Virus-like particles (VLPs) are similar to pathogenic viruses, but because they have no nucleic acid, they have excellent safety and immunogenicity and are used as a good vaccine material. However, in the selection of various structural proteins of pathogenic viruses to form VLPs, all expression systems consume a lot of time in common. Among them, the baculovirus expression system causes additional time consumption to construct the recombinant baculovirus. Therefore, there is a need for a system that can rapidly determine the structural proteins required for effective VLP production. This study aims at solving this problem by constructing a BmNPV inducible expression platform through the construction of vectors induced by BmNPV. The platform was evaluated for overexpression using EGFP. We also confirmed the formation of virus-like particles through overexpression of canine parvovirus structural proteins.

The baculovirus expression vector system (BEVS) is an effective and widely used system for the production of recombinantproteins in insect cells or larvae. However, the expression efficiency of recombinant proteins using the polyhedrin promotercould not acquire the protein yields observed for native polyhedrin. In this study, we tried to develop hyper expressionvector by the optimal combination of previously reported various enhancer factors. The selected enhancer factors for optimalexpression consists homologous region5 (hr5), VP39 promoter and burst sequences. Seven recombinant viruses were madeto compare EGFP expression level. Each recombinant viruses showed different expression levels respectively, and themost of expression level was observed with higher than those of the previous vectors. This study suggests a new optionfor hyper expression of useful recombinant protein using the BEVS.

Viral particles of Porcine epidemic diarrhea virus (PEDV) consist of a four structural proteins. Among them Spikeprotein mediated responsible for receptor binding and membrane fusion during viral infection and therefore the main targetof neutralizing antibodies. Virus-like particles (VLPs) are consisted of one or more viral structural proteins, and theirmorphologies closely resemble those of the native virus. VLPs have no virulence and can elicit robust immune responsesas compared with inactivated or live-attenuated virus vaccines. Thus, in this study, we tried two methods for VLP constructionin Bombyx mori, one is traditional method and the other is chimeric VLP method using the influenza matrix protein.Both methods could produce successfully PEDV VLPs.

The baculovirus expression vector system (BEVS) is an effective and widely used method for the production of recombinant proteins in insect cells or larvae. However, the expression efficiency of foreign proteins using the polyhedrin promoter could not obtain the protein yields observed for native polyhedrin. To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of various enhancer factor were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). As a hyper expression factor, the optimal hyper BEVS was constructed by combining Hr5 sequence, VP39 promoter and Burst sequences. Additionally, the proteins expressed by the hyper expression system was markedly increased. This study suggests a new option for higher expression of useful foreign recombinant protein using the BEVS.

The production of therapeutic proteins from transgenic animals is one of the most important successes of animal biotechnology. Endostatin is 20 KDa C-terminal fragment derived from type XVIII collagen and an endogenous inhibitor of tumor growth by inhibition of angiogenesis. In this study, we are developed knock-in vector consists of 5’ arm region (1.02 kb), human Endostatin cDNA, CMV-EGFP, and 3’ arm region (1.83 kb). To express Endostatin gene as transgene, the F2A sequence was fused to the 5’ terminal of Endostatin gene and inserted into exon 3 of the β -casein gene. If this knock-in vector is inserted into the porcine β-casein gene locus by homologous recombination, human Endostatin mRNA are expressed using the gene regulatory region of the β-casein. Also, the β-casein and Endostatin fusion protein is translated and Endostatin protein is separated by F2A self cleavage during translation. In conclusion, our knock-in vector may help to create transgenic pig expressing human Endostatin protein via the endogenous expression system of the porcine β-casein gene in the mammary gland.

Recombinant proteins including a polypeptide fusion partner, termed affinity tag, to facilitate the purification of the target polypeptides are widely used. However, the affinity tag must be removed from the target after the purification process. Recently, the Synechocystis sp. PCC6803 DnaB mini-intein (Ssp DnaB mini-intein) is widely used in Escherichia coli expression systems as the solution of this problem. The Ssp DnaB mini-intein can be induced simply by shifting of pH and temperature, offering a benefit to cleave a peptide bond without using a protease or chemical reagent. Although the utility of this novel tag is widely studied in E. coli, there is no report yet in baculovirus expression vector system (BEVS). In this study, we generated several recombinant baculoviruses to express foreign proteins with Ssp DnaB mini-intein. In conclusion Ssp DnaB mini-intein was good tag also in BEVS with more advantages.

Nucleotide metabolism in endothelium is variable between different species. Recent studies demonstrated that this variability could contribute coagulation dysfunction, even though organs of the alpha 1,3-galactosyltransferase gene knockout pig were transplanted into the primate. CD73 (ecto-5'-nucelotidase) is an enzyme at cell surface catalyzing the hydrolysis of adenosine triphosphate to adenosine, which plays role on a substance for anti-inflammatory and anti-coagulant. Thus, overexpression of CD73 in endothelial cells of the pig is considered as an approach to reduce coagulopathy. In this study, we constructed a human CD73 expression vector under control of porcine Icam2 promoter (pIcam2-hCD73), which is expressed specifically at endothelial cells, and of CMV promoter as a control (CMV-CD73). First, we transfected the CMV-CD73 vector into HEK293 cells, and then confirmed CD73 expression at cell surface by flow cytometry analysis. Next, we transfected the pIcma2-CD73 and CMV-CD73 vectors into primary porcine fibroblasts and endothelial cells. Consequence was that the pIcma2-CD73 vector was expressed only at the porcine endothelial cells, meaning that the pIcam2 promoter lead to endothelial cell-specific expression of CD73 in vitro. Finally, we nucleofected the pIcam2-hCD73 vector into passage 3 fibroblasts, and enforced hygromycin selection of 400mg/ml. We were able to obtain forty three colonies harboring pIcam2-CD73 to provide donor cells for transgenic cloned porcine production.

Brucellosis is an important and re-emerging zoonotic disease worldwide. The prevention of human infection is achieved predominantly through the control of brucellosis in agricultural animals, which in turn depends on accurate diagnosis and vaccination. However, conventional serological diagnosis of brucellosis has several limitations, and currently available vaccines for animals have several drawbacks, including the ability to cause infection in humans. Phosphoglycerate kinase (Pgk) is one of the specific proteins reactive with mouse sera in the early stage of Brucella infection, and deletion of the pgk gene in B. abortus strain 2308 resulted in extreme attenuation of this strain in vitro and in vivo. Furthermore, the B. abortus pgk mutant has been used as a live vaccine, and in challenge experiments, it induced protection that was superior to that conferred by commercial strains. In this study, the pgk gene from Brucella abortus 544 was successfully amplified and cloned into a maltose binding protein fusion protein expression vector (pMAL). The recombinant protein was expressed in Escherichia coli DH5α and purified. The immunogenicity of purified recombinant B. abortus 544 Pgk (rPgk) was evaluated by western blot analysis using Brucella-positive mouse sera. rPgk could be used as an antigenic component for future serological tests and potential vaccine development.

The genus Diadegma is a well known parasitoid group and some are known to have symbiotic virus, PDV. A novel IV was discovered from the calyx of D. fenestrale female. This virus was named as D. fenestrale Ichnovirus (DfIV). The encapsidated DfIV genome contains 65 circular DNA segments with an aggregate size of 247,191 bp. Based on BLAST analysis, a total of 120 ORFs were predicted as follows: rep; 48, cys-motif; 11, vinnexin; 10, vankyrin; 9, PRRP; 3 and other unassigned genes (39). These viral genes were expressed in lepidopteran hosts (Phthorimaea operculella and Plutella xylostella) after parasitization which means DfIV genome segments were integrated into lepidopteran hosts. This study was focused on this result. Based on gene expression profile, candidate promoter and integration motifs were selected and then, fused with eGFP as a reporter gene. Modified DfIV genome segment was ligated to a commercial containing f1 ori and Ampr gene to propagate in E. coli. We have named this fusion vector as pIN. The construction methodology of pIN and its application would be further discussed in detail.

The baculovirus expression system is one of the most popular methods used for the production of recombinant proteins but has several complex steps which have proved inherently difficult to meet a multi-parellel process. We have developed a novel recombinant bacmid, bEasyBm that enabling easy and fast generation of pure recombinant virus without any purification step. In the bEasyBm, attR recombination sites were introduced to facilitate the generation of recombinant viral genome by in vitro transposition. Moreover, extracellular RNase gene from bacillus amyloliquefaciens, barnase, was expressed under the control of Cotesia plutellae bracovirus early promoter. Therefore, only when the barnase gene was replaced to gene of interest, the bEasyBm could replicate in host insect cells. When the bEasyBm was transposed with pDualBac-EGFP and pDualBac-LUC respectively, there were no non-recombinant backgrounds were detected from unpurified BmEasy-EGFP or BmEasy-LUC stocks. In addition, the resulting recombinant virus, BmEasy-EGFP, showed comparable level of EGFP expression efficiency with the plaque-purified recombinant virus, BmEGFP, which was constructed using bBmGOZA system. Based on these results, high-throughput condition for generation of multiple recombinant viruses in a time was established.

Chicken Insulin-like Growth Factor-1 (cIGF-1), one of the most important hormone for regulating physiological function includes body growth, muscle volume, bone density, chicken cell development and metabolism. In order to find in vitro Knokdown expression of cIGF-1, this study introduced tetracycline inducible RNA interference expression system (TetRNAi system). Tet system can inductively control high expression of extrinsic genes and expression of intrinsic genes. So it has advantages such as minimized physiological side-effects any cell and low cytotoxicity. RNAi system is proving to be a powerful experimental tool for inhibition of gene expression and post-transcriptional mechanism of gene silencing. RNAi is mediated by small interfering RNA (siRNA) consisting of 19- to 23- nucleotide double-stranded RNA duplexes that promote specific endonucleolytic cleavage of mRNA targets through an RNA-induced silencing. Then, this study RNAi-based gene knockdown can be achieved by retroviral-based expression systems. Stable integration of our inducible siRNA vector allowed the production of siRNA on doxycycline induction, followed by specific down regulation of chicken IGF-1 gene. Analyses of Real-time PCR to determine expression of the cIGF-1 gene showed successful from chicken embronic fibroblast (CEF) cells with the reduced rate of an approximately 92%. Our results demonstrate the successful regulation of cIGF-1 knockdown expression in CEF cells and support the application of an tetracycline inducible RNAi expression system in transgenic Mini chicken production. This research was supported by Bio-industry Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

Human growth hormone (hGH), one of the most important hormones in medicine, is secreted from anterior pituitary gland. Its broad physiological function includes body growth, cell regeneration, increasement of muscle volume, bone density, body fat reduction, and so on. Due to the wide range of therapeutic effects, the hGH produced from E. coli has been commercialized already. In this study, we asked whether it is possible to produce recombinant hGH efficiently from various cultured mammalian cells. To meet this purpose, we chose a retrovirus vector system for transfer and expression of the hGH gene in various mammalian cells. Analyses of RT-PCR, ELISA, and Western blot to determine expression of the hGH gene showed the highest production of the hGH was determined from chicken embronic fibroblast (CEF) cells with the concentration of 8.58 μg/ml. The biological activity of the hGH was similar to the commercially available counterpart. These results suggest that mass production of hGH is possible not only in the E. coli but also in the various mammalian cells.

hFSH is a glycoprotein secreted from anterior pituitary and consists of α and β subunits. Because of its major biological functions including sperm formation in the male and for follicular growth, FSH is used to cure woman's sterility. In this study we tried to produce recombinant hFSH in vitro using a retrovirus expression vector. Two major components of the vector we constructed are: (ⅰ) a DNA fragment containing α and β genes fused by a DNA sequence coding carboxyl terminal peptide (CTP) of human chorionic gonadotropin, (ⅱ) a DNA fragment corresponding woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Evaluation of expression profile of the recombinant FSH using reverse transcription PCR and enzyme-linked immunosorbent assay (ELISA). Among three cell lines tested, HeLa cells were the best for hFSH expression (5,395 mIU/ml), then followed by chicken embryonic fibroblast (CEF) cells and Chinese hamster ovary (CHO) cells in the order of hFSH production. In addition to the amount, the FSH produced from HeLa cells was highest in terms of biological activity which was determined by measuring cAMP.

The sweet potato whitefly Bemisia tabasi is one of the most important pests of various horticultural crops. In addition, B. tabaci is a vector of many plant-pathogenic viruses and cause a serious secondary damage to crop plants. Association of plant-pathogenic virus with vector insects is known to be effective on the transmission capacity, fecundity, longevity of vectors including whiteflies. However, the interactive mechanisms between virus and vector insects are still poorly understood. Recently, a serious damage caused by virus disease together with B. tabasi emergence was identified at tomato glasshouse in Tongyoung. We detected the signals of Tomato Yellow Leaf Curl Virus (TYLCV) in tomato leaves and vector whiteflies using PCR amplification and confirmed its presence by those sequence comparison. To determine the effects of TYLCV acquisition on physiological status of vector whiteflies, transcript levels of genes that associated with metamorphosis, metabolism, stress and immune processes were compared between TYLCVinfected whiteflies and non-infected ones. Generally, the transcript levels of virus-infected whiteflies were lower than those of non-infected ones. In addition, the associations of endosymbiont levels within whiteflies were discussed in aspect of the acquisition and transmission of TYLCV.

본 연구는 돼지 B-casein 유전자 위치에서 EGFP가 발현될 수 있는 knock-in 벡터를 구축하기 위하여 실시되었다. 돼지의 B-casein 유전자를 이용하여 knock-in 벡터를 구축하기 위해 돼지의 태아 섬유아세포로부터 B-casein 유전자를 동정하였고 EGFP, SV4O polyA signal을 동정하였다. Knock-in 벡터는 5' 상동 영역 약 5 kb와 3' 상동 영역 약 2.7 kb로 구성되어있으며, positive selection marker로 neor 유전자를, negative selection marker로 DT-A 유전자를 사용하였다. 구축된 knock-in 벡터로부터 EGFP의 발현을 확인하기 위하여 생쥐 유선 세포인 HC11 세포에 knock-in 벡터를 도입하였다. 그 결과 EGFP의 발현을 HC11 세포에서 확인하였다. 이와 같은 결과로서 이 block-in 벡터는 knock-in 형질전환 돼지를 생산하는데 사용될 수 있을 것으로 생각된다.

본 연구에서는 외래 유전자의 지속적인 발현에 의한 형질 전환 개체나 세포의 생리적인 부작용을 최소화하기 위하여 hTPO 유전자의 발현을 조절할 수 있는 tetracycline-inducible retrovirus vector system을 구축하고자 하였다. hTPO 유전자는 사람의 간암세포인 HepG2에서 분리한 RNA를 주형으로 하여 RT-PCR 방법을 이용하여 확보하였으며, 이 유전자를 MLV 유래의 vector에 도입하여 pLNChTPOW를 재조합하였다. 재조합한 vector는 GP2 293 포장세포에 도입하여 바이러스를 생산하였으며, 이 바이러스를 이용하여 감염시킨 여러 표적세포에서 hTPO의 발현을 확인하였다. 또한, hTPO의 발현을 유도적으로 조절할 수 있도록 하기 위하여 hTPO를 one vector 형태의 Tet-On vector system에 도입하였으며, 발현의 유도 조건에서 보다 강한 발현을 위하여 WPRE 서열을 여러 위치에 도입하였다. 구축한 Tet system의 발현 조절 정도는 각 바이러스를 감염시켜서 구축한 CEF와 PFF 세포에서 RT-PCR과 Western blot, 그리고 ELISA 방법을 이용하여 확인하였다. 그 결과, CEF에서는 WPRE 서열이 hTPO 유전자의 3'에 위치한 경우에서, PFF에서는 WPRE가 rtTA의 3'에 위치한 경우의 vector system에서 가장 높은 발현율과 유도율을 나타내었다. 이는 Tet system에서의 hTPO 유전자 발현 조절이 매우 효율적으로 이루어지며, 세포주에 따른 의존적인 조절 양상을 보이는 것을 의미한다. 따라서 hTPO의 대량 생산을 위한 생체 반응기로서의 형질 전환 동물의 개발을 보다 효율적으로 수행하려면 적절한 Tet system이 선별적으로 적용되어야 할 것이다.

본 연구에서는 hG-CSF의 발현을 유도적으로 조절하기 위한 FIV-Tet-On lentivirus vector system을 구축하고자 하였다. hG-CSF는 호중성구 계열 세포의 증식과 분화, 생존에 영향을 미치는 물질로서, 이 유전자의 발현을 증가시키기 위하여 FIV-Tet-On vector 상의 hG-CSF나 rtTA2SM2 서열의 3' 위치에 WPRE 서열을 도입하였다. 구축된 각각의 vector는 293FT 세포에 일시적으로 transfection하여 virus를 생산하였으며, 이 virus를 일차 배양 세포인 CEF와 PFF에 감염시켰다. 각 세포에 전이된 hG-CSF의 발현 양상을 관찰하기 위하여 doxycycline을 첨가하거나 첨가하지 않은 배지에서 배양한 후 quantitative real-time PCR, Western blot과 ELISA를 이용하여 hG-CSF 유전자의 발현 정도를 비교 측정한 결과, CEF에서는 WPRE가 hG-CSF의 3' 위치에 도입된 경우에 발현량과 유도율이 가장 높은 것으로 나타났고, PFF에서는 rtTA 서열의 3'위치에 도입된 경우에 발현량과 유도율이 가장 큰 것으로 확인되었다. 이 FIV-Tet-On vector system은 형질 전환 동물의 생산이나 유전자 치료에서 문제시되는 외래 유전자의 지속적인 과다 발현에 의한 개체의 생리적인 부작용을 최소화하기 위한 해결 방법으로 제시될 수 있을 것이다.