• Applied Biological Chemistry
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• v.38 no.5
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• pp.382-386
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• 1995

The potato proteinase inhibitor II (PI-II) protein contains chymotrypsin and trypsin inhibitory site. Among several PI-II genes isolated from genomic library, amino acid sequence deduced from PI-IIT gene has 84% identity with that of the polypeptide chymotrypsin inhibitor (PCI). Therefore a gene fragment having homology with the PCI was cloned into a vector using polymerase chain reaction(PCR) from the potato proteinase inhibitor IIT gene. Two different primers were utilized for cloning; primer A contains NdeI restriction site and 30 nucleotides, which has AUG N-terminal methionine codon, primer B contains BclI restriction site and 28 nucleotides, which has TAG translation stop codon. After PCR, about 160 bp-long DNA fragment was cloned into pRT146, derivative of pUC118, and sequenced. The sequenced NdeI/BclI fragment was moved to pET3a, containing bacteriophage T7 promoter and terminator. The expressed proteins in E. coli BL2l(DE3) were determined on a polyacrylamide gel containing sodium dodecyl sulfate. The expected size of protein deduced from the sequenced gene fragment is about 6,500 dalton whose size was similar to the IPTG-induced protein (6,000 dalton) on a gel. However the expression level was much lower than expected.

• Journal of Life Science
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• v.13 no.3
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• pp.291-297
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• 2003

A human endogenous retroviral family (HERV-W) has recently been described that is related to multiple sclerosis-associated retrovirus (MSRV) sequences that have been identified in particles recovered from monocyte cultures from patients with multiple sclerosis. Two pol fragments (HWP-FB10 and HWP-FBl2) of HERV-W family were identified and analysed by the PCR approach with cDNA library of human fetal brain. They showed 89 percent nucleotide sequence similarity with that of the HERV-W (accession no. AF009668). Deletion/insertion or point mutation in the coding region of the pol fragments from human fetal brain resulted in amino acid frameshift that induced a mutated protein. Phylogenetic analysis of the HERV-W family from GenBank database indicates that the HWP-FB10 is very closely related to the AC000064 derived from human chromosome 7q21-q22. Further studies on the genetic relationship with neighbouring genes and functional role of these new HERV-W pol sequences are indicated.

• Journal of Life Science
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• v.26 no.6
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• pp.721-726
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• 2016

It has already been reported that short neuropeptide F (sNPF) stimulates feeding behaviors in a wide variety of insect species. In the present study, we cloned cDNA, encoding a sNPF receptor homologue from a silkworm, Bombyx mori, named BsNPF-R. The amino acid sequence of BsNPF-R was compared with those of sNPF-R thus far reported, which is shared with humans (36%), mice (34%), zebrafish (35%), and fruit flies (51%), respectively. A BsNPF-R protein’s mass was theoretically estimated to be 42,731 Da and it is a putative plasma membrane-penetrating protein. The mRNA expression of BsNPF-R was tested; the results showed that a strong expression was detected at the midgut, post-silk gland, Malpighian, and testis; however, a weak expression was at the fat body, hemocyte, and ovary. In addition, the synthesized sNPF of a silkworm regulated the BsNPF-R mRNA expression through the cell-based functional analysis.

• Journal of Sericultural and Entomological Science
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• v.49 no.2
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• pp.37-42
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• 2007

Lactoferrin, an ion-binding 80-kDa glycoprotein, has been suggested to have many biologic activities, such as facilitating ion absorption and having antimicrobial and anti-inflammatory effects. Several of these activities are likely to only be facilitated by human lactoferrin because they depend on the binding of human lactoferrin to specific receptor. To produce recombinant human lactoferrin to animal foods using transgenic silkworm, Bombyx mori L, we have cloned and sequenced the cDNA encoding for a human lactoferrin (HLf) from the mRNA in mammary tumor line (GI-101). As a result, the 2.5-kb fragment of HLf gene was cloned with pGEM-T vector and then this fragment was sequenced. In the nucleotide sequence analysis, single open reading frame of the 2,136-bp encoding for a polypeptide of 712 amino acid residues was detected. On the other hand, we constructed a recombinant plasmid(pPT-HLf), containing human lactoferrin gene for germ line transformation of the silkworm using a piggyBac transposon-derived vector. A nonautonomous helper plasmid encodes the piggyBac transposase. Approximately 6.7% of individuals in the G0 silkworms expressed green fluorescent protein (GFP). PCR analyses of GFP-positive silkworms (G0 and G1) revealed that independent insertions occurred frequently. Furthermore, Western blot analysis showed that the recombinant HLf expressed in hemolymph has the same molecular weight (80 kDa) as a native protein. On the basis of these experiments, expression of HLf in next generation of transgenic silkworm is now in process.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.1013-1021
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• 2005

The metagenomes of complex microbial communities are rich sources of novel biocatalysts. The gene encoding an extracellular $\alpha$-amylase from a genomic DNA of cow rumen was cloned in Escherichia coli DH5$\alpha$ and sequenced. The $\alpha$-amylase (amyA) gene was 1,893 bp in length, encoding a protein of 631 amino acid residues with calculated molecular weight of 70,734 Da. The molecular weight of the enzyme was estimated to be about 71,000 Da by active staining of a SDS-PACE. The enzyme was 21 to $59\%$ sequence identical with other amyloyltic enzymes. The AmyA was optimally active at pH 6.0 and $40\%$. The AmyA had a calculated pI of 5.87. AmyA expressed in E. coli DH5$\alpha$ was enhanced in the presence of $Mg^{2+}$ (20 mM) and $Ca^{2+}$ (30 mM) and inhibited in the presence of $Fe^{2+}$ and $Cu^{2+}$. The origin of amyA gene could not be confirmed by PCR using internal primer of amyA gene from extracted genomic DNA of 49 species rumen culturable bacteria so far. An amyh is supposed to obtained from unculturable rumen bacterium in cow rumen environment.

• Journal of Life Science
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• v.12 no.3
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• pp.264-273
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• 2002

We have isolated and artificially expressed three cDNA clones of Capsicum annuum PR5 genes for elucidating the antifungal activity against Phytophthora capsici which contracted a hot pepper root rot in field condition. Three divergent PR5 proteins from hot pepper were designated as CAPR5-1 and CAPR5-2 from susceptible cultivar (Subicho) as well as CAPR5-3 from resistant cultivar (CM331) in response to P. capsici. The cDNA similarity was found over 80% of identity among the three CAPR5s, and deduced amino acid sequence was characterized that all of CAPR5s contained 16 cysteine residues which possibly had a significant role in the structural formation. The result of genomic DNA blot showed that CAPR5-1 and CAPR5-2 existed as single copy in the Subicho genome. Three recombinant CPARs in E. coli were identified by SDS-PACE, and each expressed protein was treated on the PDA medium which contained cultured pathogens. Although three CAPR5 proteins did not affected the hyphal growth of Glomerella glycines and Colletotrichum fagenarium, CAPR5-1, CAPR5-2, and CAPR5-3 showed a specific antifungal activities against P. capsici.

• Journal of Sericultural and Entomological Science
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• v.50 no.2
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• pp.145-149
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• 2012

Cecropin is a well-studied antimicrobial peptide that play important role as key factor in insect humoral immunity. In this study, cecropin-like antimicrobial peptide was isolated and purified from the larval haemolymph of immune-challenged japanese oak silkworm, Antheraea yamamai. To isolate antimicrobial peptide, we separated and compared acidic extracted hemolymph protein bends between control and immune-challenged larvae using SDS-PAGE analysis. In the immune hemolymph extract, but not of non-immune hemolymph, we detected differential expressed peptide band with molecular mass 4,223.01 Da. To understand this peptide better, we successfully purified this peptide using cation exchange chromatography and gel permeation chromatography. Its N-terminal amino acid sequence obtained by Edman degradation evidenced a significant degree of identity with other lepidopteran cecropins. The purified A. yamamai cecropin-like peptide showed a broad spectrum of activity against fungi, Gram-negative and Gram-positive bacteria.

• Journal of Veterinary Science
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• v.22 no.4
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• pp.43.1-43.10
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• 2021

Background: The H5 avian influenza viruses (AIVs) of clade 2.3.4.4 circulate in wild and domestic birds worldwide. In 2017, nine strains of H5N6 AIVs were isolated from aquatic poultry in Xinjiang, Northwest China. Objectives: This study aimed to analyze the origin, reassortment, and mutations of the AIV isolates. Methods: AIVs were isolated from oropharyngeal and cloacal swabs of poultry. Identification was accomplished by inoculating isolates into embryonated chicken eggs and performing hemagglutination tests and reverse transcription polymerase chain reaction (RT-PCR). The viral genomes were amplified with RT-PCR and then sequenced. The sequence alignment, phylogenetic, and molecular characteristic analyses were performed by using bioinformatic software. Results: Nine isolates originated from the same ancestor. The viral HA gene belonged to clade 2.3.4.4B, while the NA gene had a close phylogenetic relationship with the 2.3.4.4C H5N6 highly pathogenic avian influenza viruses (HPAIVs) isolated from shoveler ducks in Ningxia in 2015. The NP gene was grouped into an independent subcluster within the 2.3.4.4B H5N8 AIVs, and the remaining six genes all had close phylogenetic relationships with the 2.3.4.4B H5N8 HPAIVs isolated from the wild birds in China, Egypt, Uganda, Cameroon, and India in 2016-2017, Multiple basic amino acid residues associated with HPAIVs were located adjacent to the cleavage site of the HA protein. The nine isolates comprised reassortant 2.3.4.4B HPAIVs originating from 2.3.4.4B H5N8 and 2.3.4.4C H5N6 viruses in wild birds. Conclusions: These results suggest that the Northern Tianshan Mountain wetlands in Xinjiang may have a key role in AIVs disseminating from Central China to the Eurasian continent and East African.

• The Plant Pathology Journal
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• v.35 no.4
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• pp.381-388
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• 2019

For several years, temperatures in the Korean peninsula have gradually increased due to climate change, resulting in a changing environment for growth of crops and vegetables. An associated consequence is that emerging species of insect vector have caused increased viral transmission. In Jeju Island, Korea, occurrences of viral disease have increased. Here, we report characterization of five newly collected turnip mosaic virus (TuMV) isolates named KBJ1, KBJ2, KBJ3, KBJ4 and KBJ5 from a survey on Jeju Island in 2017. Full-length cDNAs of each isolate were cloned into the pJY vector downstream of cauliflower mosaic virus 35S and bacteriophage T7 RNA polymerase promoters. Their fulllength sequences share 98.9-99.9% nucleotide sequence identity and were most closely related to previously reported Korean TuMV isolates. All isolates belonged to the BR group and infected both Chinese cabbage and radish. Four isolates induced very mild symptoms in Nicotiana benthamiana but KBJ5 induced a hypersensitive response. Symptom differences may result from three amino acid differences uniquely present in KBJ5; Gly(382)Asp, Ile(891)Val, and Lys(2522)Glu in P1, P3, and NIb, respectively.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.5
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• pp.614-628
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• 2019

Objective: The inhibitory leukocyte immunoglobulin-like receptors (LILRBs) play an important role in innate immunity. The present study represents the first description of the cloning and structural and functional analysis of LILRB1 and LILRB3 isolated from two genetically disparate chicken lines. Methods: Chicken LILRB1-3 genes were identified by bioinformatics approach. Expression studies were performed by transfection, quantitative polymerase chain reaction. Signal transduction was analyzed by western blots, immunoprecipitation and flow cytometric. Cytokine levels were determined by enzyme-linked immunosorbent assay. Results: Amino acid homology and phylogenetic analyses showed that the homologies of LILRB1 and LILRB3 in the chicken line 6.3 to those proteins in the chicken line 7.2 ranged between 97%-99%, while homologies between chicken and mammal proteins ranged between 13%-19%, and 13%-69%, respectively. Our findings indicate that LILRB1 and LILRB3 subdivided into two groups based on the immunoreceptor tyrosine-based inhibitory motifs (ITIM) present in the transmembrane domain. Chicken line 6.3 has two ITIM motifs of the sequence LxYxxL and SxYxxV while line 7.2 has two ITIM motifs of the sequences LxYxxL and LxYxxV. These motifs bind to SHP-2 (protein tyrosine phosphatase, non-receptor type 11) that plays a regulatory role in immune functions. Moreover, our data indicate that LILRB1 and LILRB3 associated with and activated major histocompatibility complex (MHC) class I and ${\beta}2-microglobulin$ and induced the expression of transporters associated with antigen processing, which are essential for MHC class I antigen presentation. This suggests that LILRB1 and LILRB3 are transcriptional regulators, modulating the expression of components in the MHC class I pathway and thereby regulating immune responses. Furthermore, LILRB1 and LILRB3 activated Janus kinase2/tyrosine kinase 2 (JAK2/TYK2); signal transducer and activator of transcription1/3 (STAT1/3), and suppressor of cytokine signaling 1 genes expressed in Macrophage (HD11) cells, which induced Th1, Th2, and Th17 cytokines. Conclusion: These data indicate that LILRB1 and LILRB3 are innate immune receptors associated with SHP-2, MHC class I, ${\beta}2-microglobulin$, and they activate the Janus kinase/signal transducer and activator of transcription signaling pathway. Thus, our study provides novel insights into the regulation of immunity and immunopathology.