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Mengshiou Lee - One of the best experts on this subject based on the ideXlab platform.

  • expression and characterization of highly antigenic domains of Chicken Anemia Virus viral vp2 and vp3 subunit proteins in a recombinant e coli for sero diagnostic applications
    BMC Veterinary Research, 2013
    Co-Authors: Guan-hua Lai, Hsijien Chen, Chihung Huang, Yiyang Lien, Jason Tc Tzen, Mingkuem Lin, Mengshiou Lee
    Abstract:

    Background Chicken Anemia Virus (CAV) is an important viral pathogen that causes Anemia and severe immunodeficiency syndrome in Chickens worldwide. Generally, CAV infection occurs via vertical transmission in young chicks that are less than two weeks old, which are very susceptible to the disease. Therefore, epidemiological investigations of CAV infection and/or the evaluation of the immunization status of Chickens is necessary for disease control. Up to the present, systematically assessing viral protein antigenicity and/or determining the immunorelevant domain(s) of viral proteins during serological testing for CAV infection has never been performed. The expression, production and antigenic characterization of CAV viral proteins such as VP1, VP2 and VP3, and their use in the development of diagnostic kit would be useful for CAV infection prevention.

  • Efficient Production of an Engineered Apoptin from Chicken Anemia Virus in a Recombinant E. coli for Tumor Therapeutic Applications
    BMC biotechnology, 2012
    Co-Authors: Mengshiou Lee, Chihung Huang, Yiyang Lien, Mengshiunn Lee, Hisjien Chen, Fang-chun Sun, Shin-huei Feng, Guan-hua Lai, Jung Chao, Jason Tc Tzen
    Abstract:

    Background: Apoptin, a nonstructural protein encoded by the VP3 gene of Chicken Anemia Virus (CAV), has been shown to not only induce apoptosis when introduced into the precursors of Chicken thymocytes, but has been found to specifically kill human cancer cells, tumor cell and transformed cells without affecting the proliferation of normal cells. This tumor-specific apoptotic characteristic of the protein potentially may allow the development of a protein drug that has applications in tumor therapy. However, several major problems, which include poor expression and poor protein solubility, have hampered the production of apoptin in bacteria. Results: Significantly increased expression of recombinant full-length apoptin that originated from Chicken Anemia Virus was demonstrated using an E. coli expression system. The CAV VP3 gene was fused with a synthetic sequence containing a trans-acting activator of transcription (TAT) protein transduction domain (PTD). The resulting construct was cloned into various different expression vectors and these were then expressed in various E. coli strains. The expression of the TAT-Apoptin in E. coli was significantly increased when TAT-Apoptin was fused with GST-tag rather than a His-tag. When the various rare amino acid codons of apoptin were optimized, the expression level of the GST-TAT-Apoptinopt in E. coli BL21(DE3) was significantly further increased. The highest protein expression level obtained was 8.33 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 4 h at 25 °C. Moreover, approximately 90% of the expressed GST-TAT-Apoptinopt under these conditions was soluble. After purification by GST affinity chromatography, the purified recombinant TAT-Apoptinopt protein was used to evaluate the recombinant protein’s apoptotic activity on tumor cells. The results demonstrated that the E. coli-expressed GSTTAT-apoptinopt showed apoptotic activity and was able to induce human premyelocytic leukemia HL-60 cells to enter apoptosis.

  • efficient production of an engineered apoptin from Chicken Anemia Virus in a recombinant e coli for tumor therapeutic applications
    BMC Biotechnology, 2012
    Co-Authors: Mengshiou Lee, Chihung Huang, Yiyang Lien, Mengshiunn Lee, Hisjien Chen, Fang-chun Sun, Shin-huei Feng, Guan-hua Lai, Jung Chao, Jason Tc Tzen
    Abstract:

    Apoptin, a nonstructural protein encoded by the VP3 gene of Chicken Anemia Virus (CAV), has been shown to not only induce apoptosis when introduced into the precursors of Chicken thymocytes, but has been found to specifically kill human cancer cells, tumor cell and transformed cells without affecting the proliferation of normal cells. This tumor-specific apoptotic characteristic of the protein potentially may allow the development of a protein drug that has applications in tumor therapy. However, several major problems, which include poor expression and poor protein solubility, have hampered the production of apoptin in bacteria. Significantly increased expression of recombinant full-length apoptin that originated from Chicken Anemia Virus was demonstrated using an E. coli expression system. The CAV VP3 gene was fused with a synthetic sequence containing a trans-acting activator of transcription (TAT) protein transduction domain (PTD). The resulting construct was cloned into various different expression vectors and these were then expressed in various E. coli strains. The expression of the TAT-Apoptin in E. coli was significantly increased when TAT-Apoptin was fused with GST-tag rather than a His-tag. When the various rare amino acid codons of apoptin were optimized, the expression level of the GST-TAT-Apoptinopt in E. coli BL21(DE3) was significantly further increased. The highest protein expression level obtained was 8.33 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 4 h at 25 °C. Moreover, approximately 90% of the expressed GST-TAT-Apoptinopt under these conditions was soluble. After purification by GST affinity chromatography, the purified recombinant TAT-Apoptinopt protein was used to evaluate the recombinant protein’s apoptotic activity on tumor cells. The results demonstrated that the E. coli-expressed GST-TAT-apoptinopt showed apoptotic activity and was able to induce human premyelocytic leukemia HL-60 cells to enter apoptosis. On expression in E. coli, purified recombinant TAT-Apoptinopt that has been fused to a GST tag and had its codons optimized, was found to have great potential. This protein may in the future allow the development of a therapeutic protein that is able to specifically kill tumor cells.

  • identification of the nls and nes motifs of vp2 from Chicken Anemia Virus and the interaction of vp2 with mini chromosome maintenance protein 3
    BMC Veterinary Research, 2012
    Co-Authors: Jaihong Cheng, Yiyang Lien, Shyangchwen Sheu, Mengshiunn Lee, Hisjien Chen, Mengshiou Lee
    Abstract:

    Background VP2 of Chicken Anemia Virus (CAV) is a dual-specificity phosphatase required for Virus infection, assembly and replication. The functions of the nuclear localization signal (NLS) and nuclear export signal (NES) of VP2 in the cell, however, are poorly understood. Our study identified the presence of a NLS in VP2 and showed that the protein interacted significantly with mini-chromosome maintenance protein 3 (MCM3) in the cell.

  • High yield expression in a recombinant E. coli of a codon optimized Chicken Anemia Virus capsid protein VP1 useful for vaccine development
    Microbial Cell Factories, 2011
    Co-Authors: Mengshiou Lee, Youcheng Hseu, Wente Chang, Hsijien Chen, Chihung Huang, Minying Wang, Mengshiunn Lee, Guan-hua Lai, Jung-yie Kao, Bang-jau You
    Abstract:

    Background Chicken Anemia Virus (CAV), the causative agent Chicken Anemia, is the only member of the genus GyroVirus of the Circoviridae family. CAV is an immune suppressive Virus and causes Anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention. Results Significantly increased expression of the recombinant full-length VP1 capsid protein from Chicken Anemia Virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive Chicken serum in an ELISA assay. Conclusions Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli , may be useful in the future for the development of subunit vaccines and diagnostic tests.

Chihung Huang - One of the best experts on this subject based on the ideXlab platform.

  • expression and characterization of highly antigenic domains of Chicken Anemia Virus viral vp2 and vp3 subunit proteins in a recombinant e coli for sero diagnostic applications
    BMC Veterinary Research, 2013
    Co-Authors: Guan-hua Lai, Hsijien Chen, Chihung Huang, Yiyang Lien, Jason Tc Tzen, Mingkuem Lin, Mengshiou Lee
    Abstract:

    Background Chicken Anemia Virus (CAV) is an important viral pathogen that causes Anemia and severe immunodeficiency syndrome in Chickens worldwide. Generally, CAV infection occurs via vertical transmission in young chicks that are less than two weeks old, which are very susceptible to the disease. Therefore, epidemiological investigations of CAV infection and/or the evaluation of the immunization status of Chickens is necessary for disease control. Up to the present, systematically assessing viral protein antigenicity and/or determining the immunorelevant domain(s) of viral proteins during serological testing for CAV infection has never been performed. The expression, production and antigenic characterization of CAV viral proteins such as VP1, VP2 and VP3, and their use in the development of diagnostic kit would be useful for CAV infection prevention.

  • Efficient Production of an Engineered Apoptin from Chicken Anemia Virus in a Recombinant E. coli for Tumor Therapeutic Applications
    BMC biotechnology, 2012
    Co-Authors: Mengshiou Lee, Chihung Huang, Yiyang Lien, Mengshiunn Lee, Hisjien Chen, Fang-chun Sun, Shin-huei Feng, Guan-hua Lai, Jung Chao, Jason Tc Tzen
    Abstract:

    Background: Apoptin, a nonstructural protein encoded by the VP3 gene of Chicken Anemia Virus (CAV), has been shown to not only induce apoptosis when introduced into the precursors of Chicken thymocytes, but has been found to specifically kill human cancer cells, tumor cell and transformed cells without affecting the proliferation of normal cells. This tumor-specific apoptotic characteristic of the protein potentially may allow the development of a protein drug that has applications in tumor therapy. However, several major problems, which include poor expression and poor protein solubility, have hampered the production of apoptin in bacteria. Results: Significantly increased expression of recombinant full-length apoptin that originated from Chicken Anemia Virus was demonstrated using an E. coli expression system. The CAV VP3 gene was fused with a synthetic sequence containing a trans-acting activator of transcription (TAT) protein transduction domain (PTD). The resulting construct was cloned into various different expression vectors and these were then expressed in various E. coli strains. The expression of the TAT-Apoptin in E. coli was significantly increased when TAT-Apoptin was fused with GST-tag rather than a His-tag. When the various rare amino acid codons of apoptin were optimized, the expression level of the GST-TAT-Apoptinopt in E. coli BL21(DE3) was significantly further increased. The highest protein expression level obtained was 8.33 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 4 h at 25 °C. Moreover, approximately 90% of the expressed GST-TAT-Apoptinopt under these conditions was soluble. After purification by GST affinity chromatography, the purified recombinant TAT-Apoptinopt protein was used to evaluate the recombinant protein’s apoptotic activity on tumor cells. The results demonstrated that the E. coli-expressed GSTTAT-apoptinopt showed apoptotic activity and was able to induce human premyelocytic leukemia HL-60 cells to enter apoptosis.

  • efficient production of an engineered apoptin from Chicken Anemia Virus in a recombinant e coli for tumor therapeutic applications
    BMC Biotechnology, 2012
    Co-Authors: Mengshiou Lee, Chihung Huang, Yiyang Lien, Mengshiunn Lee, Hisjien Chen, Fang-chun Sun, Shin-huei Feng, Guan-hua Lai, Jung Chao, Jason Tc Tzen
    Abstract:

    Apoptin, a nonstructural protein encoded by the VP3 gene of Chicken Anemia Virus (CAV), has been shown to not only induce apoptosis when introduced into the precursors of Chicken thymocytes, but has been found to specifically kill human cancer cells, tumor cell and transformed cells without affecting the proliferation of normal cells. This tumor-specific apoptotic characteristic of the protein potentially may allow the development of a protein drug that has applications in tumor therapy. However, several major problems, which include poor expression and poor protein solubility, have hampered the production of apoptin in bacteria. Significantly increased expression of recombinant full-length apoptin that originated from Chicken Anemia Virus was demonstrated using an E. coli expression system. The CAV VP3 gene was fused with a synthetic sequence containing a trans-acting activator of transcription (TAT) protein transduction domain (PTD). The resulting construct was cloned into various different expression vectors and these were then expressed in various E. coli strains. The expression of the TAT-Apoptin in E. coli was significantly increased when TAT-Apoptin was fused with GST-tag rather than a His-tag. When the various rare amino acid codons of apoptin were optimized, the expression level of the GST-TAT-Apoptinopt in E. coli BL21(DE3) was significantly further increased. The highest protein expression level obtained was 8.33 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 4 h at 25 °C. Moreover, approximately 90% of the expressed GST-TAT-Apoptinopt under these conditions was soluble. After purification by GST affinity chromatography, the purified recombinant TAT-Apoptinopt protein was used to evaluate the recombinant protein’s apoptotic activity on tumor cells. The results demonstrated that the E. coli-expressed GST-TAT-apoptinopt showed apoptotic activity and was able to induce human premyelocytic leukemia HL-60 cells to enter apoptosis. On expression in E. coli, purified recombinant TAT-Apoptinopt that has been fused to a GST tag and had its codons optimized, was found to have great potential. This protein may in the future allow the development of a therapeutic protein that is able to specifically kill tumor cells.

  • high yield expression in a recombinant e coli of a codon optimized Chicken Anemia Virus capsid protein vp1 useful for vaccine development
    Microbial Cell Factories, 2011
    Co-Authors: Youcheng Hseu, Wente Chang, Hsijien Chen, Chihung Huang, Minying Wang, Yiyang Lien
    Abstract:

    Background Chicken Anemia Virus (CAV), the causative agent Chicken Anemia, is the only member of the genus GyroVirus of the Circoviridae family. CAV is an immune suppressive Virus and causes Anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention.

  • High yield expression in a recombinant E. coli of a codon optimized Chicken Anemia Virus capsid protein VP1 useful for vaccine development
    Microbial Cell Factories, 2011
    Co-Authors: Mengshiou Lee, Youcheng Hseu, Wente Chang, Hsijien Chen, Chihung Huang, Minying Wang, Mengshiunn Lee, Guan-hua Lai, Jung-yie Kao, Bang-jau You
    Abstract:

    Background Chicken Anemia Virus (CAV), the causative agent Chicken Anemia, is the only member of the genus GyroVirus of the Circoviridae family. CAV is an immune suppressive Virus and causes Anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention. Results Significantly increased expression of the recombinant full-length VP1 capsid protein from Chicken Anemia Virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive Chicken serum in an ELISA assay. Conclusions Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli , may be useful in the future for the development of subunit vaccines and diagnostic tests.

C. P. Muller - One of the best experts on this subject based on the ideXlab platform.

  • Epidemiology of Chicken Anemia Virus in Central African Republic and Cameroon
    Virology journal, 2012
    Co-Authors: Chantal J. Snoeck, Giscard F. Komoyo, Bonya P Mbee, Emmanuel Nakouné, Alain Le Faou, Mbah P. Okwen, C. P. Muller
    Abstract:

    Background: Although Chicken Anemia Virus (CAV) has been detected on all continents, little is known about this Virus in sub-Saharan Africa. This study aimed to detect and characterize CAV for the first time in Central African Republic and in Cameroon. Results: An overall flock seroprevalence of 36.7% was found in Central African Republic during the 2008–2010 period. Virus prevalences were 34.2% (2008), 14.3% (2009) and 10.4% (2010) in Central African Republic and 39% (2007) and 34.9% (2009) in Cameroon. CAV DNA was found in cloacal swabs of 76.9% of seropositive Chickens, suggesting that these animals excreted the Virus despite antibodies. On the basis of VP1 sequences, most of the strains in Central African Republic and Cameroon belonged to 9 distinct phylogenetic clusters at the nucleotide level and were not intermixed with strains from other continent. Several cases of mixed infections in flocks and individual Chickens were identified.

  • epidemiology of Chicken Anemia Virus in central african republic and cameroon
    Virology Journal, 2012
    Co-Authors: Chantal J. Snoeck, Giscard F. Komoyo, Bonya P Mbee, Emmanuel Nakouné, Alain Le Faou, Mbah P. Okwen, C. P. Muller
    Abstract:

    Although Chicken Anemia Virus (CAV) has been detected on all continents, little is known about this Virus in sub-Saharan Africa. This study aimed to detect and characterize CAV for the first time in Central African Republic and in Cameroon. An overall flock seroprevalence of 36.7% was found in Central African Republic during the 2008–2010 period. Virus prevalences were 34.2% (2008), 14.3% (2009) and 10.4% (2010) in Central African Republic and 39% (2007) and 34.9% (2009) in Cameroon. CAV DNA was found in cloacal swabs of 76.9% of seropositive Chickens, suggesting that these animals excreted the Virus despite antibodies. On the basis of VP1 sequences, most of the strains in Central African Republic and Cameroon belonged to 9 distinct phylogenetic clusters at the nucleotide level and were not intermixed with strains from other continent. Several cases of mixed infections in flocks and individual Chickens were identified. Our results suggest multiple introductions of CAV in each country that later spread and diverged locally. Mixed genotype infections together with the observation of CAV DNA in cloacal samples despite antibodies suggest a suboptimal protection by antibodies or Virus persistence.

  • Molecular Epidemiology of Chicken Anemia Virus (CAV) in Southeastern Chinese Live Birds Markets
    Avian diseases, 2008
    Co-Authors: M. F. Ducatez, Honglin Chen, Yi Guan, C. P. Muller
    Abstract:

    Between January 2004 and December 2005, cloacal swabs from essentially healthy Chickens and silky Chickens from live birds markets in Guangdong and Hunan provinces in southeastern China were screened for Chicken Anemia Virus (CAV) by polymerase chain reaction. Phylogenetic analysis of the major structural protein VP1 sequences showed no clear genotype cluster and no correlation with the geographic origin of CAV strains. Virus evolution at the amino acid level was very slow, which corresponds to a strong negative selection of the VP1 gene in China and worldwide. A high proportion (87%) of birds was CAV positive, suggesting that many farms in the region were infected. Further investigations are necessary to evaluate the economic losses caused by CAV and the cost-benefit of vaccination.

  • Molecular epidemiology of Chicken Anemia Virus in Nigeria
    Archives of Virology, 2006
    Co-Authors: M. F. Ducatez, A. A. Owoade, J. O. Abiola, C. P. Muller
    Abstract:

    Between February 2002 and May 2004, Chicken Anemia Virus (CAV) was detected by PCR in organ samples from 14 flocks of poultry farms in Lagos, Ogun and Oyo States in Southwestern Nigeria. The farms reported low (< 5%) to high mortalities ( up to 100%) with various lesions at necropsy. The complete VP1 gene of 30 of these positive strains was sequenced. Strains that diverged by up to 4.4% on a nucleotide level differed only by up to 2.5% at the amino acid level (7 aa) as a result of clustered silent mutations. No amino acid substitutions specific for Nigerian strains were observed. Some birds had a CAV mixed infection. Genetic clustering of the VP1 gene did not correlate with differences in flock mortality but the co-infection of CAV with IBDV may be particularly lethal. This first molecular epidemiological study of CAV in Africa shows that the Nigerian strains cluster with Viruses from very diverse geographic origins and were almost as diverse (4.4%) as all other strains combined (5.8%).

Yiyang Lien - One of the best experts on this subject based on the ideXlab platform.

  • expression and characterization of highly antigenic domains of Chicken Anemia Virus viral vp2 and vp3 subunit proteins in a recombinant e coli for sero diagnostic applications
    BMC Veterinary Research, 2013
    Co-Authors: Guan-hua Lai, Hsijien Chen, Chihung Huang, Yiyang Lien, Jason Tc Tzen, Mingkuem Lin, Mengshiou Lee
    Abstract:

    Background Chicken Anemia Virus (CAV) is an important viral pathogen that causes Anemia and severe immunodeficiency syndrome in Chickens worldwide. Generally, CAV infection occurs via vertical transmission in young chicks that are less than two weeks old, which are very susceptible to the disease. Therefore, epidemiological investigations of CAV infection and/or the evaluation of the immunization status of Chickens is necessary for disease control. Up to the present, systematically assessing viral protein antigenicity and/or determining the immunorelevant domain(s) of viral proteins during serological testing for CAV infection has never been performed. The expression, production and antigenic characterization of CAV viral proteins such as VP1, VP2 and VP3, and their use in the development of diagnostic kit would be useful for CAV infection prevention.

  • Efficient Production of an Engineered Apoptin from Chicken Anemia Virus in a Recombinant E. coli for Tumor Therapeutic Applications
    BMC biotechnology, 2012
    Co-Authors: Mengshiou Lee, Chihung Huang, Yiyang Lien, Mengshiunn Lee, Hisjien Chen, Fang-chun Sun, Shin-huei Feng, Guan-hua Lai, Jung Chao, Jason Tc Tzen
    Abstract:

    Background: Apoptin, a nonstructural protein encoded by the VP3 gene of Chicken Anemia Virus (CAV), has been shown to not only induce apoptosis when introduced into the precursors of Chicken thymocytes, but has been found to specifically kill human cancer cells, tumor cell and transformed cells without affecting the proliferation of normal cells. This tumor-specific apoptotic characteristic of the protein potentially may allow the development of a protein drug that has applications in tumor therapy. However, several major problems, which include poor expression and poor protein solubility, have hampered the production of apoptin in bacteria. Results: Significantly increased expression of recombinant full-length apoptin that originated from Chicken Anemia Virus was demonstrated using an E. coli expression system. The CAV VP3 gene was fused with a synthetic sequence containing a trans-acting activator of transcription (TAT) protein transduction domain (PTD). The resulting construct was cloned into various different expression vectors and these were then expressed in various E. coli strains. The expression of the TAT-Apoptin in E. coli was significantly increased when TAT-Apoptin was fused with GST-tag rather than a His-tag. When the various rare amino acid codons of apoptin were optimized, the expression level of the GST-TAT-Apoptinopt in E. coli BL21(DE3) was significantly further increased. The highest protein expression level obtained was 8.33 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 4 h at 25 °C. Moreover, approximately 90% of the expressed GST-TAT-Apoptinopt under these conditions was soluble. After purification by GST affinity chromatography, the purified recombinant TAT-Apoptinopt protein was used to evaluate the recombinant protein’s apoptotic activity on tumor cells. The results demonstrated that the E. coli-expressed GSTTAT-apoptinopt showed apoptotic activity and was able to induce human premyelocytic leukemia HL-60 cells to enter apoptosis.

  • efficient production of an engineered apoptin from Chicken Anemia Virus in a recombinant e coli for tumor therapeutic applications
    BMC Biotechnology, 2012
    Co-Authors: Mengshiou Lee, Chihung Huang, Yiyang Lien, Mengshiunn Lee, Hisjien Chen, Fang-chun Sun, Shin-huei Feng, Guan-hua Lai, Jung Chao, Jason Tc Tzen
    Abstract:

    Apoptin, a nonstructural protein encoded by the VP3 gene of Chicken Anemia Virus (CAV), has been shown to not only induce apoptosis when introduced into the precursors of Chicken thymocytes, but has been found to specifically kill human cancer cells, tumor cell and transformed cells without affecting the proliferation of normal cells. This tumor-specific apoptotic characteristic of the protein potentially may allow the development of a protein drug that has applications in tumor therapy. However, several major problems, which include poor expression and poor protein solubility, have hampered the production of apoptin in bacteria. Significantly increased expression of recombinant full-length apoptin that originated from Chicken Anemia Virus was demonstrated using an E. coli expression system. The CAV VP3 gene was fused with a synthetic sequence containing a trans-acting activator of transcription (TAT) protein transduction domain (PTD). The resulting construct was cloned into various different expression vectors and these were then expressed in various E. coli strains. The expression of the TAT-Apoptin in E. coli was significantly increased when TAT-Apoptin was fused with GST-tag rather than a His-tag. When the various rare amino acid codons of apoptin were optimized, the expression level of the GST-TAT-Apoptinopt in E. coli BL21(DE3) was significantly further increased. The highest protein expression level obtained was 8.33 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 4 h at 25 °C. Moreover, approximately 90% of the expressed GST-TAT-Apoptinopt under these conditions was soluble. After purification by GST affinity chromatography, the purified recombinant TAT-Apoptinopt protein was used to evaluate the recombinant protein’s apoptotic activity on tumor cells. The results demonstrated that the E. coli-expressed GST-TAT-apoptinopt showed apoptotic activity and was able to induce human premyelocytic leukemia HL-60 cells to enter apoptosis. On expression in E. coli, purified recombinant TAT-Apoptinopt that has been fused to a GST tag and had its codons optimized, was found to have great potential. This protein may in the future allow the development of a therapeutic protein that is able to specifically kill tumor cells.

  • identification of the nls and nes motifs of vp2 from Chicken Anemia Virus and the interaction of vp2 with mini chromosome maintenance protein 3
    BMC Veterinary Research, 2012
    Co-Authors: Jaihong Cheng, Yiyang Lien, Shyangchwen Sheu, Mengshiunn Lee, Hisjien Chen, Mengshiou Lee
    Abstract:

    Background VP2 of Chicken Anemia Virus (CAV) is a dual-specificity phosphatase required for Virus infection, assembly and replication. The functions of the nuclear localization signal (NLS) and nuclear export signal (NES) of VP2 in the cell, however, are poorly understood. Our study identified the presence of a NLS in VP2 and showed that the protein interacted significantly with mini-chromosome maintenance protein 3 (MCM3) in the cell.

  • high yield expression in a recombinant e coli of a codon optimized Chicken Anemia Virus capsid protein vp1 useful for vaccine development
    Microbial Cell Factories, 2011
    Co-Authors: Youcheng Hseu, Wente Chang, Hsijien Chen, Chihung Huang, Minying Wang, Yiyang Lien
    Abstract:

    Background Chicken Anemia Virus (CAV), the causative agent Chicken Anemia, is the only member of the genus GyroVirus of the Circoviridae family. CAV is an immune suppressive Virus and causes Anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention.

Hsijien Chen - One of the best experts on this subject based on the ideXlab platform.

  • expression and characterization of highly antigenic domains of Chicken Anemia Virus viral vp2 and vp3 subunit proteins in a recombinant e coli for sero diagnostic applications
    BMC Veterinary Research, 2013
    Co-Authors: Guan-hua Lai, Hsijien Chen, Chihung Huang, Yiyang Lien, Jason Tc Tzen, Mingkuem Lin, Mengshiou Lee
    Abstract:

    Background Chicken Anemia Virus (CAV) is an important viral pathogen that causes Anemia and severe immunodeficiency syndrome in Chickens worldwide. Generally, CAV infection occurs via vertical transmission in young chicks that are less than two weeks old, which are very susceptible to the disease. Therefore, epidemiological investigations of CAV infection and/or the evaluation of the immunization status of Chickens is necessary for disease control. Up to the present, systematically assessing viral protein antigenicity and/or determining the immunorelevant domain(s) of viral proteins during serological testing for CAV infection has never been performed. The expression, production and antigenic characterization of CAV viral proteins such as VP1, VP2 and VP3, and their use in the development of diagnostic kit would be useful for CAV infection prevention.

  • high yield expression in a recombinant e coli of a codon optimized Chicken Anemia Virus capsid protein vp1 useful for vaccine development
    Microbial Cell Factories, 2011
    Co-Authors: Youcheng Hseu, Wente Chang, Hsijien Chen, Chihung Huang, Minying Wang, Yiyang Lien
    Abstract:

    Background Chicken Anemia Virus (CAV), the causative agent Chicken Anemia, is the only member of the genus GyroVirus of the Circoviridae family. CAV is an immune suppressive Virus and causes Anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention.

  • High yield expression in a recombinant E. coli of a codon optimized Chicken Anemia Virus capsid protein VP1 useful for vaccine development
    Microbial Cell Factories, 2011
    Co-Authors: Mengshiou Lee, Youcheng Hseu, Wente Chang, Hsijien Chen, Chihung Huang, Minying Wang, Mengshiunn Lee, Guan-hua Lai, Jung-yie Kao, Bang-jau You
    Abstract:

    Background Chicken Anemia Virus (CAV), the causative agent Chicken Anemia, is the only member of the genus GyroVirus of the Circoviridae family. CAV is an immune suppressive Virus and causes Anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention. Results Significantly increased expression of the recombinant full-length VP1 capsid protein from Chicken Anemia Virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive Chicken serum in an ELISA assay. Conclusions Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli , may be useful in the future for the development of subunit vaccines and diagnostic tests.

  • high yield expression in a recombinant e coli of a codon optimized Chicken Anemia Virus capsid protein vp1 useful for vaccine development
    Microbial Cell Factories, 2011
    Co-Authors: Mengshiou Lee, Youcheng Hseu, Wente Chang, Hsijien Chen, Chihung Huang, Minying Wang, Mengshiunn Lee, Guan-hua Lai, Jung-yie Kao, Bang-jau You
    Abstract:

    Chicken Anemia Virus (CAV), the causative agent Chicken Anemia, is the only member of the genus GyroVirus of the Circoviridae family. CAV is an immune suppressive Virus and causes Anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention. Significantly increased expression of the recombinant full-length VP1 capsid protein from Chicken Anemia Virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive Chicken serum in an ELISA assay. Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.

  • production of Chicken Anemia Virus cav vp1 and vp2 protein expressed by recombinant escherichia coli
    Process Biochemistry, 2009
    Co-Authors: Mengshiou Lee, Wente Chang, Yiyang Lien, Shin-huei Feng, Rayling Huang, Mingcheng Tsai, Hsijien Chen
    Abstract:

    Abstract Chicken Anemia Virus (CAV) is an Anemia agent of breeder and young chicks. This Virus is the cause of economic losses across the Chicken industry worldwide as a consequence of severe Anemia and immunodeficiency among the birds. Two genes of CAV encoding the VP1 and VP2 proteins were cloned and expressed in Escherichia coli BL21 (DE3). A Western blot assay using His-tag antiserum was used to assess the expression level of the CAV viral proteins in E. coli. The results demonstrated that only full-length VP2 can be successfully expressed in E. coli, but not full-length VP1. A serial of N-terminus deletions of the VP1 protein, VP1Nd30, VP1Nd60 and VP1 Nd129, were created using PCR in order to improve VP1 expression. The results demonstrated that all three of these recombinant VP1 mutant proteins can be expressed in E. coli. VP1Nd129 protein demonstrates the highest expression level compared to the other two proteins. The specificity of Nd129-VP1 and VP2 protein were confirmed by mass spectrometry. By comparing the expression level of VP1Nd129 and VP2 protein after the addition of IPTG, the results indicated that the VP1Nd129 protein gave a higher level of protein expression than VP2. The highest yields of VP1Nd129 and VP2 were 26.2 and 15.5 mg/L, respectively, after IPTG induction with 0.1 mM IPTG for 6 h, respectively. The identification of the optimized conditions for production of the CAV viral proteins VP1 and VP2 will allow them to be used in the future as an antigen for the development of vaccines and diagnostic tests.