Random mutagenesis of NS1 protein of influenza a H1N1 and docking of RNA aptamers to wild type and mutant NS1 proteins

• Main Author: Chelliah, Kumutha
• Format: Thesis
• Language: English
• Published: 2012
• Subjects: Q Science (General); QH301 Biology
• Online Access: http://eprints.utm.my/id/eprint/36995/5/KumuthaChelliahMFBB2012.pdf; http://eprints.utm.my/id/eprint/36995/
• Institution: Universiti Teknologi Malaysia

The NS1A protein is a non-structural protein from influenza A virus H1N1 strain. The protein is a multifunctional protein which is capable of blocking the defense mechanism of host immune by inhibiting the secretion of host cell IFN a/ß. Even existing vaccines cannot protect host cells against this viral infection due to constant mutations of NS1A protein. In this study, the NS1A gene which was formerly cloned in pET 32c(+) vector was successfully mutated using error-prone PCR with increased concentration of MgCl2 to 10 mM and subsequently cloned into yT&A vector and transformed into E. coli DH5a. There were four proteins that contain non-conservative mutations from sequencing which were NS1 F103LN209D, NS1 S7P, NS1 T76I and NS1 E159G mutant proteins. These proteins together with the wild-type protein were modeled using EasyModeller 2.1 and were energy minimized using GROMACS. The qualities of the structures were validated using ERRAT, PROCHECK, Verify3D and ProSA web. All the structures were of good quality and the high RMSD value shows that the mutant proteins have low structural homology to the wild-type protein. This proves that the structures were affected by point mutations. None of the mutations fell into ‘hot spot’ mutations. These proteins were subsequently docked to RNA aptamers via HEX server to analyze the binding regions and binding affinity of aptamers to proteins. The results obtained shows that the protein mutations affect the binding properties of aptamers to the mutant proteins because aptamers were docked at various regions with different binding affinities. The aptamers with the highest binding affinity towards wild-type NS1A protein and mutant proteins were selected which were aptamers 21, 174 and 176. These results were expected to be useful for potential drug design to curb future H1N1 viral infections.