Meng-Shiunn Lee

High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

BackgroundChicken 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.ResultsSignificantly 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.ConclusionsPurified recombinant VP1 protein with the genes 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.

Rapid and sensitive identification of the herbal tea ingredient Taraxacum formosanum using loop-mediated isothermal amplification.

Taraxacum formosanum (TF) is a medicinal plant used as an important component of health drinks in Taiwan. In this study, a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for authenticating TF was established. A set of four specific LAMP primers was designed based on the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA (nrDNA) of TF. LAMP amplicons were successfully amplified and detected when purified genomic DNA of TF was added in the LAMP reaction under isothermal condition (65 °C) within 45 min. These specific LAMP primers have high specificity and can accurately discriminate Taraxacum formosanum from other adulterant plants; 1 pg of genomic DNA was determined to be the detection limit of the LAMP assay. In conclusion, using this novel approach, TF and its misused plant samples obtained from herbal tea markets were easily identified and discriminated by LAMP assay for quality control.

Efficient Production of an Engineered Apoptin from Chicken Anemia Virus in a Recombinant E. coli for Tumor Therapeutic Applications

BackgroundApoptin, 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.ResultsSignificantly 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.ConclusionsOn 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.

Involvement of Calcium-Mediated Reactive Oxygen Species in Inductive GRP78 Expression by Geldanamycin in 9L Rat Brain Tumor Cells

Treatment with geldanamycin (GA) leads to an increase in [Ca2+]c and the production of reactive oxygen species (ROS) in rat brain tumor 9L RBT cells. GA-exerted calcium signaling was blocked by BAPTA/AM and EGTA. The effect of GA on [Ca2+]c was significantly reduced in the presence of thapsigargin (TG) and ruthenium red (RR). GA-induced GRP78 expression is significantly decreased in the presence of BAPTA/AM, EGTA and RR, suggesting that the calcium influx from the extracellular space and intracellular calcium store oscillations are contributed to by the calcium mobilization and GRP78 expression induced by GA. The induced GRP78 expression is sensitive to added U73122 and Ro-31-8425, pinpointing the involvement of phospholipase C (PLC) and protein kinase C (PKC) in GA-induced endoplasmic reticulum (ER) stress. The antioxidants N-acetylcysteine (NAC), BAPTA/AM, EGTA and H7 also have significant inhibitory effects on ROS generation. Finally, neither H7 nor NAC was able to affect the calcium response elicited by GA. Our results suggest that the causal signaling cascade during GA-inducted GRP78 expression occurs via a pathway that connects PLC to cytoplasmic calcium increase, PKC activation and, then, finally, ROS generation. Our data provides new insights into the influence of GA on ER stress response in 9L RBT 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

BackgroundVP2 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.ResultsAn arginine-lysine rich NLS could be predicted by software and spanned from amino acids 133 to 138 of VP2. The critical amino acids residues between positions 136 and 138, and either residue 133 or 134 are important for nuclear import in mammalian cells based on systematic mutagenesis. A NES is also predicted in VP2; however the results suggest that no functional NES is present and that this protein is CRM1 independent. It was also shown that VP2 is a chromatin binding protein and, notably, using a co-immunoprecipitation assay, it was found that VP2 association with MCM3 and that this interaction does not require DSP activity.ConclusionsVP2 contains a NLS that span from amino acids 133 to 138. VP2 is a CRM1 independent protein during nuclear export and associates with MCM3 in cells.

Glutathione-S-Transferase M1 and T1 Gene Polymorphisms and Susceptibility to the Progression of Liver Fibrosis in Hcv-Infected Patients in Taiwan

Abstract Background: This study was conducted to evaluate the influence of glutathione-S-transferase (GST) M1 and T1 polymorphisms in 184 patients with different stages of liver fibrosis and hepatitis C virus infection and 173 healthy control subjects. Methods: DNA samples were extracted from whole blood, and the polymorphisms of GSTM1 and GSTT1 were determined with PCR using fluorescence-labeled Taq Man probes. Associations between specific genotypes and progression of liver fibrosis were examined by use of the logistic regression analysis to calculate the odds ratio (OR) and 95% confidence intervals (CI). Results: Results show that no differences were found between the frequencies of GSTM1 (49.8% versus 50.2%) and GSTT1 (52.2% versus 47.8%) null genotypes in HCV-infected pa tients and healthy controls, respectively. In addition, there was also no significant relation between the frequency of GSTM1 or GSTT1 gene polymorphisms and fibrosis stage as classified by the METAVIR group. Conclusions: The combined GSTM1 and GSTT1 null genotypes showed an association between GSTM1 [-]/GSTT1 [- ] and progression of liver fibrosis. Kratak sadržaj Uvod: Ova studija sprovedena je sa ciljem da se utvrdi uticaj polimorfizama Ml i T1 glutation-S-transferaze (GST) kod 184 pacijenta sa različitim stadijumima fibroze jetre inficira- nih virusom hepatitisa C i 173 zdravih kontrolnih osoba. Metode: Uzorci DNK ekstrahovani su iz pune krvi a polimor- fizmi GSTM1 i GSTT1 određeni su pomoću PCR uz koriš- čenje fluorescentno obeleženih Taq Man sondi. Povezanost između specifičnih genotipova i progresije fibroze jetre ispi- tana je pomoću logističke regresije da bi se izračunala ve- rovatnoća (odnos šansi) i 95% intervali poverenja. Rezultati: Rezultati pokazuju da nisu pronađene razlike izme- đu učestalosti nultih genotipova GSTM1 (49,8% prema 50,2%) i GSTT1 (52,2% prema 47,8%) kod pacijenata infi- ciranih virusom hepatitisa C i zdravih kontrolnih subjekata. Pored toga, nije utvrđena značajnost odnosa između učesta- losti genetičkih polimorfizama GSTM1 ili GSTT1 i stupnja fibroze određenog na osnovu klasifikacije METAVIR grupe. Zaključak: Kombinovaní nulti genotipovi gena GSTM1 i GSTT1 ukazali su na povezanost između GSTM1 [-]/GSTT1 [-] i progresije fibroze jetre.

Development and characterization of a potential diagnostic monoclonal antibody against capsid protein VP1 of the chicken anemia virus

Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. In this study, a potential diagnostic monoclonal antibody against the CAV VP1 protein was developed which can precisely recognize the CAV antigen for diagnostic and virus recovery purposes. The VP1 gene of CAV encoding the N-terminus-deleted VP1 protein, VP1Nd129, was cloned into an Escherichia (E.) coli expression vector. After isopropyl-β-D-thiogalactopyronoside induction, VP1Nd129 protein was shown to be successfully expressed in the E. coli. By performing an enzyme-linked immunoabsorbent assay using two coating antigens, purified VP1Nd129 and CAV-infected liver tissue lysate, E3 monoclonal antibody (mAb) was found to have higher reactivity against VP1 protein than the other positive clones according to the result of limiting dilution method from 64 clones. Using immunohistochemistry, the presence of the VP1-specific mAb, E3, was confirmed using CAV-infected liver and thymus tissues as positive-infected samples. Additionally, CAV particle purification was also performed using an immunoaffinity column containing E3 mAb. The monoclonal E3 mAb developed in this study will not only be very useful for detecting CAV infection and performing histopathology studies of infected chickens, but may also be used to purify CAV particles in the future.

VP2 of Chicken Anaemia Virus Interacts with Apoptin for Down-regulation of Apoptosis through De-phosphorylated Threonine 108 on Apoptin

Chicken anaemia virus (CAV) is an important contagious agent that causes immunosuppressive disease in chickens. CAV Apoptin is a nucleoplasmic shuffling protein that induces apoptosis in chicken lymphoblastoid cells. In the present study, confocal microscopy revealed co-localisation of expressed CAV non-structural protein VP2 with Apoptin in the nucleus of MDCC-MSB1 cells and the nucleoplasmic compartment of CHO-K1 cells. In vitro pull-down and ex vivo biomolecular fluorescent complementation (BiFC) assays further showed that the VP2 protein directly interacts with Apoptin. Transient co-expression of VP2 and Apoptin in MDCC-MSB1 cells significantly decreased the rate of apoptosis compared with that in cells transfected with the Apoptin gene alone. In addition, the phosphorylation status of threonine 108 (Thr108) of Apoptin was found to decrease upon interaction with VP2. Although dephosphorylated Thr108 did not alter the subcellular distribution of Apoptin in the nucleus of MDCC-MSB1 cells, it did suppress apoptosis. These findings provide the first evidence that VP2 directly interacts with Apoptin in the nucleus to down-regulate apoptosis through alterations in the phosphorylation status of the latter. This information will be useful to further elucidate the underlying mechanism of viral replication in the CAV life cycle.