• International Journal of Industrial Entomology and Biomaterials
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• 제34권1호
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• pp.1-5
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• 2017

Bone morphogenetic protein 2 (BMP2) plays an important role in the development of bone and cartilage. It is involved in the hedgehog pathway, TGF beta signaling pathway, and in cytokine-cytokine receptor interaction. It is involved also in cardiac cell differentiation and epithelial to mesenchymal transition. In this study, We expressed human BMP2 (hBMP2) recombinant protein using Baculovirus Expression Vector System (BEVS) in Sf9 insect cells. The hBMP2 cDNA was cloned into baculovirus transfer vector, pBacgus-4x-1 and recombinant baculovirus was screened out through X-gal and GUS-fusions assay. Western blot analysis shown that molecular weight of hBMP2 recombinant protein was about 44.71 kDa.

• Animal cells and systems
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• 제16권6호
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• pp.455-461
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• 2012

Amongst the various antimicrobial peptides, lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. Here we propose the biosynthesis and refolding of recombinant lysozyme in Escherichia coli expressed in inclusion body form. The Agrius lysozyme gene was amplified using gene specific primers and then ligated into the pGEX-4T-1 vector, which contained the glutathione S-transferase (GST) gene as a fusion partner. A recombinant lysozyme was expressed in E. coli Rosetta cells using a pGEX-4T-1 expression vector, and the fusion protein was induced by ioporpyl-${\beta}$-D-thiogalactopyranoside (IPTG). The recombinant protein produced as an inclusion body was resolubilized in solubilization buffer, and the resultant solution was dialyzed in refolding buffer. After thrombin cleavage, the recombinant lysozyme was purified by ion exchange chromatography and reverse phase chromatography. The recombinant lysozyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoreactivity against the anti-Agrius lysozyme was observed by western blot analysis of this protein. The recombinant lysozyme displayed antibacterial activity against Bacillus megaterium and Micrococcus luteus, which was confirmed by the inhibition zone assay.

• Reproductive and Developmental Biology
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• 제35권1호
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• pp.115-121
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• 2011

hFSH is a glycoprotein secreted from anterior pituitary and consists of ${\alpha}$ and ${\beta}$ 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 ${\alpha}$ and ${\beta}$ 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.

• 한국미생물·생명공학회지
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• 제20권6호
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• pp.637-641
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• 1992

한국 재래식 된장.간장 발효균 Bacillus sp. SSA3 균주의 expression vector를 개발하기 위해 Bacillus sp. SSA3의 chromosomal DNA로부터 유전자의 promoter 부위를 cloning 하였다. Recombinant plasmid를 제작하기 위하여 Bacillus sp. SSA3의 chromosomal DNA을 HindIII로 절단한 단편을 pGR71 plasmid의 CAT gene과 pUC18 plasmid의 $\beta$-galactosidase gene의 전방에 삽입시킨 후, E.coli JM109에 형질전환하였다.E. coli JM109의 chloramphenicol 내성 clones으로부터 6 recombinant plasmid를 선별하였다. 이들 선별된 plasmid는 Bacillus sp. SSA3의 expression vector로 사용시 각 재조합 plasmid 중에 삽입된 promoter의 염에 대한 영향의 정도를확인하기 위해 10% NaCl이 첨가된 LB medium상에서 배양하였을 때, 이들 중 Bacillus sp. SSA3의 4 clones은 융합 CAT gene의 발현이 강하게 감소되었으나 2 clones은 약하게 저해되었다.

• BMB Reports
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• 제41권10호
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• pp.739-744
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• 2008

Turnip yellow mosaic virus (TYMV) is a positive strand RNA virus that infects mainly Cruciferae plants. In this study, the TYMV genome was modified by inserting an extra subgenomic RNA promoter and a multiple cloning site. This modified TYMV was introduced into Nicotiana benthamiana using a Agrobacterium-mediated T-DNA transfer system (agroinfiltration). When a gene encoding $\beta$-glucuronidase or green fluorescent protein was expressed using this modified TYMV as a vector, replication of the recombinant viruses, especially the virus containing $\beta$-glucuronidase gene, was severely inhibited. The suppression of replication was reduced by co-expression of viral silencing suppressor genes, such as tombusviral p19, closteroviral p21 or potyviral HC-Pro. As expected, two subgenomic RNAs were produced from the recombinant TYMV, where the larger one contained the foreign gene. An RNase protection assay revealed that the recombinant subgenomic RNA was encapsidated as efficiently as the genuine subgenomic RNA.

• Journal of Microbiology and Biotechnology
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• 제14권1호
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• pp.1-14
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• 2004

More than twenty years have passed since the approval of the first recombinant DNA product for therapeutic use (recombinant human insulin, 1982). However, the biotechnology industry is still facing a shortage of manufacturing capacity due to the increasing demand of therapeutic proteins. This demand has prompted the search for a growing number of biological production systems but, nevertheless, the Gram-negative bacterium Escherichia coli remains one of the most attractive production hosts. This review highlights the most important features and developments of plasmid vector design, emphasizing the different reported strategies for improving the expression and secretion of heterologous proteins using the cellular machinery of E. coli.

• Journal of Microbiology and Biotechnology
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• 제2권2호
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• pp.122-128
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• 1992

Post-translational modifications of the nucleocapsid protein of bovine coronavirus (Quebec strain) were investigated. Coronavirions were radiolabelled in vivo with inorganic $[^{32}P]$orthophosphate and analysed by SDS-PAGE, followed by autoradiography. A single polypeptide with a migration rate of 55 KDa was identified by metabolic phosphate labelling, demonstrating that the nucleocapsid protein of bovine coronavirus was a phosphoprotein. A gene encoding the nucleocapsid protein was inserted immediately downstream from the polyhedrin promoter of Autographa californica nuclear polyhedrosis baculovirus. Spodoptera frugiperda cells infected with this recombinant baculovirus synthesized a 55 KDa polypeptide, as demonstrated by immunoprecipitation with anti-nucleocapsid monoclonal antibody. The recombinant nucleocapsid protein synthesized in Spodoptera cells could also be labelled by $[^{32}P]$orthophosphate. Phosphoamino acid analysis showed that both serine and threonine residues were phosphorylated in authentic, as well as in recombinant nucleocapsid proteins, with a relative phosphorylation ratio of 7:3. Our studies demonstrated that the nucleocapsid protein of bovine coronavirus was a serine and threonine-phosphorylated protein and that Spodoptera insect cells were able to properly phosphorylate the relevant foreign proteins.

• 생명과학회지
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• 제11권4호
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• pp.385-391
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• 2001

Filamentous hemagglutinin (FHA) is considered as an essential immunogenic component for incorporation into acellular vaccines against Bordetella pertussis, the causative agent of whooping cough. Classically, antipertussis vaccination has employed an intramuscular route. An alternative approach to stimulate mucosal and systemic immune responses is oral immunization with recombinant live vaccine carrier strains of Salmonella typhimurium. An attenuated live Salmonella vaccine sgrain($\Delta$cya $\Delta$crp) expressing recombinant FHA(rFHA) was developed. Stable expressionof rFHA was achieved by the use of balanced-lethal vector-host system. which employs an asd deletion in the host chromosome to impose in obligate requirement for diaminopimelic acid. The chromosomal $\Delta$asd mutation was complemented by a plasmid vector possessing the asd$^{+}$ gene. A 3 kb DNA fragment encoding immuno dominant regionof FHA was subcloned in-frame downstream to the ATG translation initiation codon in the multicopy Asd$^{+}$ pYA3341 vector to create pYA3457. Salmonella vaccine harboring pYA3457 expressed approximately 105kDa rFHA protein. The 100% maintenance of [YA3457 in vaccine strain was confirmed by stability examinations. Additionally, a recombinant plasmid pYA3458 was constructed to overpress His(8X)-tagged rFHA in Essherichia coli. His-tagged rFHA was purified from the E. coli strain harboring pYA3458 using Ni$^{2+}$-NTA affinity purification system.>$^{2+}$-NTA affinity purification system.

• 대한의생명과학회지
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• 제8권3호
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• pp.107-113
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• 2002

Enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 have been shown to colonize the intestinal epithelial cell by the attaching and effacing (AE) mechanism. The AE lesion is mediated by an intimin, of which production and expression are controlled by a 3-Kb eaeA gene located EHEC chromosomal DNA. If the eaeA gene is mutated, EHEC O157:H7 strains lose capacity of adhesion to intestinal epithelial cells. In this study, a 891 bp of the 3'-end region of a gamma intimin was amplified by polymerase chain reaction (PCR). The PCR product was inserted into pSTBlue-1 cloning vector and transformed into DE3 (BL21) competent cell. After plasmid mini-preparation and restriction enzyme digestion of eaeA/891-pSTBlue-1 vector, target eaeA gene was re-inserted into pET-28a expression vector and was transformed. Then the expression of recombinant eaeA/891 (891 bp) gene was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG). The expression of the 40-KDa recombinant protein was identified in SDS-PAGE and confirmed by immunoblotting using the His.Tag$^{\circledR}$ and T$_{7}$.Tag$^{\circledR}$ monoclonal antibody. This recombinant protein expressed by eaeA gene could be applied in further studies on the mechanisms of E. coli O157:H7 infection and the development of recombinant vaccine.

• Journal of Genetic Medicine
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• 제3권1호
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• pp.33-37
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• 1999

Recombinant adenovirus vector systems with strong promoters have been used to achieve high level production of recombinant protein. However, this overexpression system cause some problems such as disturbance of cell physiology and increment of cellular toxicity. Here, we showed a tetracycline-regulated adenovirus expression vector system. Our results showed that the expression level of transgene(p-53) was high and easily regulated by tetracycline. In addition, the maximal gene expression level of the tetracycline-controlled gene expression system was higher than that of the wild type CMV promoter system. Therefore, tetracycline-regulated adenoviral vector system could be applicable for regulatory high-level expression of toxic gene. Also, this system will be useful for functional studies and gene therapy.