Problem statement: Dengue fever, which was caused by Dengue virus infection, had became a major public health problem in the tropic and subtropical countries. Dengue virus (DENV) had four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4), based on their immunogenic in the human body. Preventive measure will be necessary to decrease the prevalence of dengue fever, by developing modern vaccine. Approach: This research was focused on in silico study of dengue virus vaccines, by using envelope (E) protein of DENV-2 and DENV-3 as their backbones. T cell epitope prediction was determined by using MULTIPRED server and B cell epitope prediction was determined by using Conformational Epitope Prediction server (CEP). Homology modeling study of E DENV-3 protein as the vaccine backbone had produced six dengue vaccine peptides (HMM Vaccine 1-6). Moreover, homology modeling study of E DENV-2 protein as vaccine backbone had produced six dengue vaccine peptides (ANN vaccine 1-6). Results: The BLAST analysis of HMM and ANN vaccines had produced 93% and 91% identity, respectively. The Ramachandran Plot of both vaccines had shown less than 15% non glycine residue in the disallowed region, therefore it showed the solid stability of the proteins. The VAST analysis of E DENV-3 backbone vaccines had determined, that HMM4 and HMM6 had the highest structure similarity with native E DENV-3. HMM4 and HMM6 had the highest VAST score of 64.5. Moreover, the VAST analysis of E DENV-2 backbone vaccines had determined, that ANN1, ANN3, ANN4, ANN5 and ANN6 had the highest structure similarity with native E DENV-2. ANN1, ANN3, ANN4, ANN5 and ANN6 have the highest VAST score of 64.7. Conclusion/Recommendation: It could be inferred from this research that HMM4; HMM6; ANN1; ANN3; ANN4; ANN5; and ANN6 were the best in silico vaccine design, based on their similarity with native E DENV Proteins. This research could be applied for the wet laboratory and computerised vaccine design
Candida Antarctica Lipase B (CALB) is extensively studied in enzymatic production of biodiesel, pharmaceutical products, detergents and other chemicals. One drawback of using CALB is its relatively low optimum temperature at 313 K (40°C). The objective of this research is to design CALB mutant with improved thermostability by introducing extra disulfide bond. Molecular dynamic simulation was conducted to get better insight into the process of thermal denaturation or unfolding in CALB. Thermal denaturation of CALB was accelerated by conducting simulation at high temperature. Molecular dynamic simulation of CALB was performed with GROMACS software package at 300-700 K. Prediction of possible mutation was done using "Disulfide by Design TM " software. Selection of mutated residues was based on flexibility analysis of CALB. From those analyses, three mutants were designed, which are Mutant-1 (73LeuCys/151AlaCys), Mutant-2 (155TrpCys/294GluCys) and Mutant-3 (43ThrCys/67SerCys). Parameters that were used to compare the thermostability of mutant with wild type enzyme were Root Mean Square Deviations (RMSD), Solvent Accessible Surface Area (SASA), Radius of gyration (Rg) and secondary structure. Molecular dynamic simulation conducted on those three mutants showed that Mutant-1 has better thermostability compared to wild type CALB. We proposed the order of mutant thermostability improvement as follows: Mutant-1, Mutant-2 and Mutant-3, with Mutant-1 having better potential thermostability improvement and Mutant-3, the least stable.
Problem statement: Hyaluronidase is an enzyme which catalyze hydrolysis of Hyaluronan (HA). Hyaluronan is important in cell migration during embryonic development, cellular proliferation and differentiation and has a structural role in connective tissues. Hyaluronidase deficiency is correlated with mucopolysaccharidosis IX. In human, hyaluronidase is encoded by HYAL1 gene. The mutation study of HYAL1 gene was carried out by many researchers, but until now, mutation study of HYAL1 still in progress and limited due to the lack of primer used in amplification of selected DNA sequence of HYAL1 gene and maximum length limitation imposed by DNA sequencer. Approach: The search for three pairs of primers for human exon 1 segmented amplification of HYAL1 gene was conducted and evaluated. The first step was to acquire HYAL1 gene sequence and then had it aligned with human chromosome 3 genomic contig sequence. Exon 1 of HYAL1 gene was found to be located at nt 201-1711 of the acquired human chromosome 3 genomic contig. Online Primer3 program was used to design three pairs of primers. The selected pairs of primer had been subjected to BLASTn operation for selectivity examination while onlineNetPrimer operation was carried out for examination of secondary structures. Results: The search for primers to amplify three different fragments of exon 1 of HYAL1 gene yielded three selected pairs of primers, namely forward primer 5-TGACCCCCTACAAAAGCTCA-3 (20 bp) and reverse primer 5-AAGTCTCCGATTCCCCCACT-3 (20 bp) for amplifying nt 1-551 of HYAL1 gene, forward primer 5-AGTCCTGTGGGAGATGGCAGA-3 (21 bp) and reverse primer 5-CGGTAAATGTCCTTGGTGTCC-3 (21 bp) for amplifying nt 355-1053 of HYAL1 gene and forward primer 5-GCCATACCTGCTCCTGACTT-3 (20 bp) and reverse primer 5-ACAAGGTGGGCAGGTTACAG-3 (20 bp) for amplifying nt 956-1511 of HYAL1 gene. When using these primers, nt 1-46 of amplified product of the first pair of primers and nt 555-584 of amplified product of the third pair of primers must not be considered because those are not part of exon 1 of HYAL1 gene. The results from both operations and trial to real samples using these primers indicated that all three pairs of primer were satisfactory for use. Conclusion/Recommendations: The three pairs of primers could be used to amplify specified segments of HYAL1 gene
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