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目的研究乙型肝炎病毒(HBV)基因型在不同民族慢性乙型肝炎(CHB)患者中是否存在进化差异及临床意义。方法应用聚合酶链反应(PCR)对中国云南少数民族地区50例CHB患者(25例少数民族CHB患者与对照组25例汉族CHB患者)HBV前S2/S(preS2/S)和C(core,C)基因进行扩增,将PCR扩增产物克隆到载体pBS(pBluescriptⅡSK,pBS)上进行测序分析。结果50例患者HBVpreS2/S基因全长846bp,GC含量占49.1%,编码281个氨基酸(aa);C基因全长552bp,GC含量占46.1%,编码183个aa。并在GenBank登录注册(AY517619、AY517620、AY517488、AY517489、AY517598、AY517599)。通过与GenBank数据库中HBV基因及亚型序列比较,发现全部患者HBV基因与ayw1亚型具有高度同源性,preS2/S基因aa同源性为97.5%～98.6%,C基因aa同源性为94.5%～97.8%。所有患者S基因在“a”抗原决定簇aa第122位为精氨酸(AGA)、第160位为赖氨酸(AAA)。发现全部患者HBV基因型均为B型(属ayw1亚型),未发现其他基因型。HBV准种发生频率为4%。25例少数民族CHB患者之间与对照组25例汉族患者之间HBV基因型B差异比较,差异无统计学意义(P>0.05)。所有患者preS2/S基因均存在aa124精氨酸(R)→赖氨酸(K124)(1.1%)、172亮氨酸(L)→脯氨酸(P172)(1.3%)、306蛋氨酸(M)→苏氨酸(T306)(1.5%)和361异亮氨酸(I)→蛋氨酸(M361)(1.6%)的aa替代,其发生替代的频率>1%。但所有患者发生aa145甘氨酸(G)→精氨酸(R)(0.4%)替代的频率<1%。50例患者C基因在T细胞和B细胞表位存在27～63、80～110、135～153位间aa变异,其中45例患者C基因由于nt1979A→G,nt2012T→A,nt2088G→T,nt2304C→A和nt2339A→G的变异,分别引起V27、N38、V63、Q135和A147的aa替代,其发生替代的频率>1%。而另外5例重型CHB患者C基因由于nt2159A→G,nt2189A→C的变异,分别引起甘氨酸(G87)和亮氨酸(L97)的aa替代。所有患者preS2/S和C基因中未发现碱基的插入或缺失。结论中国云南少数民族地区HBV流行毒株为基因型B(属ayw1亚型,包括发现25个少数民族CHB患者为基因型B),基因型B与不同民族无相关性。HBV病毒载量可作为判定CHB患者预后的重要参数。
Objective To investigate the evolutionary difference and clinical significance of hepatitis B virus (HBV) genotypes in patients with different ethnic groups of chronic hepatitis B (CHB). Methods Pre-S2 / S (preS2 / S) and C (core) were detected in 50 CHB patients (25 minority CHB patients and 25 CHB patients in the control group) in Yunnan ethnic minority areas by polymerase chain reaction (PCR) C) gene was amplified, and the PCR amplification product was cloned into vector pBS (pBluescript II SK, pBS) for sequencing analysis. Results The full length of HBVpreS2 / S gene was 846bp in 49 patients, accounting for 49.1% of the total, encoding a polypeptide of 281 amino acids. The full length of C gene was 552bp and the content of GC was 46.1%, encoding 183 aa. And registered in GenBank (AY517619, AY517620, AY517488, AY517489, AY517598, AY517599). Compared with the HBV genotypes and subtypes in GenBank database, all the patients were found to have high homology with ayw1 subtype, the homology of preS2 / S aa was 97.5% -98.6%, and the homology of a gene of a was 94.5% ~ 97.8%. All patients with S gene in the “a” epitope aa 122 arginine (AGA), the first 160 for the lysine (AAA). All patients were found HBV genotype B (ayw1 subtype), found no other genotypes. HBV quasispecies occur at a frequency of 4%. There was no significant difference in HBV genotype B between 25 minority CHB patients and 25 control Han patients (P> 0.05). All patients had aa124 arginine (R) → lysine (K124) (1.1%), 172 leucine (L) → proline (P172) (1.3%), 306 methionine ) → threonine (T306) (1.5%) and 361 isoleucine (I) → methionine (M361) (1.6%) with a substitution frequency> 1%. However, all patients had <1% replacement with aa145 glycine (G) -> arginine (R) (0.4%). Fifty patients had aa mutation in T cell and B cell epitopes between 27-63, 80-110 and 135-153, of which 45 patients had nt1979A → G, nt2012T → A, nt2088G → T, nt2304C → A and nt2339A → G, causing aa substitutions of V27, N38, V63, Q135 and A147, respectively, with substitution frequency> 1%. The other 5 cases of heavy CHB patients due to nt2159A → G, nt2189A → C mutation, respectively, caused by glycine (G87) and leucine (L97) aa substitution. No base insertion or deletion was found in the preS2 / S and C genes in all patients. CONCLUSION: HBV genotype B (genotype ayw1, including 25 genotypes of CHB found genotype B) in ethnic minority areas of Yunnan, China has no correlation with genotype B. HBV viral load can be used as an important parameter to determine the prognosis of patients with CHB.