Synthesis of Glutamate-Zinc-Aluminium-Layered Double Hydroxide Nanobiocomposites and Cell Viability Study

A layered compound of zinc-aluminium layered double hydroxide (LDH) to be used as a host for a guest amino acid, glutamate was synthesized. Different parameters were used and optimized to form amino acid-intercalated pure phase materials. The resulting Bio-Inorganic Nanohybrid (BINH) was chosen for further characterization. BINH exhibits the glutamate to be in vertical or perpendicular orientation to the inorganic layer. Cytotoxicty test indicates that the IC50 value was observed at 3.125 μg/ml. Results from this study will be used in the development of a new delivery system for therapeutic agents comprising amino acids or peptides

Nipah virus glycoprotein: production in baculovirus and application in diagnosis

A method for serological diagnosis of Nipah virus (NiV) is described. DNA encoding truncated G protein of NiV was clone into the pFastbac HT vector, and the fusion protein to His-Tag was expressed in insect cells by recombinant baculovirus. The resulting His-G recombinant fusion protein was purified by affinity chromatography and used as the coating antigen for serological testing by in direct enzyme-linked immunosorbant assay (ELISA). When tested against a panel of swine serum samples, the recombinant G protein-based ELISA successfully discriminated all 40 samples previously determined to be serum neutralizing test (SNT) positive from 11 SNT negative samples. The data show that the recombinant G protein exhibit the antogenic epitopes and conformation necessary for specific antigen-antibody recognition. The main advantage of the recombinant G protein-base NiV ELISA compared to and ELISA using whole virus antigen is the use of single antigenic protein instead of inactivated whole virus which is required to be prepared under high risk and cost. This test is suitable for routine diagnosis of NiV and also for epidemiological surveys as it allows highly reliable testing of a large number of sera rapidly

Cell rupture of recombinant escherichia coli using high pressure homogenizer

Cell rupture is one of the earlier steps in downstream processing which are required for the recovery of biological products that are located inside cells. Cells could be disrupted either by using chemicals or mechanical method. In this study, cell rupture was carried out by mechanical force using high pressure homogenizer (HPH). The aim of this study is to identify optimal conditions of HPH to disrupt the cell wall of recombinant Escherichia coli harboring nucleocapsid (NP) gene of Newcastle disease virus (NDV). The optimized conditions were achieved by manipulating the independent variables of HPH such as pressure, pump speed and number of cycles through an optimization process. The efficiency of the cell disruption was determined by estimating the percentage of cell rupture as well as the amount of NP protein released from the cell lysis. Through the means plot analysis of Minitab Software (Version 14.12), pressure was recognized as the main factor for achieving the highest cell rupture and the release of NP protein. The optimized conditions for obtaining the highest NP protein yield were by operating three cycles of cell rupture, homogenizer pressure of 800 bars and pump speed of 7 psi

Detection and differentiation of velogenic and lentogenic Newcastle disease viruses using SYBR Green I real-time PCR with nucleocapsid gene-specific primers

SYBR Green I real-time PCR was developed for detection and differentiation of Newcastle disease virus (NDV). Primers based on the nucleocapsid (NP) gene were designed to detect specific sequence of velogenic strains and lentogenic/vaccine strains, respectively. The assay was developed and tested with NDV strains which were characterized previously. The velogenic strains were detected only by using velogenic-specific primers with a threshold cycle (Ct) 18.19 ± 3.63 and a melting temperature (Tm) 86.0 ± 0.28 °C. All the lentogenic/vaccine strains, in contrast, were detected only when lentogenic-specific primers were used, with the Ct value 14.70 ± 2.32 and Tm 87.4 ± 0.21 °C. The assay had a dynamic detection range which spans over a 5 log10 concentration range, 109–105 copies of DNA plasmid/reaction. The velogenic and lentogenic amplifications showed high PCR efficiency of 100% and 104%, respectively. The velogenic and lentogenic amplifications were highly reproducible with assay variability 0.45 ± 0.31% and 1.30 ± 0.65%, respectively. The SYBR Green I real-time PCR assay detected successfully the virus from tissue samples and oral swabs collected from the velogenic and lentogenic NDV experimental infection, respectively. In addition, the assay detected and differentiated accurately NDV pathotypes from suspected field samples where the results were in good agreement with both virus isolation and analysis of the fusion (F) cleavage site sequence. The assay offers an attractive alternative method for the diagnosis of NDV

Rapid and non-radioactive detection method of microsatellites in Mystus nemurus: a refined technique

A simple and rapid method of DNA microsatellite isolation based on the Random Amplified Microsatellites (RAMs) PCR technique was used in this study. The work presented here is part of a continuous effort in refining and perfecting the technique for more rapid, effective and optimum productivity in single locus microsatellite marker development for the River catfish, Mystus nemurus. The current refined protocol for microsatellite isolation was able to detect a total of 135 microsatellite regions resulting in 42 unique genomic sequences being submitted to GenBank. This refined technique is able to reduce the total time required from peR cloning till sequencing specific microsatellite regions to less than three and a half months

Newly developed microsatellite markers of Mystus nemurus tested for cross-species amplification in two distantly related aquacultured catfish species

The work reported here is an attempt to explore the possibility of DNA microsatellite loci transfer (cross-species amplification) to other economically important aquacultured catfish species other than its source species. A total of 25 new microsatellite loci developed for riverine catfish, Mystus nemurus were successfully cross-amplified in two distantly related catfish species within the suborder Siluroidei. Five out of the 19 loci that successfully cross-amplified in Pangasius micronemus were polymorphic, while for Clarias batrachus, cross-amplification was successful using 17 polymorphic loci. The observed heterozygosities were high for all the three catfishes. The results indicated that microsatellite loci could be as polymorphic in non-source species as in the source species

Expression of a thermostable xylanase gene from Bacillus coagulans ST-6 in Lactococcus lactis

Aims: The aim of the study is to evaluate whether xylanase can be used as a potential reporter gene for cloning and expression studies in Lactococcus. Methods and Results: The 750 bp xylanase gene was amplified and subcloned into the unique NheI restriction enzyme site of pMG36e and subsequently transformed into competent Escherichia coli XLI-blue MRF cells and Lactococcus lactis cells. Bacterial culture containing pMG36e-Xy has an enzyme activity of 390 μg xylose ml−1 culture 30 min−1, respectively, when compared with 40 μg xylose ml−1 culture 30 min−1 for the negative control (plasmidless strain). Conclusions: The thermostable xylanase gene was successfully expressed in both E. coli and L. lactis. The activity of xylanase can be easily detected by the formation of visible clearing zones around the transformed colonies on Remazol Brilliant Blue-Xylan (RBB-Xylan) agar media. However, there were some significant differences in the optimum growth temperature and plasmid stability in the new clones. Significance and Impact of the Study: The constructed reporter vector has the potential to be used as a reporter system for Lactococcus as well as E. coli, and it is an addition to the pool of lactococcal vector systems

Successful transfer of plasmid DNA into in vitro cells transfected with an inorganic plasmidMg/Al-LDH nanobiocomposite material as a vector for gene expression

The delivery of a full plasmid, encoding the green fluorescent protein gene into African monkey kidney (Vero3) cells, was successfully achieved using nanobiocomposites based on layered double hydroxides. This demonstrated the potential of using the system as an alternative DNA delivery vector. Intercalation of the circular plasmid DNA, pEGFP-N2, into Mg/Al-NO−3 layered double hydroxides (LDH) was accomplished through anion exchange routes to form the nanobiocomposite material. The host was previously synthesized at the Mg2+ to Al3+ molar ratio Ri = 2 and subsequently intercalated with plasmid DNA. Size expansion of the interlamellae host from 8.8 ° A in LDH to 42 °A was observed in the resulting nanobiocomposite,indicating stable hybridization of the plasmid DNA. The powder x-ray diffraction (PXRD)results, supplemented with Fourier-transform infrared (FTIR) spectroscopy, compositional and electrophoresis studies confirmed the encapsulation episode of the biomaterial. In order to elucidate the use of this resulting nanobiocomposite as a delivery vector, an MTT assay was performed to determine any cytotoxic effects of the host towards cells. The intercalated pEGFP-N2 anion was later successfully recovered through acidification with HNO3 after treatment with DNA-degrading enzymes, thus also showing the ability of the LDH host to protect the intercalated biomaterial from degradation. Cell transfection studies on Vero3 cells were then performed, where cells transfected with the nanobiocomposite exhibited fluorescence as early as 12 h post-treatment compared to naked delivery of the plasmid itself

Production of a panel of monoclonal antibodies against the nucleocapsid protein of heat resistant Newcastle disease virus

A panel of six monoclonal antibodies against the nucleocapsid protein (NP) of Newcastle disease virus (NDV) strain AF2240 was produced by immunization of Balb/c mice with purified NP. Isotyping test revealed three of the monoclonal (mAbs) were IgG1 and the others were IgG2a. All of the mAbs recognized linearized NP epitopes. The reactivity of these mAbs with local isolates revealed that three mAbs recognized conserved antigenic sites and the others bind to antigenic sites which undergone considerable changes