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Sample: from the School of BABS, UNSW table of contents abstract i table of contents ii acknowledgements v list of tables vi list of figures vii list of abbreviations viii 1 INTRODUCTION 1 1.1 hepatitis C virus 1 1.1.1 discovery 1 1.1.2 epidemiology 2 1.1.3 pathogenesis 2 1.1.4 treatment 3 1.2 molecular biology 3 1.2.1 structure of genome 3 1.2.2 genetic variation 6 1.2.3 genotypic differences 8 1.3 RNA dependent rna polymerase activity 9 1.3.1 polymerase function 9 1.3.2 model systems of HCV replication 11 1.3.3 genotype specific studies 11 1.3.4 biochemical properties 12 1.4 kunjin virus RNA dependent RNA polymerase 13 1.5 conclusion 15 1.6 aims and hypothesis 16 2 MATERIALS AND METHODS 17 2.1 HCV positive sera samples 17 2.2 RNA extraction 17 2.3 cDNA synthesis 17 2.4 HCV primer design and usage 18 2.5 nested polymerase chain reaction (nPCR) 21 2.5.1 reaction and cycling conditions 21 2.5.2 PCR product purification 22 2.6 agarose gel visualisation 22 2.7 DNA sequencing 22 2.8 DNA sequence and phylogenetic analysis 23 2.9 kunjin virus plasmid 23 2.10 KUN primer design and usage 23 2.11 cloning PCR products 24 2.11.1 restriction digest 24 2.11.2 ligation 24 2.11.3 transformation 24 2.11.4 colony PCR 24 2.12 protein expression 25 2.13 sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) 26 2.14 western blot 26 2.15 plasmid extraction 26 2.16 production of cell stock 27 3 RESULTS 28 3.1 thirteen samples are confirmed to be HCV 5’-UTR positive 28 3.2 eight samples are confirmed to be HCV NS5B positive 28 3.3 sequence and phylogenetic analysis of 9 HCV NS5B positive samples 29 3.4 amplification of full-length NS5B products 31 3.5 evaluation of set 3 primers 32 3.6 amplification of half-length NS5B products 34 3.7 sequence analysis of 7 half-length NS5B products 36 3.8 alternative method for cDNA synthesis 37 3.9 five NS5B products are amplified by nPCR 39 3.10 sequence and phylogenetic analysis of 5 full-length NS5B products 39 3.11 construction of kunjin clone 41 3.12 protein expression of HCV and KUN clones 41 4 DISCUSSION 46 4.1 sample integrity confirmed by 5’UTR nPCR and the NS5B nPCR 46 4.2 genotypes of 9 samples confirmed by phylogenetic analysis 46 4.3 amplification of full-length NS5B products 47 4.4 evaluation of set 3 primers 48 4.5 amplification of half-length NS5B products 49 4.6 alternative method for cDNA synthesis 50 4.7 sequence analysis of HCV genotype 3a NS5B sequences 50 4.8 construction of kunjin clone 52 4.9 protein expression of HCV and KUN clones 52 4.10 future directions and conclusions 54 5 REFERENCES 55 (Tan, 2004,pp.ii-iv) (FM] e y … † ˜ ™ ¡ ¤ µ ¹ É Ì Ú Þ í ò äën°Ôq{q{”•ëÝÌÝÀ±À©™‰~v~v~v~v~v~v~v~v~vlvlv~v~veahð\û hð\û\^Jhð\ûCJmH sH hð\ûmH sH hð\û5\mH sH h¸6Ðhð\û5CJ\mH sH h¸6Ðhx5CJ\mH sH hxmH sH h¸6Ðhx56]mH sH h¸6Ðhx5mH sH hx5OJPJQJ\nH tH hxOJPJQJnH tH 'hë=—hx5CJ$OJPJQJnH tH &()[ \ ] „ … † ˜ ™ ¤ ¹ Ì Þ ò 6 H ] r „ œ ¸ Ò ð ùùùùù÷÷÷ïïííííííííííííííííí$a$gdð\û ÆàÀ!8b…¥Öè?UkŽ½æ ) ? m ‰ Ê ç ú (C‘¦ÁýýýýýýýýýýýýýýýýýýýýýýýýýýýýýÁâï/j³å:rŸÏ>oÁ4W‰¶ñEo€•ýýýýýýýýýýýýýýýýýýýýýýýýýýôô¤¤gdð\û,1h°‚. °ÆA!°"°# $ %°°Ä°ÄÄ‚˜˜˜˜˜˜˜˜˜666666666vvvvvvvvv666666>666666666666666666666666666¨6666666666¸666666666666hH66666666666666666666666666666666666666666666666666666666666666666°66666666662ÀÐàð 0@P`p€ÀÐàð2(Øè 0@P`p€ÀÐàð 0@P`p€ÀÐàð 0@P`p€ÀÐàð 0@P`p€ÀÐàð 0@P`p€ÀÐàð 0@P`p€8XøV~_HmH nH sH tH H`ñÿHNormal CJOJQJ_HaJmH sH tH „„Heading 1,Char$ &F¤ð¤<@&55CJ KH OJPJQJ\^JaJ mH nHsH tHuˆˆHeading 2, Char$ &F¤ð¤<@&756CJOJPJQJ\]^JaJmH nHsH tHuvv Heading 3$ &F¤ð¤<@&15CJOJPJQJ\^JaJmH nHsH tHurr Heading 4$ &F¤ð¤<@&-5CJOJPJQJ\aJmH nHsH tHutt Heading 5 &F¤ð¤<@&356CJOJPJQJ\]aJmH nHsH tHunn Heading 6 &F¤ð¤<@&-5CJOJPJQJ\aJmH nHsH tHu`` Heading 7 &F¤ð¤<@&OJPJQJmH nHsH tHuff Heading 8 &F¤ð¤<@&%6OJPJQJ]mH nHsH tHud d Heading 9 &F¤ð¤<@&#CJPJ^JaJmH nHsH tHuDA òÿ¡DDefault Paragraph FontZióÿ³ZTable Normal :Vö4Ö l4Öaö_H(k ôÿÁ(No List†þò†-Style Heading 1 + Before: 0" First line: 0"„„^„`„|þ1|-Style Heading 3 + Arial Line spacing: Doubledàd@d TOC 1,TOC CHEN ¤x¤x$5;CJPJ\mH nHsH tHuTT TOC 2 „ð^„ð&:CJOJPJQJmH nHsH tHu.. 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