The baculovirus expression system (BES) utilize the p10 or polyhedrin promoter, a very late promoter that exhibits strong transcriptional activity primarily at the end of viral infection, to produce useful recombinant proteins. The burst sequence of the very late promoter is essential for strong transcription, and VLF-1 is a transcription factor that binds specifically to the burst sequence, and it has been reported that it can regulate the amount and timing of expression of protein by the very late promoter. Recently, a VLF-1 constitutively expressing cell line was constructed to increase the production of the target protein, but the effect was minimal. In this study, to find the optimal VLF-1 expression conditions to increase target protein production efficiency, we controlled the expression of VLF-1 through various promoters and evaluated the target protein expression efficiency by the p10 promoter accordingly.

Canine parvovirus (CPV) remains a leading infectious cause of death in canines, especially in young puppies. Though vaccination is being carried out regularly, immunization failures occur, and puppies may be exposed to infection. Virus-like particles (VLPs) act like a subunit vaccine, mimicking the structure of authentic viruses. Therefore, VLPs have the potential to be used as vaccine candidates. Since Viral Protein 2 (VP2), a major structural protein of CPV, is the crucial antigen for CPV, the purpose of this study was to produce a recombinant VP2 of new canine parvovirus-2a using the baculovirus expression system in SF9 insect cells. The results revealed that recombinant VP2 assembles to form VLPs with antigenic properties similar to those of natural CPV, the recombinant VLP can produce a hemagglutination assay (HA) titer (1:210) in SF9 cells. Expression of the recombinant 6-His-tagged VP2 in SF9 cells was confirmed by western blotting. These findings suggest that the recombinant VP2 expressed in this study could be used as an efficient subunit vaccine against CPV infection.

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.

The baculovirus-insect cell expression system has been widely used method for the recombinant protein expression. The present study has several limitation. In this study, we constructed vectors consisting of transcriptional enhanced factor and promoter that improve the expression level. To confirm the usefulness of these vector system, Human papillomavirus (HPV) VLPs have been expressed by baculovirus hyper expression system. HPV VLPs were purified using a CaptoTM Core 700 (GE Healthcare Life Sciences) chromatography approach. Baculovirus hyper expression system production efficiency was influenced by the HPV VLPs production. HPV VLPs vaccination to BALB/c mice induced the generation of antibody confirmed by ELISA. This study could provide improvements on the vaccine production for the development of VLP vaccines high expression of useful heterologous proteins.

깨다시 꽃게(Ovalipes punctatus)는 갑각류로서 우리나라에서 잡히는 매끈 꽃겟속, 주름 꽃 겟속, 톱날 꽃겟속, 민 꽃겟속, 두갈래 민꽃겟속 들 중에 하나이다. 대부분의 꽃게는 가공되지 않은 상태로 찜 또는 찜육 등 반 가공 형태로 산업화 되었지만 최근에 게로부터 생리활성을 나타내는 펩타이드를 생산하는 연구가 발표되고 있다. 본 연구는 항산화 기능성을 나타내는 펩타이드를 선별하고 생산 최저공정 확립에 연구를 수행 하였다. 사용된 효소 alcalase, bromelain, flavourzyme, neutrase, papain, protamex들 중에서 bromelain으로 생산된 꽃게육 단백질 가수분해물이 가장 높은 활성을 보여 주었다. 꽃게육 단백질의 bromelain 가수분해물의 펩타이드들의 분자량 분포는 500-3,200 Da로서 7 종류의 이상의 펩타이드들로 구성되었다. 가수분해물의 구성아미노산 분포는 항산화 기능성에 관련된 소수성 아미노산은 전체 42.54%를 차지하였다. 가수분해물의 최적 생산 수율 조건을 확립하기 위하여 공정 조건, 효소 반응 온도 40-60℃, pH 6-8, 효소의 농도 1–3%(w/v)로 표면반응 분석법을 수행한 결과 효소 반응온도 55℃, 반응 pH 6.5, 효소의 양은 3%(w/v)에서 결정되었다. 최적 조건에서 단백질 가수분해도는 최대 71.60%에 도달하였다.

In this study, PAT protein of genetically modified maize was prepared from the recombinant E. coli strain BL21 (DE3), and mice were immunized with the recombinant PAT protein. After cell fusion and cloning, two hybridoma cells (PATmAb-7 and PATmAb-12) were chosen since the monoclonal antibodies (Mabs) produced by them were confirmed to be specific to PAT protein in the indirect enzyme-linked immunsorbent assay (ELISA) and western blot tests. There were no cross-reactions of either Mabs to other GM proteins or to the extracts of non-GM maize. The ELISA based on the PATmAb-7 can sensitively detect 0.3 ng/g PAT protein in corn. These results indicate that the developed Mabs can be used as bio-receptors in the development of immunosensors and biosensors for the rapid and simple detection of GM corn adulterated in foods.

식물 및 동물성 단백질 유래 펩타이드 형태의 단백질 가수분해물들은 항산화, 고혈압 완화, 면역조절, 진통완화 및 항균작용 등 생리활성이 있는 것으로 알려져 왔다. 본 연구는 건조 해삼으로부터 해삼육 슬러리를 제조하고 flavourzyme 프로티아제를 이용하여 단백질 가수분해 최적공정을 수행하였 다. 이어서 생산된 펩타이드의 항산화 특성을 연구하였다. 효소반응 최적공정은 표면반응 분석법을 이 용하여 수행을 하였고 공정의 범위는 반응온도 40-60 ℃, 반응 pH 6-8, 효소의 농도 0.5-1.5%(w/v) 이었다. 해삼의 단백질 최적 효소가수분해 공정조건은 효소 반응온도 48–50 ℃, 반응 pH 7.0–7.2, 효소 의 양은 1.0-1.1%(w/v)에서 결정 되었다. 이때 단백질 가수분해 수율은 43-45%에 도달하였다. 생산된 대부분 가수분해물의 분자량들은 전형적인 펩타이드인 분자량 500-3,500Da로 분포되었다. 펩타이드들 은 항산화 능력은 금속 킬레이션 능력(IC50, 1.25 mg/mL), DPPH 소거능(IC50, 3.40 mg/mL), 슈퍼옥사 이드 라디칼 소거능(IC50, 10.3 mg/mL), 하이드록시 라디칼 소거능(IC50, 22.11 mg/mL) 순서로 저해농 도가 낮음을 보여 주었다. 따라서 해삼 단백질 가수분해물은 건강 기능 식품소재로서 활용할 가치가 높 을 것으로 기대를 한다.

For people who have a food allergy the only way to manage the allergy is to avoid the food allergen. The mackerel is one of the major food allergens, but no immunoassay for the rapid and simple detection of mackerel has been reported. The objectives of this study are to develop and characterize monoclonal antibodies (MAbs) specific to mackerel using thermal stable-soluble proteins (TSSP) as an immunogen and to characterize the MAbs by indirect enzyme-linked immunosorbent assay (iELISA). The mice immunized with mackerel TSSP and showing high titer were used for cell fusion and cloning. The characterization of MAbs produced from hybridoma cells obtained was confirmed by indirect ELISA and western blot. Four MAbs were confirmed to be specific to mackerel without crossreaction to other marine products and livestock products in the both methods. The iELISA and western blot based on the MAbs can sensitively detect 1% mackerel protein in other marine products. These results support that immunochemical methods based on the MAb produced could be used as rapid means to detect low levels of mackerel and to identify mackerel adulterated in food.

The present in vitro study was conducted to examine the effect of buffer solubility of eight protein feeds (coconut meal, distillers grain, sesame meal, perilla meal, soy source cake, rape seed meal, soybean meal and lupine) on the fermentation characteristics, degradability of dry matter (DM) and crude protein (CP), and methane (CH4) production by rumen microbes. Buffer extraction increased pH (P<0.05 ~ p<0.001) of the culture solution but tended to lower ammonia- N concentration for all protein feeds. Total volatile fatty acids(VFAs) and each VFAs concentrations in all incubation was decreased by buffer extraction (P<0.01 ~ P<0.001). Also, molar proportion of acetate in 1h (P<0.001), 3h (P<0.01) and 12h (P<0.05) incubations and molar proportion of propionate in 1h (P<0.001), 3h (P<0.01), 6h (P<0.05) and 12h (P<0.05) were decreased by buffer extraction. But molar proportion of butyrate in 1h (P<0.001), 3h (P<0.01) and 6h (P<0.05) were increased by buffer extraction. The in vitro effective degradability of dry matter (P<0.001) and CP (P<0.001) was decreased by buffer extraction. The methane production (P<0.01~P<0.001) in all incubation was decreased by buffer extraction. The results from in the current study might be useful for diet formulation to improve the feed efficiency of the ruminant animals without massive loss of major nutrients.

To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of polyhedrin fragments were investigated by fusion expression them with the enhanced green fluorescence protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the minimal region for self-assembly and the KRKK nuclear localization signal (NLS). The increase of EGFP production by fusion expressions was confirmed through protein and fluorescence intensity analyses. The importance of nuclear localization for enhanced production of EGFP was shown by the mutation of the NLS within the fused polyhedrin fragment. Among the fusion expressed protein in cytoplasm, the most hyper-expression was observed in the fusion of amino acids 32 to 59 of polyhedrin. Polyhedrin fragment fusion expression with classical swine fever virus E2 protein also resulted hyper-enhanced expression of E2 protein. However, the fusion expression of porcine circovirus ORF2 with polyhedrin fragment did not show significant enhance of ORF2 production. These results suggested that the enhancement of foreign protein production when fused with polyhedrin is caused by the enhanced stability of expressed protein.

Human protein C (hPC) is a regulator of homeostasis, suggesting its potential use as a therapy for many disease states. Protein C is a zymogen of a serine protease that is activated by thrombin. Protein C, also known as autoprothrombin ⅡA and blood coagulation factor ⅪⅤ, is a zymogenic (inactive) protein, the activated form of which plays an important role in regulating blood clotting, inflammation, cell death and maintaining the permeability of blood vessel walls in humans and other animals. hPC is a 62 KD disulfide- linked heterodimer consisting of a 21 KD light chain and a 41 KD heavy chain which circulates as an inactive zymogen in plasma. In this study, we focus on generation of hPC transgenic mice. hPC transgenic mice were produced by using micro-injection method. The hPC cDNA was cloned into pBC1 vector under goat β-casein promoter. One-cell stage embryos microinjected were transferred to 24 recipient mice on day 1 of the estrus cycle. We screened 61 mice by the PCR. Four line transgenic mice were identified and confirmed expression of protein C gene in mammary gland and several organ. We also analyzed the expression of mRNA and protein through the northern blot and western blot in mammary gland of hPC transgenic mice. hPC was localized in the alveolar epithelial cell by immunohistochemistry. Now, we are collecting the milk from the 2 found lines. After then, we are checking the activity of hPC produced from mice milk.

본 시험에서 건초(티머시, 알팔파 및 클라인)와 짚류(톨페스큐 및 볏짚)의 buffer 용해도와 단백질 분획이 실시되었으며, 조사료 자원의 buffer 추출이 in vitro 발효 성상, 분해율 및가스(CO2 및 CH4) 생성량에 미치는 효과를 조사하였다. 다른 조사료에 비해 총 단백질 중buffer 가용성 조단백질과 A fraction은 알팔파건초에서 각각 61% 및 41.77%로 가장 높았으며 볏짚에서 가장 낮았다(각각 42.8% 및19.78%). 총단백질 중 B1 fraction은 조사된 조사료간 비교적 큰 차이를 보이지 않았으나 B2fraction에서는 다른 조사료(6.34~8.85%)에 비하여 톨페스큐짚(10.05%) 및 클라인 건초(12.34)%에서 다소 높은 수준을 보였다. 총 단백질 중B3 fraction이 차지하는 비율은 톨페스큐짚에서38.49%로 가장 높았으나 다른 조사료 자원 간에는 큰 차이가 없었으며, C fraction의 경우 볏짚에서 가장 높은 비율(15.05%)을 보였다. 모든 사료에서 배양 개시 후 3시간(P<0.01) 및 6시간(P<0.05)에서 buffer 추출 전에 비해 추출후 배양액의 pH가 증가되었으며, 배양 6시간(P<0.05) 및 12시간(P<0.001)에서 다른 사료에비해 티모시 건초 및 알팔파 건초로부터의 pH가 낮았다. 배양액의 암모니아 농도는 모든 배양시간에서 가용성 물질의 추출 전 후에 다른조사료에 비해 알팔파 건초에서 가장 높았으나모든 사료의 추출효과는 배양 3시간(P<0.01)에서만 나타났다. 배양액의 총 VFA 농도는 배양24시간까지 알팔파 건초에서 가장 높았던 반면톨페스큐짚과 볏짚에서 가장 낮았다. 또한 모든 조사료에서 buffer 추출 전에 비하여 추출후에 총 VFA 농도가 감소되었다(P<0.01~P<0.001). Acetic acid (C2)의 조성 비율에서는 배양 6시간까지 추출 전에 더 높았으나(P<0.001)사료 간 차이는 없었다. Propionic acid (C3) 조성 비율 역시 배양 개시 후 3, 24 및 48시간(P<0.001)에 추출 전에 더 높았으며, 6 및 12시간의 배양액에서 대부분 건초(티모시, 알팔파 및 클라인)와 짚류(톨페스큐짚 및 볏짚) 간차이가 있는 것으로 조사되었다(P<0.05). 그러나 butyric acid (C4) 조성비율의 경우 대부분의배양시간에서 사료 간 차이는 없었다. 건물에서의 분해율 관련 parameter 중 a 값은 조사된전체 조사료에서 buffer 추출 전이 추출 후에비해서 높았으며(P<0.001), 다른 조사료에 비해톨페스큐짚과 볏짚에서 크게 낮았다(P<0.05).또한 b 값의 경우 역시 추출 전에 비해 추출후에서 현저히 낮았으나(P<0.001) 사료 간 차이는 없었다. 볏짚을 제외한 조사료에서 추출후에 비해 추출 전의 건물 유효분해율(EDDM)이 더 높았다(P<0.001). 조단백질에서의 a, b및 c 값은 추출 전에 비해 추출 후에서 현저히낮았으나(P<0.05) 사료 간 차이는 없었다. 조단백질 유효분해율(EDCP)에서는 다른 조사료 종류에 비해 톨페스큐짚과 볏짚에서 낮았다(P<0.05). 한편, NDF의 경우 a 값과 b 값(P<0.01)및 NDF 유효분해율(EDNDF, P<0.001)은 추출후에 비해 추출 전에 더 높았으나(P<0.01) 사료 간 차이는 보이지 않았다. 반추위미생물에의해 사료분해과정 중 생성되는 CO2 량도 24시간 배양까지는 추출 전에 더 많았으며(P<0.05~P<0.001), 톨페스큐짚과 볏짚에 비해 건초형태의 조사료로부터의 CO2 생성량이 더 많았다(P<0.05~P<0.01). 메탄가스(CH4) 생성량 역시 모든 배양시간에서 추출 전에 비해 추출 후에 크게 감소되었으며(P<0.01~P<0.001), 12~24시간을 제외하고는 짚류에 비해 건초에서 현저히 높은(P<0.05) 것으로 나타났다. 본 시험의 결과를 종합하면, 조사료 자원에 대한 buffer용해도와 단백질의 분획이 in vitro VFA 농도와 분해율 및 gas (CO2 및 CH4) 발생량 간 상호 밀접한 관계를 보이는 것으로 여겨진다. 이에 따라 조사료 이용 효율 개선을 위해 조사료자원에 대한 buffer 용해도와 단백질 분획을 반추동물 TMR 조제에 활용할 필요가 있는 것으로 여겨진다.

Pigs may be considered as a suitable organ source for its characteristics in xenotransplantation if significant immunological barriers can be overcome. However, xenograft could be rejected by T cells, especially CD8+ cytotoxic T lymphocytes (CTL)-mediated response, because these elements show great cytotoxicity against xenograft by recognizing Swine Leukocyte Antigen (SLA)-I. Human cytomegalovirus (HCMV) encodes unique short (US) 11 gene, which interferes with cellular immune responses by inducing rapid degradation of newly synthesized heavy chains (HC) of MHC class I from endoplasmic reticulum (ER) to the cytosol. In this study we established two US11 clonal cell lines by transfection into minipig fetal fibroblasts and confirmed the integration of US11 gene by PCR and FISH. The reduction of Swine Leukocyte Antigen (SLA)-I which was expressed on the cell surface by US11 was also detected by flow cytometry assay. The level (14.6 % to 21.2%) of SLA-I expression in US11 clonal cell lines was decreased relative to the control. The reconstructed embryos were produced with these clonal cells and transferred to nine surrogate gilts. Ultrasound examination of recipient surrogates on days 35 after embryo transfer confirmed established pregnancies in two recipients. One recipient delivered one piglet with normal birth weight. PCR analysis revealed that transgene vector was integrated in the offspring genome. Transgene-expression analysis and CTL assay are currently underway. The present results show that transgenic pig was produced with US11 cDNA for controlling cell-mediated rejection. This result indicated that grafts of transgenic pigs expressing human cytomegalovirus protein US11 could control the cellular immune response to xenografts, and create a window of opportunity to facilitate xenograft survival. This research was supported by the BioGreen 21 Program (#20110301-061-541- 001-05-00), Rural Development Administration, Republic of Korea.