흰불나방 核多角體病 바이러스의 製劑化에 관한 試驗

陳炳來,金槿榮,姜錫權 한국잠사학회 1987 한국잠사곤충학회지 Vol.29 No.2

The intent of this research is to acquire some basic informations about formulation of the viral pesticide, Hyphanlria cunea nuclear polyhedrosis virus and its virulence under field condition. The nuclear polyhedrosis virus was formulated as wettable powder using spreader, sticker and U.V. protector. The formulated product and aqueous virus were diluted with water at the concentration of 1 X 106 PIB/ml and sprayed on mulberry leaves in the field. The leaves were fed with 3rd instar larvae of H. cunea to determine the inactivation period of the viral pesticides. The aqueous virus was completely inactivated on 5th day after spray, while the formulated one showed a spare mortality to the larvae even on 20th day after spray. In field application test, The formulated and aqueous virus were sprayed on individual mulberry tree and 3rd instar laevae of H. cunea were fed on the trees. The mortality of the larvae one day after spray of the formulated and aqueous virus were about 50% and 40%, respectively. The formulated virus exhibited a persistent virulence to the larvae up to 9th day after spray, which the mortality was approximately 30%. The residual virulence of the formulated and aqueous virus was extended up to 14th day and 2nd day after spray, respectively.

곤충 핵다각체병 바이러스 다각체 단백질의 소화액에 의한 분해

진병래,박범석,우수동,김주읍,강석권 한국산업미생물학회 1993 한국미생물·생명공학회지 Vol.21 No.6

담배거세미나방 핵다각체병 바이러스(Spodoptera litura nuclear polyhedrosis virus : SINPV)의 다각체에 오염되어 있는 alkaline protease를 불활화시키기 위하여 100℃에서 20분간 처리하고 SDS-PAGE로 다각체 단백질을 분석했을 때, 31 kDa의 band와 그 중합체만이 관찰되었다. 그러나 alkaline protease를 불활화시킨 다각체를 중장 소화액으로 처리했을 경우에는 처리시간이 경과함에 따라 다각체 단백질은 일정한 pattern으로 분해되었다. 한편, 100℃ 에서 5분간 처리한 중장 소화액에 alkaline protease를 불활화시킨 다각체를 장시간 처리하였을 때에는 다각체 단백질이 저분자화되지 않으므로서 다각체 단백질을 분해시키는 alkaline protease가 중장 소화액으로부터 유래된 것임을 확인하였다. SINPV의 다각체 단백질을 분해시키는 중장 소화액의 alkaline protease의 활성을 조사하기 위하여 peptide mapping한 결과 papain과 비슷한 활성을 나타내었으며, 누에(Bombyx mori ; Bm) 그리고 흰불나방(Hyphantria cunea ; Hc)NPV 다각체 단백질의 peptide map을 서로비교한 결과 3종 NPV의 다각체 단백질 간에는 서로 유사성이 있음을 알 수 있었다. The alkaline protease in the polyhedra preparation of Spodoptera litura nuclear polyhedrosis virus was successfully inactivated by heating at 100℃ for 20 minutes. SDS-PAGE analysis indicated that heat inactivated polyhedra is composed of major proteins of 31kDa and presumptive its polymer protein of 62 kDa. However, this polyhedra was converted into several smaller molecular weight proteins when treated with midgut juice, but not by treatment with heat-inactivated midgut juice. When peptide map of polyhedral protein of Spodoptera litura(SI), Bombyx mori(Bm) and Hyphantria cunea(Hc) nuclear polyhedrosis viruses(NPVs) was compared, activity of alkaline protease in midgut juice of S. litura were similar to that of papain. Peptide mapping of polyhedral proteins of three NPVs showed homologous pattern.

家蠶 培養細胞에서 核多角體病 바이러스의 多角體 蛋白質 合成과 DNA 復製

陳炳來,朴範錫,柳江善,諸蓮鎬,姜錫權 한국잠사학회 1991 한국잠사곤충학회지 Vol.33 No.1

Bombyx mori nuclear polyhedrosis virus (BmNPV) was successfully multiplied in the nuclei of BmN4 cells cultured with insect Grace's medium. By electron microscopic observation, the virons had a single nucleocapsid in an envelope. Polyhedral protein synthesis of BmNPV in BmN4 cells was detected at 18 hr p.i. and polyhedral protein was a single-polypeptide with a M.W of 30 kd. At 48 hr p.i. polyhedra formation was observed by inverted microscope and electron microscope. Genome analysis of BmNPV by restriction endonucleases was not revealed the difference between virus produced in vivo and that in vitro.

누에 核多角體病 바이러스에 의한 發見 벡터 시스템 開發

陳炳來 한국잠사학회 1995 한국잠사곤충학회지 Vol.37 No.2

파밤나방 핵다각체병 바이러스의 생화학적 특성

진병래,박범석,재연호,강석권 한국응용곤충학회 1991 한국응용곤충학회지 Vol.30 No.2

국내에서 분리된 파밤나방 핵다각체병바이러스(Spodoptera exigua nuclear polyhedrosis virus: SeNPV)의 생화학적인 특성을 규명하기 위하여 몇가지 실험을 행하였다. SeNPV는 하나의 envelopeso내에 다수의 nucleocapsid가 존재하는 MNPV(multiple embeded NPV)형태였다. 다각체단백질은 분자량 30kb의 단일 band로 나타났으며, Spodoptera litura NPV와 Bombyx mori NPV의 다각체단백질 항체에 반응하여 뚜렷한 침강선을 형성하였다. 비리온 단백질을 은염색한 결과, 많은 수의 minor band들이 포함된 49개의 band로 나타났으며, 바이러스 DNA를 분리하여 여러종의 제한효소에 의한 대략적인 genome size는 약 110kb 였다. Biochemical characteristics of Spodoptera uigua nuclear polyhedrosis virus (SeNPV) isolated in Jinju were studied. SeNPV contained a number of nucleocapsids within a viral envelope embeded in polyhedra. The polyhedral protein of SeNPV was composed of a single polypeptide with a M. W. of 30kd. Double-immunodiffusion test showed that the polyhedral protein of SeNPV had common antigenic determinants with SINPV and BmNPV. Virion proteins of SeNPV were resolved into 49 polypeptides by silver staining after SDS-PAGE. The approximate genome size of SeNPV by restriction endonuclease analysis was 1l0kb.

Bombyx mori 세포주와 Spodoptera frugiperda 세포주의 분자생물학적 표식자

진병래,제연호,강석권 한국잠사학회 1996 한국잠사곤충학회지 Vol.38 No.1

To investigate the molecular biological marker in insect sells, BmN-4 and Sf-9 cells were analysed by SDS-PAGE and random amplification of polymorphic DNA. The results showed that the pattern of total cell protein and random amplification of polymorphic DNA were distinguished between BmN-4 and Sf-9 cell, suggesting that the unique major bands were useful as molecular biological marker in BmN-4 and Sf-9 cells.

담배나방 핵다각체병바이러스의 형태 및 생화학적 특성

진병래,박현우,우수동,김우진,김우진,박범석,강석권 한국응용곤충학회 1995 한국응용곤충학회지 Vol.34 No.3

담배와 고추의 주요해충인 담배나방 [Helicoverpa assulta(Guenee)]의 미생물적 방제법을 개발하기 위하여, 아직 국내에서 보고되지 않은 담배나방 핵다각체병 바이러스 담배나방 사충으로 부터 분리하고, 바이러스의 전자현미경 관찰 및 바이러스 DNA의 제한효소 분석 등을 통한 생화학적 특성을 조사하였다. 담배나방 핵다각체병 바이러스의 다각체는 구형에 가까운 20면체로서 크기는 평균 약 1.0$\mu$m 정도이고, 다수의 바이러스 입자가 다각체 단백질에 포매되어 있었다. 포매된 바이러스 입자는 한개의 봉상 nucleocapsid가 하나의 envelope 내에 매립된 형태의 SNPV(single embedded nuclear polyhedrosis virus)로, 형태는 전형적인 봉상으로서 그 크기는 약 $65nm\times300nm$ 였다. 또한 다각체 단백질의 SDS-PAGE 분석 결고, 분자량은 약 31 Kd이었으며, 담배나방 핵다각체병바이러스 DNA를 분리하여 EcoRI. HindIII 등 수종의 제한효소로 처리분석한 결과, 그 genome의 크기는 약 120Kb 정도였다. 본 핵다각체병바이러스는 담배나방 유충에만 병원성을 나타내 기주특이성을 보였다. A nuclear polyhedrosis virus isolated from the oriental tobacco budworm larvae, Helicoverpa assulta (Guenee) was characterized by electron microscopy, SDS-PAGE, restriction endonuclease analysis and cross infectivity. The shape of a polyhedron was $1.0\mu\textrm{m}$ in average with icosahdral outline, and the virus particle was $65nm\times300nm$ in average with rod-shape. The nuclear polyhedrosis virus was contained a single nucleocapsid within a viral envelope embedded in a polyhedron. The polyhedral protein was composed of a single polypeptide with a M.W. of 31 Kd. The genome size of the virus by restriction endonuclease analysis was about 120 Kb. Among several nuclear polyhedrosis viruses, the nuclear polyhedrosis virus from Helicoverpa assulta (HaNPV) and Autographa california nuclear polyhedrosis virus (AcNPV) were infected the oriental tobacco budworm larvae.

害蟲病原 바이러스의 分離同定과 바이러스 農藥 開發 : Ⅲ. Some basic studies on mass production of hyphantria cunea nucler polyhedrosis for the Ⅲ. 바이러스 製劑化를 위한 흰불나방 核多角體病 바이러스의 量産에 관한 基礎硏究

陣炳來,金英勳,金槿榮,姜錫權 서울大學校 農科大學 1985 서울대농학연구지 Vol.10 No.1

It is the intent of this research to acquire some basic informations about in vivo mass production of Hyphantria cunea muclear polyhedrosis virus for the viral pesticide. There are many factors influencing virus production through mass rearing of host larvae, but some important ones,1.e., diet for rearing, haversting time of infected larvae, and virus concentration, were examined in these expriments because those factors may play a significant role in the production system by affecting the growth of host larvae and virus yields. The 4th and 5th instar larvae of H. cunea, separately fed on artificial diet and mulberry leaves, were used for the investigation of pathogencity, comparison of changes in body weight of host larvae and virus yields on each dav after infection. 1. In the virulence of H. cunea nuclear polyherosis virus, LD?? values were 8.76x10?,1.98x10?,and 9.3x10? 5,1.5x10? PIB/ml for the 4th and 5th instar larvae of H. cunea separately fed on artificial diet and mulberry leaves, respectively. LD?? of H. cunea was 10 times higher in the larvae fed on mulberry leaves than the artificial diet. The larvae inoculated in the 5th instar were found more resistant than fourth instar to virus infection. Considerable number of larvae infected in 5th instar survived and pupated but larvae infected in the 4th instar did not survie at the concentration of 1.09x10?. 2. The body weight of H.cunea harvae infected in the 4th instar were found more reduced than the 5th instar. The increasing rate of the body weight of infected larvae fed on artificial diet was lower as compared with larvae fed on mulberry leaves. 3. The maximal virus yields of H.cunea larvae infected with 1.09x10? PIB/ml at the 5th instar were 1.3x10? PIB and 1.1x10? PIB per g of final larval weight, using mulberry leaves and artificial diet rearing, respectively. From the above results, it was demonstrated that more virus was obtained from larvae inoculated in later instars than earlier instars with sufficient concentration virus lead to 100% mortality.

들민달팽이(Deroveras varians)의 배자발육과 난황단백질

진병래,손흥대,박혜진,조은숙 東亞大學校附設 農業生命科學硏究所 1999 農業生命資援硏究 Vol.8 No.1

들민달팽이의 배자발육은 해부현미경으로 관찰하였다. 산란 후 4일이 경과면서 발육이 급격하게 진전되어, 10일이 지나면서 난 내에서 들민달팽이 유충의 형태를 갖추고, 12일째 부화하였다. 들민달팽이의 배자발육에 따른 난황단백질의 변화는 산란 1일째, 3일째 및 6일째 알의 난황단백질을 전기영동방법으로 분석하였다. 그 결과 배자발육과 함께 급격히 감소되는 난황단백질 관련 밴드를 관찰할 수 있었다. We characterized embryogenesis and egg proteins of Variable field Slug, Deroceras varians. The embryogenesis was observed by light microscopy. The eggs were hatched at the twelfth day after oviposition. The egg proteins of the first day, third day and sixth day after oviposition were analyzed by native-and SDS-PAGE analyses. The results showed that yolk proteins are gradually decreased during embryogenesis.

국내에서 분리한 미국흰불나방 과립병바이러스의 전체 게놈 염기서열 분석

최재방,허원일,배성민,신태영,이준범,제연호,진병래,우수동 한국응용곤충학회 2012 한국응용곤충학회 학술대회논문집 Vol.2012 No.05

미국흰불나방은 가로수에 커다란 피해를 주고 있는 해충으로, 우리나라에서는 연간 2회 발생한다. 우리나라 곤충바이러스 연구는 1974년 미국흰불나방 (Hyphantria cunea)에서 베큘로바이러스(baculovirus)를 분리하여 병원성이 높은 것을 확인하면서 해충 종합적 방제를 위해 연구가 시작되었다. 본 연구에서는 미국 흰불나방 자연 사충으로부터 과립병바이러스(granulovirus)를 분리하고 바이러스 의 특성을 이해하기 위해 게놈 분석을 시행하였다. 미국흰불나방 과립병바이러스 의 전체염기서열의 분석은 454 pyrosequencing 방법을 이용하였고, 크기는 114.556bp 로 판명되었다. 또한 G+C 함량은 전체염기서열의 39.30 %를 차지하였 고, 130개의 open reading frame (ORF)를 갖는 것으로 나타났다. 130개의 ORF 중 106개의 ORF가 기존에 보고된 베큘로바이러스와 상동성을 갖는 것으로 확인되었 다. 기존에 전체염기서열이 분석된 다른 베큘로바이러스들과 계통분석을 수행한 결과, 과립병바이러스 중 씨자무늬거세미방나방 과립병바이러스와 근연관계를 갖는 것으로 확인되었다.