【作者】 张玲玲；

【导师】 孟祥伟；

【作者基本信息】 吉林大学 ， 内科学， 2005， 硕士

【摘要】 乙型肝炎病毒(hepatitis B virus ,HBV) 感染在我国是一个严重的公共卫生问题,HBV 变异研究在乙肝发病机理及防治上有重要意义,衡量基因变异与否的标准是以标准参照序列为参照的。现存HBV标准参照序列多是借用国外全序列,而我国HBV基因全序列仅有少数几例,我国是HBV 高流行区,其毒株与欧、美和日本等低流行区存在区别。本试验旨在通过对东北地区HBV 相关慢性肝病患者用RFLP 方法进行HBV 基因分型,测定S 区基因序列,探讨HBV S 基因变异的特点,从而对建立我国乃至本地区HBV 基因库提供重要参考资料。采用Sanger 双脱氧链终止法进行HBV S 区DNA 测序分析结果表明,S基因PCR-RFLP 方法与S 基因序列测定在HBV 基因分型中相比较,具有同样的特异性和准确性。在HBV 相关性慢性肝病病人中,S 基因存在序列多态性,多处存在核苷酸不同而氨基酸同义,部分核苷酸变异导致所编码的氨基酸发生变异。更多还原

【Abstract】 In 1965, hepatitis B virus surface antigen(HBsAg )was founded in the serum of Australian indigene by Blumberg, this was regarded as the start of molecular biological study of hepatitis B virus(HBV). Later, the development of study on HBV is fast in the genomic structure , gene mutation ,epidemiologic trait, pathogenesis, gene therapy and vaccine ,and so on. HBV is a major causative agent of acute and chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Hepatitis B virus has infected substantial populations throughout the world. In China, HBV infection is highly prevalent ;there are about 50%-70% percent of people has infected with HBV and approximately 100 million (8%-12%) carriers of HBsAg. Therefore, the study on polymorphism of HBV S gene sequence, which is related to immune escape mutants and resistant to HBV vaccine, is very important to prevention and cure of HBV infection. In our study, twenty-one serum samples were evaluated from patients with chronic HBV infection. HBV DNA extracted from the 21 serum samples were amplified by nested polymerase chain reaction (nest-PCR) to obtain the conserved S gene sequences which were genotype-specific. The amplification mixture was2.5μl of 2.5 mM deoxyribonucleotide triphosphate, 2.5μl of 10×PCR buffer, 0.5μl of Takara TaqTm , 10 pmol of sense and antisense primers in a 25μl reaction volume .The amplification profile was 2 min at 960C, followed by 25 cycles at 940C for 15 s(denaturation), 45 s at 600C(annealing) and 45 s at 720C(extension). Using restriction fragment length polymorphism (RFLP) which was described by Mizokami, the second-round PCR product was digested by the restriction enzymes, AlwI, HphI, NciI, NlaIV and EarI to be genotyped. In the 21 samples the following genotypes were observed : 12 C (HBV 1---HBV 12)and 9 B (HBV 13—HBV 21).In addition, the S genes from the 21 patients were amplified by nested PCR with other two pairs primers, under the same conditions described above. All the PCR products were sequenced by dideoxy-chain-termination method, the S gene nucleotide sequences were acquired and the corresponding amino acid sequence was deduced using DNASIS software. Moreover, they were compared to representative S gene sequences obtained from GenBank and the homology was obtained using BLAST program at the NCBI WWW site .It showed that the genotype of 12 strains were C and the homology of HBVS gene were 98%--100%, the genotype of 9 strains were B and the homology of HBV S gene were 99%--100%. The results of RFLP HBV genotyping methods was coincide with that of S gene sequence and this confirmed that HBV S gene RFLP genotyping method was differential and accurate. Analyzing the nucleotide sequences and the corresponding amino acid sequence , it showed that 8 strains (HBV 1-8)belong to subtype C/adr and 4 strains (HBV 9-12)to subtype C/adw, 9 strains (HBV 13-21)belong to serotype B/adw.The amino acids of antigen determinant ‘a’of two strains(HBV1、HBV16) changed. Compared the S gene sequences of the subtype C/adr , the subtype C/adw and the subtype B/adw to the consensus sequence of HBV Y18856 、HBV Y18857 and HBV AF100309 obtained from GeneBank, respectively, the homology of nucleotide sequence were 99.27%--100%,98.53%—99.71% and 99.56%--100% .Moreover , 98.23%--100%,97.34%--99.56% and 98.67%--100% homology were at amino acid level .In 8 strains for subtype C/adr, there were sixteen point mutations in nucleotide sequence and nine of these caused amino acid residue substitution. In 4 strains for serotype C/adw, there were seventeen point mutations in nucleotide sequence and ten of these caused amino acid residue substitution. There were eleven point更多还原