Kanokwan Poomputsa

Generation of recombinant influenza virus using baculovirus delivery vector

A recombinant baculovirus vector containing mammalian cell-active promoters and transcription terminators was used to deliver a mutated influenza NS gene into Vero cells. In addition to the influenza NS gene, the baculovirus contained a reporter gene expression cassette (Green fluorescent protein, GFP), allowing to monitor the Vero cell transduction efficiency. More than 90% of Vero cells were expressing GFP 24-48 h post transduction. After infecting baculovirus transduced cells with influenza helper virus, progeny of attenuated influenza virus carrying the recombinant NS gene could be selected. Baculovirus delivery was highly reproducible and efficient in Vero cells. This new method for influenza gene delivery could contribute to influenza virus research and vaccine development.

Improved sensitivity of influenza A antigen detection using a combined NP, M, and NS1 sandwich ELISA.

A new modified triple-antigen detection test was developed for the direct detection of the influenza A virus. The nucleoprotein (NP), matrix (M), and non-structural (NS1) proteins were used as target antigens because they are abundant in infected cells. Monoclonal antibodies specific to the NP, M, and NS1 proteins were generated. The antibody pairs were selected and evaluated for their reactivity individually and in combination in the triple-antigen detection using sandwich ELISA. Triple-antigen detection demonstrated a higher sensitivity than individual antigen detection when tested with both the H1N1 and H3N2 influenza A viruses. This was illustrated by the 4-fold lower limit of detection of the triple-antigen test than the individual antigen detection test. The findings demonstrated that the sensitivity of influenza A antigen detection was improved with the triple-antigen detection system as compared to individual antigen detection. Therefore, this technique could be a useful tool for the direct detection of cell-associated influenza A antigen. Furthermore, it could provide a basis for the development of a rapid triple-antigen test for influenza A diagnosis.

Biochemical and physiological changes during chlorophyll degradation in lime (Citrus aurantifolia Swingle cv. ‘Paan’)

Summary Lime (Citrus aurantifolia Swingle cv. ‘Paan’) native to South East Asia, has a distinct flavour and quality characteristics. Maintenance of the green colour in the peel of lime is a desirable quality attribute during storage. Post-harvest chlorophyll degradation in lime was studied in fruit stored at room temperature (30°C) at a relative humidity (RH) of 70 – 85%. Within 7 d of storage, the total chlorophyll content decreased to 53.9% of its initial level. The highest chlorophyllase activity (1.68 units mg–1 protein) was observed after 4 d of storage and declined thereafter. Peroxidase activity differed from chlorophyllase activity and increased significantly to 6.25 units mg–1 protein after 9 d of storage, (i.e., at the late maturity stage). Respiration rate and 1-aminocyclopropene-1-carboxylic acid (ACC) oxidase activity did not significantly affect the chlorophyll degradation process in lime.The total nitrogen content of lime peel was inversely correlated with its chlorophyll content. A higher soluble protein content was observed in yellow peel than in mature green peel. A colour index chart was developed for maturity stages 1–4 in lime based on peel colour changes from mature green to full-yellow, and its correlation with chlorophyll content and fruit quality attributes.

Recombinant baculovirus mediates dsRNA specific to rr2 delivery and its protective efficacy against WSSV infection

White spot syndrome virus (WSSV) is a major causative agent in shrimp farming. Consequently, RNAi technology is an effective strategy to prevent WSSV infection in shrimp especially dsRNA targeting to rr2 of WSSV. In an effort to develop dsRNA expression in shrimp for control of WSSV infection, we developed a recombinant baculovirus expressing recombinant VP28 as the gene delivery system to carry a gene encoding dsRNA specific to rr2 for triggering the RNAi process in shrimp. The results showed that the recombinant baculovirus harboring VP28 was able to express VP28 indicated by Western blot with polyclonal antibody specific to VP28. VP28 transcript was detected in shrimp hemocytes after co-culture hemocytes with the recombinant baculovirus displaying VP28. In addition, we found that shrimp injected with the recombinant baculovirus displaying VP28 and encoding dsRNA synthetic gene specific to rr2 (Bac-VP28-dsrr2) showed the lowest cumulative mortality (33%) at 14days post infection (dpi) when compared to shrimp injected with baculovirus displaying VP28 (Bac-VP28) (64% cumulative mortality) (p<0.05). According to the results, shrimp injected with Bac-VP28-dsrr2 also showed significantly lower WSSV copies than shrimp injected with Bac-VP28 (p<0.05) along with the down-regulation of rr2 expression at 1, 3 and 7dpi. In conclusion, the Bac-VP28-dsrr2 was effective in prevention of WSSV infection. Therefore, the results obtained here can be applied to the prevention of WSSV infection by mixing the recombinant baculovirus with shrimp feed in the future.

Design and Generation of Humanized Single-chain Fv Derived from Mouse Hybridoma for Potential Targeting Application

Single-chain variable antibody fragments (scFvs) are attractive candidates for targeted immunotherapy in several human diseases. In this study, a concise humanization strategy combined with an optimized production method for humanizing scFvs was successfully employed. Two antibody clones, one directed against the hemagglutinin of H5N1 influenza virus, the other against EpCAM, a cancer biomarker, were used to demonstrate the validity of the method. Heavy chain (VH) and light chain (VL) variable regions of immunoglobulin genes from mouse hybridoma cells were sequenced and subjected to the construction of mouse scFv 3-D structure. Based on in silico modeling, the humanized version of the scFv was designed via complementarity-determining region (CDR) grafting with the retention of mouse framework region (FR) residues identified by primary sequence analysis. Root-mean-square deviation (RMSD) value between mouse and humanized scFv structures was calculated to evaluate the preservation of CDR conformation. Mouse and humanized scFv genes were then constructed and expressed in Escherichia coli. Using this method, we successfully generated humanized scFvs that retained the targeting activity of their respective mouse scFv counterparts. In addition, the humanized scFvs were engineered with a C-terminal cysteine residue (hscFv-C) for site-directed conjugation for use in future targeting applications. The hscFv-C expression was extensively optimized to improve protein production yield. The protocol yielded a 20-fold increase in production of hscFv-Cs in E. coli periplasm. The strategy described in this study may be applicable in the humanization of other antibodies derived from mouse hybridoma.

Optimization of recombinant dengue envelope protein production in insect cell culture in 2.5 l fermenter

The baculovirus expression vector system (BEVS) has been used to express many fuctionally authentic recombinant proteins in insect cells. Insect cells are used as hosts for recombinant baculovirus infection which results in recombinant protein production. In this study, recombinant dengue envelope protein production. In this study, recombinant dengue envelope protein was produced from insect cell (SF-9) infected with recombinant dengue baculovirus. Conditions involve the recombinant dengue envelope protein production i.e insect cell medium, insect cell growth phase, cell density, amout of recombinant baculovirus used to infect cells and optimal harvesting time were investigated. The results obtained when study in shake flask (250ml) showed that optimal medium for Sf-9 growth was the mixture of SF-900II and TC100 at ratio 1:1 supplemented with 10% FCS. The optimal growth phase and cell density were at early log phase and 1x10 6 cells/ml, respectively. Optimal multiplicity of infection was 5 (MOI =5) and harvest time was 3 days post infection. The information obtained in this study was then used for large scale production in a 2.5 L stirred tank reactor performed in 1 L working volume. It was found that the optimal agitation rate and dissolved oxygen for insect cell growth was at 90 rpm and 60-80% air saturation, respectively. Maximum recombinant dengue envelope protein production was 13 mg/L which was higher than the production from shake flask.

Recombinant neuraminidase pseudotyped baculovirus: a dual vector for delivery of Angiotensin II peptides and DNA vaccine

Baculovirus is a promising vaccine deliver vector due to its biosafety profiles, gene transfer efficiency, ability to display small foreign antigens on its surface, strong adjuvant activities, etc. A dual vector for peptide antigens and a DNA vaccine delivery was constructed. In this vector, a tetrameric glycoprotein neuraminidase (NA) from influenza A virus (H5N1) serves as a baculovirus surface protein to improve baculovirus transduction efficiency and a partner for displaying the target peptide antigen. Nucleotides encoding target peptides could be fused to a full length NA gene, at the lower part of its head structure, integrated into Autographa californica multinucleopolyhedrovirus genome and expressed under the control of a White Spot Syndrome Virus IE-1 shuttle promoter. Angiotensin II (AngII) peptides, a potent vasoconstrictor that causes high blood pressure, was our target antigen. The recombinant NA-AngII pseudotyped baculovirus had the AngII peptides fused to the NA and displayed on its surface. In vitro studies revealed that this recombinant baculovirus successfully delivered AngII peptides, as DNA vaccine, into human HEK293A cells. A single subcutaneous injection of the recombinant NA-AngII pseudotyped baculovirus into moderately high blood pressure rats at 4 × 109 pfu/rat, stimulated anti-AngII antibody production and their systolic blood pressure (SBP) levels were found to have decreased. In addition, a single intranasal immunization at 8 × 108 pfu/rat, raised anti-AngII antibodies in a rat and its SBP was also reduced. The recombinant neuraminidase pseudotyped baculovirus is a potential vector for AngII peptide antigen and DNA vaccine for subcutaneous or intranasal immunization for treatment of hypertension.

In vitro production of Spodoptera exigua multiple nucleopolyhedrovirus with enhanced insecticidal activity using a genotypically defined virus inoculum

Defective virus accumulations during baculovirus passages in insect cell culture are impediments to large scale baculovirus production. A genotypically defined virus inoculum comprises of stable genotypes was proposed for production of a Thailand isolated SeMNPV in Se-UCR1 insect cells. Targeted genotypes were from wild-type SeMNPV containing naturally mixed genotypes. Plaque assays, PCR screening and XbaI restriction analysis were employed for genotype purification, genotype selection and genome analysis, respectively. A selective marker was pif2 encoded per os infection factor which predominantly deleted, along with the adjacent pif1, in defective viruses. A purified, genetically stable pif2+ (and pif1+) genotype, namely SeThpif2+, was the first tryout. SeThpif2+ occlusion bodies (OBs) possessed insecticidal activity but at lower level than the wild-type. When the SeThpif2+ was co-infected with another purified, genetically stable pif1- (and pif2-) genotype, SeThpif2-, at ratio of 3:1, respectively, mixed genotypes OBs had 2.8 times greater insecticidal activity than the SeThpif2+ alone. Dilution of deleterious PIF1 of SeThpif2+ by the pif1 deletion genotypes, SeThpif2-, was the key for this enhanced activity. A promising approach was described for SeMNPV production in vitro using the virus inoculum whose genotypes compositions were designed to mimic virus interactions in the wild-type, to generate per oral infective baculovirus.

Chemical Pretreatment-Independent Saccharifications of Xylan and Cellulose of Rice Straw by Bacterial Weak Lignin-Binding Xylanolytic and Cellulolytic Enzymes

ABSTRACT Complete utilization of carbohydrate fractions is one of the prerequisites for obtaining economically favorable lignocellulosic biomass conversion. This study shows that xylan in untreated rice straw was saccharified to xylose in one step without chemical pretreatment, yielding 58.2% of the theoretically maximum value by Paenibacillus curdlanolyticus B-6 PcAxy43A, a weak lignin-binding trifunctional xylanolytic enzyme, endoxylanase/β-xylosidase/arabinoxylan arabinofuranohydrolase. Moreover, xylose yield from untreated rice straw was enhanced to 78.9% by adding endoxylanases PcXyn10C and PcXyn11A from the same bacterium, resulting in improvement of cellulose accessibility to cellulolytic enzyme. After autoclaving the xylanolytic enzyme-treated rice straw, it was subjected to subsequent saccharification by a combination of the Clostridium thermocellum endoglucanase CtCel9R and Thermoanaerobacter brockii β-glucosidase TbCglT, yielding 88.5% of the maximum glucose yield, which was higher than the glucose yield obtained from ammonia-treated rice straw saccharification (59.6%). Moreover, this work presents a new environment-friendly xylanolytic enzyme pretreatment for beneficial hydrolysis of xylan in various agricultural residues, such as rice straw and corn hull. It not only could improve cellulose saccharification but also produced xylose, leading to an improvement of the overall fermentable sugar yields without chemical pretreatment. IMPORTANCE Ongoing research is focused on improving “green” pretreatment technologies in order to reduce energy demands and environmental impact and to develop an economically feasible biorefinery. The present study showed that PcAxy43A, a weak lignin-binding trifunctional xylanolytic enzyme, endoxylanase/β-xylosidase/arabinoxylan arabinofuranohydrolase from P. curdlanolyticus B-6, was capable of conversion of xylan in lignocellulosic biomass such as untreated rice straw to xylose in one step without chemical pretreatment. It demonstrates efficient synergism with endoxylanases PcXyn10C and PcXyn11A to depolymerize xylan in untreated rice straw and enhanced the xylose production and improved cellulose hydrolysis. Therefore, it can be considered an enzymatic pretreatment. Furthermore, the studies here show that glucose yield released from steam- and xylanolytic enzyme-treated rice straw by the combination of CtCel9R and TbCglT was higher than the glucose yield obtained from ammonia-treated rice straw saccharification. This work presents a novel environment-friendly xylanolytic enzyme pretreatment not only as a green pretreatment but also as an economically feasible biorefinery method.