In this study, Pleurotus ostreatus No.42 was cultured in glucose-peptone-yeast-wheat bran medium using a previously reported novel rotary draft tube bioreactor. Versatile peroxidase (VP), a lignin-degrading enzyme, was isolated from a pellet-type mycelium culture grown in the medium for seven days. The VP was purified by sequentially applying ultra-filtration, DEAESepharose CL-6B column, and Mono Q column. SDS-PAGE analysis revealed the molecular weight of VP to be 36.4 KDa with an isoelectric point of 3.65. The amino acid sequence was confirmed as VTCATGQTT. The purified VP was observed to possess the property of not only oxidizing Mn ions but also decomposing veratryl alcohol, a non-phenolic compound. The catalytic ability of VP is a subject for future research.

Canine parvovirus-2 (CPV-2) has been reported worldwide as a major pathogen associated with acute hemorrhagic enteritis. The disease is a major infectious cause of death, particularly in young dogs. The earliest type of CPV-2 was replaced with three main subspecies, CPV-2a, CPV-2b, and CPV-2c, within a few years. Vaccination is carried out regularly, but the emergence of antigenic variants and the influence of maternal antibodies have limited the efficacy of commercial vaccines. New vaccines, such as the subunit vaccine, have been developed for alternative, safe, and effective vaccination. The baculovirus expression vector system (BEVS) is an excellent eukaryotic expression system with a high-level expression of foreign proteins and the ability of post-translational modification. Therefore, it is used widely to produce recombinant protein and subunit vaccines. In this study, the VP2 protein of CPV-2b cloned in the gateway vector system was generated using a baculovirus expression system in Spodoptera frugiperda (SF9) insect cells. Hemagglutination assay (HA) titers (24) were obtained, and the expression was detected in 6-His tagged VP2 and monoclonal antibody (mAb) against CPV-2 by western blotting. The VP2 protein of CPV-2b expressed in this study may provide a basis for a clinical diagnosis and vaccination applications for CPV-2.

Foot-and-mouth disease (FMD) is a highly contagious vesicular disease that affects cloven-hoofed animals, causing substantial economic losses to the livestock industry. The causative FMD virus (FMDV) comprises four structural proteins (VP1, VP2, VP3, and VP4) and several non-structural proteins. Among the capsid proteins, VP4 is the most conserved, making it an attractive target as a diagnostic and vaccine antigen, regardless of FMDV serotype. In this study, we attempted to express the VP4 protein N-terminally fused to a glutathione S-transferase (GST) tag in Escherichia coli. Whereas VP0 and VP2 proteins were expressed in the soluble fraction, we failed to detect VP4, even in the insoluble fraction. To investigate the effect of VP4 C-terminal amino acid residues on protein expression, we constructed three VP4 mutants fused to GST, among which the mutant in which the C-terminal 15 amino acid residues had been deleted showed the highest level of protein expression. Furthermore, protein expression was observed even in the mutant in which three amino acid residues (DKK) had been fused to the C terminus. However, unlike the other two mutants, the wild-type VP4 mutant was poorly expressed, thereby indicating that the C-terminal amino acid residues could play a pivotal role in determining expression of the VP4 protein in E. coli.

본고는 이동 동사 ‘来/去’의 문법화 양상과 이에 따른 상(相) 의미를 분석했다. ‘来/ 去’는 [VP+来/去]의 연동문을 기반으로 ‘이동’이라는 본 의미에서 방향성, 시간성, 결 과성, 상태 변화 등으로 의미 추상화가 진행되었고, 이에 따라 [완결], [계속]과 같은 상(相) 의미를 획득하였다. 또한, 명·청 시기까지 문장 끝에 위치하여 사건의 발생, 변화를 나타내는 “사태조사(事态助词)”로 사용되었는데, 이는 현대 중국어 문미 ‘了’ 와 유사한 성분으로 발화자의 관점에서 이미 발생한 사건을 관망하는 완료상의 속성 인 “현재 관련성”을 지니고 있다. 본고는 ‘来/去’가 지닌 사태조사로서의 의미 또한 본 의미 ‘이동’에서 시작된 문법화의 산물임을 확인할 수 있었다.

A solid-phase competition enzyme-linked immunosorbent assay (ELISA), recombinant VP2 (rVP2) protein, and monoclonal antibody (mAb) were developed for the specific and sensitive detection of porcine parvovirus (PPV) antibodies in pig sera. A total of 1,544 sera samples were collected from breeding pig farms located in the Gyeongsangbuk-do Province in the Republic of Korea. The optimal operating conditions of SC-ELISA were as follows. The concentration of rVP2 proteins coated on the wells was 4 μg/mL, the swine sera were diluted 1:2, and the HRP-conjugated PPV VP2 mAb (9A8 clone) was used at 500 ng/mL. These results suggest that the SC-rVP-ELISA assay may be a valuable alternative to the current diagnostic tools used to detect PPV-specific monoclonal antibodies and broadly monitor PPV infections in domestic pigs at different breeding stages.

Viral protein 2 (VP2), which is the structural protein of parvovirus, can produce virus-like particles (VLPs) by a self-assembly process in vitro, making VLPs attractive vaccine candidates. VP2 of canine parvovirus (CPV) is responsible for neutralizing antibodies in immunized animals. In this study, VP2 protein of canine parvovirus-2c was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells. The results show that VP2 proteins assembled into virus-like particles (VLPs) with antigenic properties similar to those of natural CPV and a high hemagglutination (HA) titer (1:27). The recombinant 6-His-tagged VP2 protein with a molecular mass of about 65 kDa was detected by anti-His antibody and anti-PPV serum. This study provides a foundation for the application of VP2 protein in the clinical diagnosis of CPV and in the vaccination against CPV.

Viral protein 2 (VP2) of porcine parvovirus (PPV) is responsible for inducing neutralizing antibodies in immunized animals. It is the major viral structural protein. In this study, novel subunit vaccines against PPV based on virus-like particles (VLPs) formed from VP2 proteins (PPV 13-7 Korean strain) were expressed in an insect baculovirus cell system and purified using Ni-NTA affinity column chromatography. These VP2 proteins assembled into virus-like particles (VLPs). They showed antigenic properties similar to those of natural PPV. In addition, they showed high hemagglutination (HA) titers (211 for PPV 13-7 Korean strain). This study provides a foundation for the application of the difference immunization of recombinant protein in the diversity of PPV VP2 genes and in vaccination against PPV in the future.

경계성(界性)이란 인류가 객관세계 사물의 경계(Boundary)를 인식하는 한 방식으로 유경계성(有界, boundary)과 무경계성(無界,unboundary)으로 분리된다. 인지적 경험에 근거하여 사물에 대한 ‘경계 설정’이 가능하면 그 사물은 유경계성을 띠고, 이 ‘경계 설정’이 불가능하다면 그 사물은 무경계성을 띤다. 인지언어학자들은 경계성 이론을 언어현상에 응용하여 설명하기 시작하였는데 본 연구는 이러한 언어현상의 경계성 이론에 입각하여 중국어 사동문법의 인지적 분석에 응용해 보았다. 특별히 현재 상용되고 있는 다양한 중국어 사동형태 가운데 그 형식상 동일한 유형의 사동구조로 취급되고 있는 ‘NP1+VP+NP2’ 구조와 ‘NP1+VP(‘V1V2’) +NP2’ 즉, 결과보어 사동구조에 착안하여 구조상 존재하는 문법적 차이를 근거로 ‘VP’와 ‘V1V2’가 술어로서 한정짓게 되는 경계성의 차이를 탐구하였다. 아울러 NP2와 了의 수반으로 인해 경계화 짓는 특성 및 자질이 어떻게 나타나는지 인지적 분석으로 접근하였다.

Rotaviruses are enteric pathogens causing acute watery dehydrating diarrhea in humans and animals. The importance of group C rotavirus (GpC-RV) infections has not been established as the studies on the GpC-RV have been hampered by the lack of an in vitro culture system. However, diarrheal diseases associated with GpC-RV have been gradually increasing worldwide. In this study, VP6 gene of bovine GpC-RV Korean isolate was expressed, and monoclonal antibodies (mAbs) against VP6 were produced and characterized. The VP6 gene was cloned and expressed based on a baculovirus expression system. Indirect fluorescence antibody (IFA), polymer chain reaction (PCR), and Western blot assays were used to confirm expression of VP6 gene synthesized by the recombinant baculovirus. Eleven mAbs against VP6 were produced using expressed VP6. Cross-reactivity of the mAbs was assessed with recombinant VP6 proteins from porcine GpC-RV and human GpA-RV, or different serotypes of group A rotavirus strains by IFA test. Some mAbs reacted with intact porcine GpC-RV Cowden strain as well as bovine GpC-RV VP6 recombinant baculoviruses, but not with human and animal GpA-RV strains. The VP6-specific mAbs might be useful to develop immunodiagnostic tests such as rapid diagnostic kit, IFA and enzyme-linked immunosorbent assay (ELISA) for detection of GpC-RV.

본 논문은 잉여부정현상 가운데 하나인 ‘难免(不)VP’ 구문을 연구하였다. 통시적 고찰을 통 해 ‘难免’의 문법화 과정을 분석하고, 공시적 관점을 결합하여 형식과 의미의 비대칭 양상을 분류하였으며, 문법화 과정을 이끄는 기제 및 통사 층위에서 의미를 부여할 수 없었던 잉여 성분 ‘不’의 인지, 화용 층위에서 역할을 모색해보았다. 먼저 ‘难免’은 동사에서 부사로 4단계 문법화를 거쳤다. 첫 번째 단계는 ‘难免’이 단독으로 술어로 활용되는 시기로 문법화는 시작 되지 않았다. 두 번째 단계는 체언성 또는 용언성 목적어가 후행하기 시작하면서 통사구조 관계가 술목구조로 변하는 시기이다. 여전히 동사성이 강하게 나타나며, 이때 ‘难免’은 ‘难以 避免, 很难避免’으로 확장 가능하다. 또한 후행하는 성분들은 객관적인 조건하에 추측 가능한 결과를 나타내었다. 세 번째 단계는 부사화가 시작되어 과도기적 단계를 거치는 시기로 용언 성 목적어가 후행하는 빈도가 급증하였다. 술목관계에서 술어로 활용되는 ‘难免’의 통사 위치 는 문법화되기 쉬운 부사어 자리이이며, 또한 후행하는 VP의 의미 변화 등과 더불어 외재하 는 구조형식의 변화로 문법화가 빠르게 진행되었다. 이때 ‘难免’은 동사 또는 부사로 볼 수 있었다. 네 번째 단계는 부사화가 성숙되고 완성되는 단계이다. ‘难免’의 두 형태소는 원래의 의미를 소실하고 완전히 결합하여 더 이상 ‘难以避免, 很难避免’으로 확장할 수 없었다. 또한 재분석과 화용강화를 통해 잉여성분을 포함한 부정형식 ‘难免不VP’가 나타나고, ‘难免’은 완 전히 부사화 되었다. 문법화가 완성된 후 ‘难免(不)VP’ 구문은 후행성분 VP와의 통사, 의미관 계 속에서 긍정형식 또는 부정형식으로만 활용 가능한 경우와 긍정형식과 부정형식이 의미상 차별이 모호해지는 3가지 비대칭 양상으로 나타났다. 또한 ‘难免VP’ 구문과 잉여부정 ‘难免不 VP’ 구문은 전경과 배경의 역전 현상을 통해 ‘难免VP’ 구문은 VP의 발생은 피할 수 없다는 것이 전경이 되어 VP를 지향하고, ‘难免不VP’ 구문은 VP의 발생을 희망하지 않는 화자의 심 리 상태가 전경이 되어 ‘不VP’를 지향함을 알 수 있었다. 객관적으로 동일한 사태(VP)를 주 관적으로 해석해낸 것이다. 따라서 ‘难免(不)VP’ 구문은 개념화자의 인지적 경향성과 인지적 전략에 따라 전경과 배경의 시점이 역전되는 현상을 기저로 통시적 문법화 과정에서 재분석 으로 나타나는 것이며, 또한 ‘不’를 통해 화자가 보다 적극적이고 의도적으로 주관적 의미를 부여하고, 무표 형식인 ‘难免VP’ 구문에 대립되는 유표 형식인 ‘难免不VP’ 구문을 통해 주관 화가 실현된 표현으로 ‘不’는 주관화의 표지로 이해할 수 있었다.

Norovirus (NoV) is an etiologic agent of human and animal acute gastroenteritis and is a member of the family Caliciviridae. NoV is classified based on nucleotide sequences of the VP1 gene into at least six genogroups (GI-GVI), among which GI, GII, and GIV are known to infect humans and GII is the most prevalent genogroup. In this study, VP1, the full gene of GII human NoV, was cloned from a human fecal sample and expressed using a baculovirus expression system. Human NoV VP1-specific monoclonal antibodies (MAbs) were produced using expressed recombinant VP1. Expressed VP1 in the recombinant virus was confirmed by polymerase chain reaction (PCR), indirect fluorescence antibody (IFA) test, and Western blot analysis. Eight hybridomas secreting VP1-specific MAbs against human GII NoV were generated and characterized. All of the MAbs produced in this study reacted with human GII NoV VP1-recombinant baculoviruses but not with other non-human calicivirus recombinant baculoviruses. These MAbs reacted specifically with human NoV GII.4-2009 virus-like particles (VLPs), and some MAbs showed cross-reactivity with other GII.4 variant VLPs. Expressed human GII NoV VP1-recombinant protein and MAbs specific to this protein can be used as useful reagents for detecting and characterizing human NoV.

Rotaviruses are double-stranded RNA viruses of the family Reoviridae, a highly diverse family of pathogens of humans and animals. In this study, we identified the lapine rotavirus from diarrheic feces of rabbits by polymerase chain reaction. In order to determine the genetic characteristics of the Korean strain, the sequences of the VP4, VP7, and NSP4 genes were determined and compared with those of reference sequences. Results of sequence and phylogenetic analyses showed that our strain was a G3P [3] rotavirus carrying the group C gene encoding NSP 4 proteins. This is the first report of an outbreak and molecular characterization of lapine rotavirus in Korea.

Canine parvovirus (CPV2) is one of the most virulent virus causing acute hemorrhagic enteritis and myocarditis in dogs. Infection mainly caused by the ingestion of virus through the mucosal route. Therefore, induction of mucosal immunity is essential in prevention of Canine Parvovirus (CPV2) infection. For safe and effective delivery of viral antigens to the mucosal immune system, a novel surface antigen display system for lactic acid bacteria using the poly-γ-glutamic acid synthetase A protein (pgsA) of Bacillus subtilis as an anchoring matrix was applied in order to display CPV2 antigen on the surface of the recombinant L. casei. Recombinant fusion proteins comprised of pgsA and the capsid protein (VP2-S1) showed stable expression in Lactobacillus casei. Surface localization of the fusion protein was verified by cellular fractionation analyses. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and mucosal IgA, as demonstrated by ELISA using recombinant VP2-S1 proteins. Mice receiving intranasal immunization mounted higher antibody response than those receiving oral immunization. These results indicate that mucosal immunization with recombinant L. casei expressing CPV2 VP2-S1 protein on its surface provides an effective means for elicitation of strong antibody responses against CPV 2 VP2-S1.