Generation of blasticidin resistance gene knockout plasmid (PGKO-BlastR) for scavenger receptor class B type 1 (SR-B1) gene / Faten Haziemah Mohd Nadzri

HEPATITIS C virus (HCV) infection is a liver disease caused by the hepatitis C virus. HCV infection is the major contribution factor of liver transplantation in many countries. There is currently no vaccine for hepatitis C; however, research in this area is ongoing. This study was performed to gain insight about the significant role of Scavenger Receptor Class B type 1 (SR-Bl) in attachments and entry in HCV infection and to re-amplify both left and right homology arms of SR-Bl and blasticidin resistance gene (Blast) for SR-Bl gene. This study also aims to generate production of blastacidin resistance gene knockout plasmid (pGKO-BlastR) for SR­ B 1 gene. Several methods were used to achieve the objectives of this study. The initial step is re-amplification of homology arms of SR-Bl and BlastR by using Polymerase Chain Reaction (PCR). The result was obtained which are lkb for both homology arms and l .5kb for blasticidin resistance gene (BlastR). The PCR products were used to generate production of plasmid gene knock-out blasticidin resistance (pGKO-BlastR) by using fusion PCR methods. Fusion PCR methods were used to link The three fragments namely left homology arms, and right homology arms of SR-Bl with the BlastR from previous PCR products lead us in production of pGKO­ Blast". Gradient PCR technique was used in fusion PCR method to determine the optimum annealing temperature and 65°C was chosen as the best optimum annealing temperature. Based on the result, there were band size appeared between 2kb and 3kb. This shows that, there are possibilities that two big fragments fuse together either LHA + Blast or Blasf +RHA. The targeted size of full fusion PCR product is 3.5kb but if the size about 2.5kb it might show the partial fusion PCR product. There are also multiple bands appeared which could come from impurities of the sample from previous PCR product and might be the crucial factor in order to obtain a clear and fine band yield on the agarose gel. It is suggested to purify the PCR products using gel extraction kit before proceed to the next reaction. All PCR reactions were performed with Q5 High Fidelity 2x Master Mix using the conditions specified by the manufactures. The analysis of all PCR products has been done with 1 % agarose gel electrophoresis method. All samples were compared with lkb DNA ladder marker. Hopefully, the production pGKO-BlastR could assists and supports other research studies on understanding of HCV entry mechanism.