METHODS AND RESULTS: The Rhizomucor pusillus proteinase (RPP) gene was sub-cloned into a pALF expression vector. The recombinant pALF-RPP vector was then electro-transferred into Lactococcus lactis. Finally, the milk coagulation ability of recombinant L. lactis carrying a RPP gene was evaluated. Nucleotide sequencing of DNA insertion from the clone revealed that the RPP activity corresponded to an open reading frame consisting of 1218 bp coding for a 43·45 kDa RPP protein. The RPP protein assay results indicated that the highest RPP enzyme expression with 870 Soxhlet units (SU) per ml and 7914 SU/OD were obtained for cultures which were incubated at pH 5·5 and 30°C. Interestingly, milk coagulation was observed after 205 min of inoculating milk with recombinant L. lactis carrying the RPP gene.
CONCLUSION: The recombinant L. lactis carrying RPP gene has the ability to function as a starter culture for acidifying and subsequently coagulating milk by producing RPP as a milk coagulant agent.
SIGNIFICANCE AND IMPACT OF THE STUDY: Creating a recombinant starter culture bacterium that is able to coagulate milk. It is significant because the recombinant L. lactis has the ability to work as a starter culture and milk coagulation agent.
METHODS: In this study, new binders derived from shark VNAR domains library were investigated. Following immunization of a wobbegong shark (Orectolobus ornatus) with three recombinant malaria biomarker proteins (PfHRP2, PfpLDH and Pvaldolase), a single domain antibody (sdAb) library was constructed from splenocytes. Target-specific VNAR phage were isolated by panning. One specific clone was selected for expression in Escherichia coli expression system, and study of binding reactivity undertaken.
RESULTS: The primary VNAR domain library possessed a titre of 1.16 × 106 pfu/mL. DNA sequence analysis showed 82.5% of isolated fragments appearing to contain an in-frame sequence. After multiple rounds of biopanning, a highly dominant clone specific to PfHRP2 was identified and selected for protein production in an E. coli expression system. Biological characterization showed the recombinant protein expressed in periplasmic has better detection sensitivity than that of cytoplasmic proteins. Assays of binding activity indicated that its reactivity was inferior to the positive control mAb C1-13.
CONCLUSIONS: Target-specific bacteriophage VNARs were successfully isolated after a series of immunization, demonstrating that phage display technology is a useful tool for selection of antigen binders. Generation of new binding reagents such as VNAR antibodies that specifically recognize the malaria biomarkers represents an appealing approach to improve the performance of RDTs.
RESULT: The screening of six physical conditions by Plackett-Burman Design has identified pH, inoculum size and incubation time as exerting significant effects on lipase production. These three conditions were further optimised using, Box-Behnken Design of Response Surface Methodology, which predicted an optimum medium comprising pH 6, 24 h incubation time and 2% inoculum size. T1 lipase activity of 2.0 U/mL was produced with a biomass of OD600 23.0.
CONCLUSION: The process of using RSM for optimisation yielded a 3-fold increase of T1 lipase over medium before optimisation. Therefore, this result has proven that T1 lipase can be produced at a higher yield in P. guilliermondii.
Methods: Infectivity, pathogenicity, and real-time dissemination of aerosolized NiV in Syrian hamsters was evaluated using NiV-Malaysia (NiV-M) and/or its recombinant expressing firefly luciferase (rNiV-FlucNP).
Results: Both viruses had an equivalent pathogenicity in hamsters, which developed respiratory and neurological symptoms of disease, similar to using intranasal route, with no direct correlations to the dose. We showed that virus replication was predominantly initiated in the lower respiratory tract and, although delayed, also intensely in the oronasal cavity and possibly the brain, with gradual increase of signal in these regions until at least day 5-6 postinfection.
Conclusion: Hamsters infected with small-particle aerosolized NiV undergo similar clinical manifestations of the disease as previously described using liquid inoculum, and exhibit histopathological lesions consistent with NiV patient reports. NiV droplets could therefore play a role in transmission by close contact.