基于基因序列的乙型肝炎病毒基因分型方法的比较

Comparison of three different gene sequence-based genotyping methods for hepatitis B virus

采用3种不同的生物信息分析法对HBV S基因序列进行分析以鉴定HBV基因型,并比较分析各方法的优缺点。采集10例HBV慢性感染者血清,抽提HBV DNA,PCR扩增HBV S基因并测序。分别采用Clustal X软件和Bioedit软件计算S基因核苷酸和氨基酸同源性;DNAstart软件、Clustal X软件和Mega 4软件绘制S基因遗传进化树;以及在线Genotyping软件,将10例S基因序列与不同型及亚型HBV S序列比较以鉴定基因型及亚型。S基因核苷酸和氨基酸同源性计算结果显示1,3,4,5,7,8,9,10号标本与B2AF121243同源性最高,核苷酸同源性分别是98.0%,99.0%,98.8%,99.1%,99.2%,99.5%,99.5%,99.6%,而2号标本与C2 M38636同源性最高,核苷酸同源性为97.7%,6号标本与C4GQ377630同源性最高,核苷酸同源性为97.3%;构建系统树法鉴定结果为1,3,4,5,7,8,9,10号标本与B2AF121243进化距离最近,而2号标本与C2M38636进化距离最近,6号标本与C4GQ377630进化距离最近;Genotyping比对发现1,3,4,5,7,8,9,10号标本属于B基因型,2,6号标本属于C基因型。结论S基因核苷酸及氨基酸同源性的计算法可有效用于乙型肝炎病毒基因分型,是对现有分型方法系统树构建法以及在线Genotyping法的补充。

To investigate three different bioinformatics methods that have been used for analyzing the S gene sequence to determine the genotype of hepatitis B virus. S gene was amplified by PCR from HBV DNA ex- tracted from the serum samples of ten patients with HBV infection. Clustal X software and Bioedit software were included in method 1 to caculate the identity of nucleotide and amino acid of S gene DNAstart,Clust- al X and Mega 4 （method 2） were used to construct phylogenetic tree of S gene and online Genotyping of vrial gene （method 3） were adopted to compare the S gene of 10 serum samples,with S gene asthe refer- ence sequence of different genotype and subgenotype HBV to determine their genotype and subgenotype. Method 1 showed that samples 1, 3, 4, 5, 7, 8, 9, 10 had highest homologue with reference sequence AF121243 （genotype B2） with the nucleotide identity of 98.0% ,99.0% ,98.8% ,99.1% ,99.2% ,99.5%, 99.5% and 99.6% respectively Sample 2 had the highest homologue with reference sequence M38636 （genotype C2） with nucleotide identity of 97.7% Sample 6 with GQ377630 （genotype C4） with the nucle- otide identity of 97.3%. Method 2 showed that the samples 1,3,4,5,7,8,9,10 were in same clade of AF121243（genotype B2） with the shortest genetic distance,where sample 2 was in a clade of M38636 （gen-otype C2） and sample 6 was in a clade of GQ377630 （genotype C4） with the shortest genetic distance. With Method 3,it was found that the samples 1,3,4,5,7,8,9,10 belonged to genotype B and the samples 2,6 were in genotype C. Conclusion The nucleotide and amino acid identity calculation, phylogenetic tree con- struction and online genotyping of viral genes can be used to determine the genotype of hepatitis B virus, as a complimentary to the normal genotyping methods.