyl dioctadecyl ammounium bromide(dda) or received a vacci nation. Four, eight and twelve weeks after the last immunization, serum from three mice in each group was pooled, specific A) IgG, B) IgG, and C) IgGa levels against M. tuberculosis CFP were determined. Each bar represents the mean of triplicate values ± standard error of the mean. :4wk, :8wk, and :wk after the last immunization 46

6 Tuberculosis and Respiratory Diseases Vol. 58. No., Feb, 5 째에서는증가된양상을나타내지못하였다. 재조합단백질 NdK를면역주사한마우스에서는면역주사후 8주째에생리식염수 (P<.) 와 DDA(P<.5) 를면역주사한마우스와비교하여 IFN-γ 의생성이증가되었으며 와재조합단백질 NdK를면역주사한마우스간의비교에서는증가된양상을나타내지못하였다. Hsp7에대한 IFN-γ 생성능에서는, 마지막면역주사후 8주째에 를면역주사한마우스에서생리식염수와 DDA를면역주사한마우스와비교하여통계적으로 IFN-γ 의생성이증가되었고 (P<.), 주째에서는증가된양상을나타내지못하였다. 재조합단백질 Hsp7 을면역주사한마우스에서는면역주사후 8주째와 주째에생리식염수와 DDA를면역주사 한마우스와비교하여 IFN-γ 의생성이증가되었고 (P<.), 와재조합단백질 Hsp7을면역주사한마우스간의비교에서는면역주사후 주째에재조합단백질 Hsp7을면역주사한마우스에서 IFN-γ 의생성이증가되었다 (P<.). 결핵균 CFP(culture filtrate protein) 에대한 IFN-γ 생성능에서는, 를면역주사한마우스에만면역주사후 8주째 (P<.) 와 주째 (P<.) 에서 IFN-γ 의생성이증가되었다 (Fig.5). 3. 결핵균에대한방어면역효능결핵균에대한방어면역효능에서는생리식염수를면역주사한마우스의결핵균수와비교한결과, 신뢰 A AK B NdK 3 3 IFN-r (pg/ml) * IFN-r (pg/ml) saline DDA AK * saline DDA NdK C Hsp7 D CFP IFN-r (pg/ml) IFN-r (pg/ml) saline DDA Hsp7 saline DDA AK NdK Hsp7 Figure 5. IFN-γ production by splenocyte of immunized mice. Spleen cells prepared from mice eight and twelve weeks after last immunization. Cells were pooled from three mice per group and restimulated in vitro with recombinant proteins or M. tuberculosis CFP. Each point represents the mean of triplicate values ± standard error of the mean. *, P<.5;, P<. compared to controls or -immunized mice. :8wk, and :wk after the last immunization 47

7 SH Lee, et al.: Protective efficacy of recombinant proteins AK, NdK, and Hsp7 against Mycobacterium tuberculosis infection in mice Table. Protective efficacy of recombinant proteins emulsified in DDA in a mouse model of tuberculosis. Vaccine groupa Mean log CFU of M. tuberculosis ± SEM (n=3-4) b Exp Exp Lung Spleen Lung Spleen Saline 5.6± ± ± ±.5 DDA 5.85±.4 4.6± ± ± ±.5 4.3±.6(P=.5) c 5.54±.34(P=.6) 4.84±.(P=.59) AK 5.7± ±.3 6.5± ±. NdK 5.57± ± ± ±.54 Hsp7 5.6± ±.5 5.6±.8(P=.5) 4.95±.37 a. C57BL/6 mice were immunized once s.c with ( x 6 CFU) or injected three times with the experimental vaccines emulsified in DDA. b. Number of bacteria isolated from the lung and spleen 6 weeks (Experiment ) and 8 weeks (Experiment ) after aerosol challenge. c. P values are given for bacterial loads different from those in control mice. 수준 95% 범위 (P<.5) 에서유의성이없어서결핵에대한재조합단백질들의백신후보물질로서의효능성을나타내지못하였다. 그러나생리식염수를면역주사한마우스와비교하여 를면역주사한마우스에서공기감염후, 6주째비장에서통계학적유의성경향을보였고 (P=.5), 재조합단백질 Hsp7 을면역주사한마우스에서도공기감염후 8주째폐에서유의성경향을나타내었다 (P=.5)(Table ). 고찰다수의실험들에의해결핵균의 CF (culture filtrate), 세포벽, 및세포질에포함된단백질들이높은면역원을지니고있어백신에이용할후보물질로서판명되고있다 5-7. 이들은 ESAT-6, 85 complex, 38kDa 등의단백질로서여러유형의백신연구에이용되고있다. 새로운백신후보물질의탐색을목적으로, 본실험은마우스에대한공기감염모델을이용하여결핵균에내재된 AK, NdK와 Hsp7 의결핵균에대한방어면역효능을측정하였다. 각단백질에대한 IgG 항체생성능은 를면역주사한마우스와비교하여재조합단백질을면역주사한마우스에서높게나타났으나, 결핵균 CFP에대한 IgG 항체생성능에서는 Hsp7을면역주사한마우스에서만높은생성능을나타내었다. 이는 CFP내에분포하는 AK와 NdK의양이매우적기때문인것으 로예측되었다. 또한 CD4+ T 세포의분화방향을알아보기위하여 IgG isotype 중 IgG과 IgGa 에대한생성능을확인한결과, IgG에대해서높은생성능을나타내어 Th 세포로분화되는것으로판단되었다. 그러나, 시간이경과함에따라 를주사한마우스는 IgGa/IgG 비율을비추어볼때 Th/Th 세포의혼합된양상으로분화하는것으로나타내었다. Lindbland 등은 C57BL/6J 마우스모델로결핵균 CFP와여러면역보강제를이용한백신실험에서 DDA를주사한마우스에서강한 IgGa 반응이나타나 Th 세포를자극한다고보고하였으나 8, 이전의보고에서는항원농도가높음에따라 IgG 의생성이두드러지게나타나 Th 세포를자극한다고보고하였다 9. Martin 은 C57BL/6 마우스모델을이용한백신실험에서 IgGa 생성능을조사하는점에대해서문제제기를하였는데 Igh-b allele 를포함한마우스 (C57BL /6, C57BL/, SJL등 ) 들은 IgGa의유전자가제거되어있고상품화된 anti-igga 혈청과반응을하지않는 IgGc isotype 을발현하기때문에 IgGa 생성능을측정하는것은불가능하다고보고하였다. 이러한내용을기초로, 본실험에서도 IgGb 항체생성능을측정하였으나오히려 IgGa 항체생성능에비해낮게나타내었다. IFN-γ 생성능의결과에서는재조합단백질들을면역주사한마우스에서각항원에대한 IFN-γ 생성이 48

8 Tuberculosis and Respiratory Diseases Vol. 58. No., Feb, 5 대조군과비교하여증가되었으나결핵균 CFP에대해서는 IFN-γ 생성이낮게나타나이들재조합단백질과 DDA 를이용한면역반응은세포성면역반응이아닌체액성면역반응으로일어나는것으로확인하였다. 면역효과를증가시키기위하여면역보강제 (adjuvant) 로세포성면역반응을유도하고낮은독성을지닌 DDA (dimethyl dioctadecylammonium bromide) 를선택하였다. 이면역보강제는결핵균의단백질백신연구에이용되고있으며 4,8,9,, 여러면역보강제와결핵균항원을이용한백신실험에서인체내에주로이용되는 Alum에비해결핵균에대한방어면역효능이높은것으로나타났다. 그러나본실험에서는결핵균에대한방어면역효능을나타내지못하였다. 이러한결과는백신후보물질로서의단일항원의면역원능과그에따른면역보강제의선택에따른것으로판단된다. 면역보강제의선정을목적으로한실험들에서는결핵균 CFP와 DDA를이용하여면역주사한마우스에서결핵균에대한방어면역효능을나타내었다 8,9. 그러나동일한면역보강제와단일항원을이용한백신실험에서는단백질의종류에따라상이한결과를나타내었는데, 항원 85B를이용한실험에서는결핵균에대한방어면역효능을나타낸반면, 항원 ESAT-6 를이용한실험에서는방어면역효능을나타내지못하였다. Brandt 등은항원 ESAT-6 의약한면역원능에대한보완책으로 DDA와 Th 면역반응을유도하는다른면역보강제를첨가함으로써방어면역효능을나타내었다 4. 결핵균 AK와 NdK는현재까지구조적특성및대사적기능에대한연구보고가있으며, 아직까지면역학적항원성에대한연구보고는없는실정이다. 최근에는재조합단백질 NdK와 ATP를대식세포에처리하였을때대식세포의사멸이유도된다는보고가있어병원성세균의감염경로등에중요한역할을할것으로예측되고있다 8. 결핵균 Hsp7은 proteomics 의발달에따라결핵균항원후보물질로서거론되어왔으며, Hsp7을과발현하는결핵균은 in vivo상에서생존율이감소하는것으로나타나면역반응에영향을미치는것으로보고되었다. 최근에는 Hsp7 등 Heat shock protein의항원 제시 (antigen-presentation) 기능이부각되면서감염된생체내에서분리한 Hsp-peptide 복합체를면역주사한뒤, 결핵균을감염시킨결과에서결핵균에대한방어면역효능을나타낸보고가있다 3. 본실험에서도재조합단백질 Hsp7을면역주사한마우스의폐에서결핵균에대한방어면역효능이 를면역주사한마우스와유사한결과를나타내었다. 이는 Hsp7의단일항원으로서의면역원능보다는다른기능에의한결과로판단된다. 현재재조합단백질백신연구는복합항원과복합면역보강제를이용한방법 4,, 새로운면역보강제의효능비교 4, 접종경로의다양화 5 및 cytokine 의첨가방법 6 등을이용한결핵균에대한방어면역효능을실시하고있다. 본실험에사용한재조합단백질들과 DDA를이용한면역효과는세포성면역반응이아닌체액성면역반응을유도함으로인해결핵균에대한방어면역효능을나타내지않았으므로, 단일항원으로서의면역원능이없거나혹은취약한것으로판단되었다. 따라서혼합단백질혹은다른 T세포면역보강제의사용에의한추시가필요하다. 요약배경 : 최근결핵에대한새로운백신개발은초회면역방법및추가면역방법을이용하는방향으로연구되고있다. 본실험은새로운백신후보물질로서의가능성을알아보기위하여결핵균 adenylate kinase (AK), nu cleoside diphosphate (NdK) 및 heat shock protein 7 (Hsp7) 의결핵균에대한방어면역효능을측정하였다. 방법 : 재조합단백질들을정제하기위하여중합효소연쇄반응으로증폭한결핵균유전자단편들을 E.coli expression vector, pqe3에클로닝한후, Ni-NTA resin을이용하여정제하였다. DDA와재조합단백질들을마우스에면역주사하고면역반응생성유무를확인하기위하여항체와 IFN-γ 생성능을측정하였다. 면역주사한마우스에결핵균을공기감염시킨후, 폐 49

9 SH Lee, et al.: Protective efficacy of recombinant proteins AK, NdK, and Hsp7 against Mycobacterium tuberculosis infection in mice 와비장을분리하여결핵균생균수실험을하였다. 결과 : 재조합단백질 AK, NdK 와 Hsp7을면역보강제인 DDA를이용하여면역주사한결과에서, 생리식염수혹은 DDA를면역주사한마우스에비교하여재조합단백질을면역주사한마우스에서는각항원에대해항체와 IFN-γ 생성능이높게나타났으나결핵균에대한효과적인방어면역효능은나타나지않았다. 결론 : 마우스를모델로한결핵균에대한방어면역효능실험에서, 면역보강제 DDA를이용한재조합단백질 AK, NdK 및 Hsp7 을면역주사한경우에는결핵균의성장을효과적으로조절하지못하였다. 혼합단백질혹은다른 T세포면역보강제의사용에의한추시가필요하다. 참고문헌. Olsen AW, Andersen P. A novel TB vaccine: strategies to combat a complex pathogen. Immunol Lett 3; 85:7-.. Fine PE. Variation in protection by : implications of and for heterologous immunity. Lancet 995;346: Agger EM, Anderson P. A novel TB vaccine; towards a strategy based on our understanding of failure. Vaccine ;: Markaryan A, Zaborina O, Punj V, Chakrabarty AM. Adenylate kinase as a virulence factor of Pseudomonas aeruginosa. J Bacteriol ;83: Munier-Lehmann H, Burlacu-Miron S, Craescu CT, Mantsch HH, Schultz CP. A new subfamily of short bacterial adenylate kinase with the Mycobacterium tuberculosis enzyme as a model: a predictive and experimental study. Proteins 999;36: Chakrabarty AM. Nucleoside diphosphate kinase: role in bacterial growth, virulence, cell signalling and polysaccharide synthesis. Mol Microbiol 998;8: Chen Y, Morera S, Mocan J, Lascu I, Janin J. X- ray structure of Mycobacterium tuberculosis nucleoside diphosphate kinase. Proteins ;47: Chopra P, Singh A, Koul A, Ramachandran S, Drlica K, Tyagi AK, Singh Y. Cytotoxic activity of nucleoside diphosphate kinase secreted from Myco bacterium tuberculosis. Eur J Biochem 3;7: Britton WJ, Hellqvist L, Basten A, Raison RL. Mycobacterium leprae antigens involved in human responses: identification of four antigens by mono clonal antibodies. J Immunol 985;35: Engers HD, Houba V, Bennedsen J, Buchanan TM, Chaparas SD, Kadival G, et al. Results of a World Health Organization-sponsored workshop to characterise antigens recognised by mycobacterium-specific mo noclonal antibodies. Infect Immun 986;5:78-.. Britton WJ, Hellqvist L, Basten A, Inglis AS. Im munoreactivity of a 7kD protein purified from Mycobacterium bovis bacillus Calmette-Guerin by monoclonal antibody affinity chromatography. J Exp Med 986;64: Adams E, Garsia RJ, Hellqvist L, Holt P, Basten A. T cell reactivity to the purified mycobacterial antigens p65 and p7 in leprosy patients and their household contacts. Clin Exp Immunol 99;8: Jungblut PR, Schaible UE, Mollenkopf HJ, Zimny- Arndt U, Raupach B, Mattow J, et al. Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis strains: towards functional genomics of microbial pathogens. Mol Microbiol 999;33: Brandt L, Elhay M, Rosenkrands I, Lindblad EB, Andersen P. ESAT-6 subunit vaccination against Mycobacterium tuberculosis. Infect Immun ;68: Rosenkrands I, Weldingh K, Jscobsen S, Hansen CV, Florio W, Gianetri I, et al. Mapping and identification of Mycobacterium tuberculosis proteins by two-dimensional gel electrophoresis, microse quencing and immunodetection. Electrophoresis ; : Rosenkrands I, King A, Weldingh K, Moniatte M, Moertz E, Adersen P. Towards the proteome of Mycobacterium tuberculosis. Electrophoresis ;: Covert BA, Spencer JS, Orme IM, Belisle JT. The application of proteomics in defining the T cell antigens of Mycobacterium tuberculosis. Proteomics ;: Lindbland EB, Elhay MJ, Silva R, Appelberg R, Andersen P. Adjuvant modulation of immune res ponses to tuberculosis subunit vaccines. Infect Immun 997;65: Adersen P. Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins. Infect Immun 994;6: Martin RM, Lew AM. Is IgGa a good Th marker in mice? Immunol Today 998;9:49. 5

10 Tuberculosis and Respiratory Diseases Vol. 58. No., Feb, 5. Weinrich-Olsen A, van Pinxteren LA, Meng Okkels L, Birk Rasmussen P, Andersen P. Protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen 85b and east-6. Infect Immun ;69: Stewart GR, Snewin VA, Walzl G, Hussell T, Tormay P, O'Gaora P, et al. Overexpression of heat-shock proteins reduces survival of Mycobacterium tuber culosis in the chronic phase of infection. Nat Med ;7: Colaco CA, Bailey CR, Keeble J, Walker KB. (Bacille Calmette-Guerin) HcpCs(heat-shock protein- peptide complexes) induce T-helper responses and protect against live challenge in a murine aerosol challenge model of pulmonary tuberculosis. Biochem Soc Trans 4;3: Hogarth PJ, Jahans KJ, Hecker R, Hewinson RG, Chanbers MA. Evaluation of adjuvants for protein vaccines against tuberculosis in guinea pig. Vaccine 3;: Doherty TM, Olsen AW, van Pinxteren L, Andersen P. Oral vaccination with subunit vaccines protects animals against aerosol infection with Mycobacterium tuberculosis. Infect Immun ;7:3-. 5

11 SH Lee, et al.: Protective efficacy of recombinant proteins AK, NdK, and Hsp7 against Mycobacterium tuberculosis infection in mice 6. Silva RA, Pais TF, Appelberg R. Effects of inter leukin- in the long-term protection conferred by a Mycobacterium avium subunit vaccine. Scand J Immunol ;5:

항체 포털 서비스 Preparation Peptide 총 4주 소요 / 473,000 ~ 511,000 Free of charge 2~3 days 243,000 ~ 281,000 의뢰서 작성 Peptide epitope prediction 유전자 정보

항체 포털 서비스 Preparation Peptide 총 4주 소요 / 473,000 ~ 511,000 Free of charge 2~3 days 243,000 ~ 281,000 의뢰서 작성 Peptide epitope prediction 유전자 정보 Preparation Service AbFrontier Custom service 장점 실험 담당자와의 원활한 1:1 커뮤니케이션 (신속한 feedback을 통한 맞춤형 제작 서비스) 고객연구센터 - 개별 서비스의 진행 상황 및 결과 즉시 확인 http://center.abfrontier.com (e-mail & SMS 전송) 회원제 -연구자의 프로젝트 보안