Identification of the NLS and NES motifs of VP2 from chicken anemia virus and the interaction of VP2 with mini-chromosome maintenance protein 3

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>An 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.</p> <p>Conclusions</p> <p>VP2 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.</p

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

<p>Abstract</p> <p>Background</p> <p>Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus <it>Gyrovirus </it>of the <it>Circoviridae </it>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.</p> <p>Results</p> <p>Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an <it>E. coli </it>expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different <it>E. coli </it>strains. The expression of CAV VP1 in <it>E. coli </it>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 <it>E. coli </it>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.</p> <p>Conclusions</p> <p>Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in <it>E. coli</it>, may be useful in the future for the development of subunit vaccines and diagnostic tests.</p

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

Anemia infecciosa aviar: caracterización lesional y del antígeno vírico en pollos broiler

El 27 de agosto de 2015 entraron 16320 pollitos broiler (machos y hembras) en una nave (Nave A) y 16116 en otra (Nave B). Las bajas durante la primera y segunda semana de vida fueron 364 en la nave A y 354 en la B. A partir del día 14 de vida (tercera semana) se incrementaron considerablemente las bajas (672 y 539) y en la cuarta y quinta semana se contabilizaron 628 y 438. A los 18 días de vida muchos de estos pollitos presentaban pododermatitis y edema en las alas, por lo que se decide remitir 6 animales para la necropsia. Las lesiones observadas eran compatibles con anemia infecciosa aviar, causada por un circovirus, “chicken anemia virus” (CAV)

Development of On-line Laser Power Monitoring and Stabilizing System

自雷射被發明以來，雷射已經被應用在許多領域，如：材料加工、通訊、量測、生醫工程、國防工業等等。在雷射材料加工的過程中，雷射輸出功率是很重要的一個參數，但雷射加工時間短，而目前現有的雷射功率計因為反應時間過長，無法在短時間內精確量測出雷射的功率。為了進行雷射功率即時監測，本研究利用CMOS(Complementary metal-oxide-semiconductor)工業相機進行雷射功率線上量測系統的開發，期望能夠在雷射加工時，達到即時量測雷射功率的目的；並且針對雷射功率易受環境溫度影響而功率輸出不穩定的現象，以回授控制(Feedback control)的方式進行雷射功率的穩定控制。 本研究將經過分光、減光的雷射光束以CMOS工業相機直接擷取光點影像，計算影像中的雷射光點亮度值，利用曲線配適(Curve fitting)的方式推算雷射功率。得到即時雷射功率值後，計算量測功率值與預設功率值的誤差，以控制雷射Q開關(Q-switch)Duty time的方式，利用PID控制器進行回授控制，達到雷射功率輸出的穩定控制。 由實驗結果可知，本研究系統在連續量測模式下，平均量測誤差僅約3%，反應時間比起熱電堆式雷射功率計至少縮短3.6秒以上；利用觸發訊號的觸發量測模式下，使CMOS相機能夠同步擷取間歇式的加工出光，進行同步量測，平均量測誤差小於3%，且能夠量測最短20毫秒(Millisecond)的出光。 回授控制功能中，PID控制器能夠在1秒以內補償雷射系統之功率誤差，且過衝不到3%、幾乎沒有穩態誤差。在鍍銅PCB基板的鑽孔加工實驗中，在脈衝頻率30kHz下鑽孔，控制器能夠將孔徑長軸因功率下降的誤差由38.4%改善至1.8%，長寬比誤差由20%降至5%。在50kHz下鑽孔，控制器能夠將孔徑長軸因功率下降的誤差由100%改善至22.3%，長寬比誤差由100%降至5.8%。Since the laser was invented, laser has been applied in many fields such as material processing, communication, measurement, biomedical engineering, defense industries and etc. Laser power is an important parameter in laser material processing. However, since the response time of current laser power meters is too long, they cannot measure laser power accurately in a short time. To monitor the laser power in laser material processing, this study utilize a CMOS(Complementary metal-oxide-semiconductor) camera to develop an on-line laser power monitoring system. Also, this study applies a feedback control to stabilize laser power in order to solve the problem that laser power is easily affected by the environment temperature. In this study, CMOS camera captures images of incident laser beam after it is split and attenuated. By comparing the average brightness of the beam spots and measurement results from laser power meter, laser power can be estimated. Moreover, laser power is stabilized by a PID controller which controls the “Duty time” of the laser Q-switch. Under continuous measuring mode, the average measuring error is about 3% in average, and the response time is at least 3.6 second shorter than thermopile power meters; under trigger measuring mode which enables the CMOS camera to synchronize with intermittent laser output, the average measuring error is less than 3%, and the shortest response time is 20 millisecond. For power stabilizing function, PID controller can fully compensate power disturbances within 1 second, the overshoot is less than 3%, and no steady-state error is noticed. In the PCB drilling experiments, the PID controller is able to reduce the error of major axis lengths of drilled holes from 38.4% to 1.8% and the error of aspect ratio from 20% to 5% while drilling at 30 kHz PRF under power disturbance. While drilling at 50 kHz PRF, PID controller reduces the error of major axis lengths from 100% to 77.7% and the error of the aspect ratio from 100% to5.8% under power disturbance

Functional Characterization of VPX protein and Polyprotein of Avian Infectious Bursal Disease Virus (IBDV) on Virus-like Particles Assembly and Proteolytical Cleavage in Insect Cells

VP2蛋白是傳染性華氏囊病毒主要的結構蛋白，亦是誘發宿主產生中和性抗體的免疫原。本研究主要是利用桿狀病毒-昆蟲細胞表現系統，進行VP2前驅物VPX蛋白的表現，藉以探討此蛋白之似病毒粒子的組裝與其免疫保護的特性。由於已在VPX蛋白的C端先行融合一段由六個組氨酸所組成的His-tag，因此在昆蟲細胞表現及親和性管柱純化的過程中，發現Hi-5細胞對VPXH蛋白的C端區域有很明顯的降解作用，至於Sf9細胞中，此降解現象並不明顯。經由穿透式電子顯微鏡的觀察，證實VPXH蛋白至少可形成三種特殊結構，分別為管柱狀結構及兩種粒徑分別為20-25 nm及30-35 nm大小不同的球狀粒子。經其組成分析顯示，20-25 nm的正二十面球狀粒子主要由VPXH蛋白C端降解產物即VP2-like蛋白所組成，而30-35 nm的球狀粒子主要由VPXH蛋白及VP2-like蛋白所組成。根據此一結果，證實VPXH與VP2之間蛋白-蛋白交互作用力的存在，且由雞隻免疫試驗證實VPXH蛋白與VP2蛋白相同，同樣具有誘發宿主產生中和性抗體的能力，因此可供作疫苗開發時其抗原選擇的來源之一。而本研究另以Hi-5細胞進行IBDV多蛋白的表現時，發現Hi-5細胞對VPX蛋白獨特的裂解作用，可用以增加IBDV多蛋白中VPX蛋白的成熟，使VPX蛋白轉變為VP2蛋白的效率提高，以促進IBDV似病毒顆粒的組裝。相對地，當以Sf9細胞表現IBDV多蛋白時，不僅VPX蛋白的裂解作用被抑制，且IBDV似病毒顆粒組裝的效率亦明顯下降。因此，以Hi-5細胞進行IBDV似病毒顆粒的生產與組裝機制的研究，相當具有潛力。此外，在IBDV似病毒顆粒組裝形成的同時，亦會衍生組裝形成另一種IBDV次病毒粒子，其粒徑約為23 nm。經其組成分析，顯示IBDV次病毒粒子主要是由VP2蛋白組成，並推測IBDV次病毒粒子形成的主因與VP2蛋白的過量表現有關。即當感染細胞的MOI愈高時，VP2蛋白的產生愈多，其IBDV次病毒粒子的組裝愈明顯。反之，感染細胞的MOI趨低時，IBDV似病毒顆粒的組裝形成相對於IBDV次病毒粒子的組裝，則愈明顯。VP2 is a major structural protein of infectious bursal disease virus(IBDV). It has been demonstrated as the major host-protective immunogen of IBDV and contains the antigenic regions responsible for eliciting the virus neutralizing antibodies in the host. In this study, the precursor protein (VPX) of infectious bursal disease virus (IBDV) host immunogen VP2 protein was expressed in insect cells, including Sf9 and Hi-5 cells, to examine its regenerated particle types and the immunogenicity induced by those particles. Since the expressed protein, VPXH, was engineered a His-tag, consisting of six histidine residues, at their C-terminal end. When expressed in Hi-5 cells, rVPXH was efficiently processed at its C-terminus by cellular proteases to yield VP2-like proteins whose molecular weight was similar to that of VP2. However, proteolytical processing of VPXH in Sf9 cells was hampered. The expressed rVPXH was purified using immobilized metal-ion affinity chromatography (IMAC). Under TEM observation of Ni-NTA purified VPXH, at least three architectures of particles were observed, including the tubular structure and two of spherical structure of isometric particle structure and a new one of icosahedral particles, with a size of approximately 20-25nm and 30-35 nm in diameter, respectively. After separation of rVPXH formatted particles, chromatographic results indicate that the expressed rVPXH protein and very few of VP2-like protein formed isometric particle structure and very few of twisted tubular structure, as well as icosahedral particles formed by the degraded products of rVPXH protein, VP2-like protein. Finally, we also demonstrated that when susceptible chickens were vaccinated with the IMAC-purified rVPXH protein (40 g/bird), virus-neutralizing antibodies were induced. This indicated that those particles are highly immunogenic. Based on our results, we found that Hi-5 harbors excellent ability of proteolytic cleavage of VPX. Therefore, this study our effort also to investigate the proteolytic processing of IBDV polyprotein in insect cell. When IBDV polyprotein was expressed in insect cells, higher productivity of IBDV VLP was observed in Hi-5 cells. Moreover, the accumulation of matured VP2 and VLPs assembly was exhibited rather efficiently than in Sf9. In addition to IBDV-like particles assembled in Hi-5 cells, some of particulated subviral particles with 23 nm in diameter were also assembled. Chromatographic results show that the IBDV subviral particle was formed by VP2 protein. When a higher multiplicity of infection(MOI) strategy was used, accumulation of VP2 protein is more significantly. The excess VP2 protein resulted in formation of subviral particles. However, at low MOI, the relative productivity of IBDV VLP and subviral particles increased in batch culture. Our results demonstrate that Hi5, harboring excellent ability of proteolytic cleavage of recombinant protein, the efficiency of IBDV-like particles production is superior to Sf9 culture. These finding therefore may provide a methodological improvement using Hi-5 cells for optimal production of IBDV VLP as an effective IBDV vaccine against infectious bursal disease or as for crystallization to study IBDV structure.中文摘要………………………………………………………………..I 英文摘要……………………………………………………………...II 第一章、文獻回顧……………………………………………………..1 第二章、昆蟲細胞中傳染性華氏囊病毒VP2蛋白前驅物VPX之蛋 白裂解與似病毒粒子組裝特性之分析……………………11 一、中文摘要……………………………….…………………….12 二、英文摘要…………………………………………………….13 三、緒論…………………………………………………...…….15 四、材料與方法………………………………………………….17 五、結果………………………………………………………….27 六、圖表…………………………………………………………32 七、討論………………………………………………………….45 第三章、利用昆蟲細胞株Hi-5細胞的蛋白降解活性進行傳染性華氏囊病毒似病毒粒子組裝之研究……………………………. 49 一、中文摘要……………………………………………………50 二、英文摘要……………………………………………………51 三、緒論…………………………………………………………52 四、材料與方法…………………………………………………54 五、結果…………………………………………………………61 六、圖表…………………………………………………………65 七、討論…………………………………………………………76 第四章、綜合討論………………………..………………………….82 參考文獻……………………………………………………………...86 附錄…………………………………………………………………...9

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

Discrimination of Four Cinnamomum Species with Physico-Functional Properties and Chemometric Techniques: Application of PCA and MDA Models

Discrimination of highly valued and non-hepatotoxic Cinnamomum species (C. verum) from hepatotoxic (C. burmannii, C. loureiroi, and C. cassia) is essential for preventing food adulteration and safety problems. In this study, we developed a new method for the discrimination of four Cinnamomum species using physico-functional properties and chemometric techniques. The data were analyzed through principal component analysis (PCA) and multiclass discriminant analysis (MDA). The results showed that the cumulative variability of the first three principal components was 81.70%. The PCA score plot indicated a clear separation of the different Cinnamomum species. The training set was used to build the discriminant MDA model. The testing set was verified by this model. The prediction rate of 100% proved that the model was valid and reliable. Therefore, physico-functional properties coupled with chemometric techniques constitute a practical approach for discrimination of Cinnamomum species to prevent food fraud

The rapid and sensitive detection of edible bird's nest (Aerodramus fuciphagus) in processed food by a loop-mediated isothermal amplification (LAMP) assay

Edible bird's nest (EBN) is a well-known and precious traditional Chinese herbal material (CHM). Because of this, preventing the adulteration of EBN efficiently and precisely is crucial to protect consumers' interests and health. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of EBN using specifically designed LAMP primers. The results demonstrated that the identification of EBN by LAMP assay was specific and rapid (within 1 h). It had no cross-reaction with EBN adulterants, including white fungus, egg white and pig skin, in different ratios. The relative detection limit was 0.01% EBN in the adulterants. Moreover, the sensitivity of LAMP in authenticating EBN was 10−8 μg, it showed higher sensitivity than that of conventional PCR with 105 fold. When genomic DNAs extracted from boiled or steamed EBN samples were used as templates, LAMP for EBN detection was not affected and was reproducible after heat processing. In conclusion, the LAMP assay established herein could be applicable for authenticating EBN and for identifying commercial EBN products in herbal markets. Keywords: Edible bird's nest (EBN), Loop-mediated isothermal amplification (LAMP), Authenticatio