江浙沪地区乙肝病毒B、C基因型S基因突变及选择压力分析

目的系统分析江浙沪三地的乙肝病毒B、C基因型S基因突变和选择压力情况,以期为乙型肝炎的防治提供理论依据。方法采用NCBI数据库提供的乙肝病毒序列,分为B、C基因型两组,分析突变情况,同时采用Datamonkey进行选择压力分析和Bioedit进行氨基酸置换熵值分析。结果S基因226个氨基酸位点突变分析中,产生突变有位点122个,位点突变率C基因型(45.58%)高于B基因型(30.09%)(χ~2=11.523,P=0.001);总突变率C基因型(1.36%)高于B基因型(0.80%)(χ~2=46.642,P=0.000);α决定簇突变率C基因型(2.40%)高于B基因型(0.96%)(χ~2=20.524,P=0.000)。B基因型α决定簇突变率高于总突变率(χ~2=0.735,P=0.391);C基因型α决定簇突变率高于总突变率(χ~2=44.467,P=0.000)。B基因型氨基酸突变最多的位点为200、213、161和21位点,C基因型氨基酸突变最多的位点为126、68、3、53和194位点。两个基因型dN/dS均值均小于1。B基因型没有发现正向选择位点,发现8个负向选择位点。C基因型发现7个正向选择位点,17个负向选择位点。B基因型仅发现一个易突变位点(200位),C基因型也仅发现一个易突变位点(126位),大多数氨基酸位点熵值0.4。结论江浙沪地区S基因突变率水平较低,其中C基因型位点突变率、α决定簇突变率和总突变率高于B基因型,α决定簇突变率高于总突变率。C基因型经历外界环境免疫选择压力和自身进化压力双重影响,自身进化压力强于外界环境免疫选择压力;而B基因型主要遭受自身进化压力。尤其要格外关注C基因型的进化进程。     

表面抗原和表面抗体检测双阳性CD重组型乙型肝炎病毒全基因组序列分析

西藏地区藏族人群乙型肝炎病毒(Hepatitis B virus,HBV)感染率较高,而针对感染者血清中HBV表面抗原(Hepatitis B surface antigen,HBsAg)和HBV表面抗原抗体(Hepatitis B surface antibody,HBsAb)双阳性的研究一直进展缓慢,尚无明确的研究结论。为探讨西藏地区藏族人群慢性HBV感染者血清中HBsAg和HBsAb双阳性与基因组核苷酸/氨基酸突变的关系,本研究在西藏选取7个地区作为研究区域,进行多阶段抽样,选取样本进行HBV血清五项指标检测,筛选HBsAg和HBsAb均为阳性的患者血清共24份作为双阳性组,以年龄和乙型肝炎e抗原(HBeAg)等感染指标进行匹配,选取96份HBsAg阳性,HBsAb阴性患者血清作为对照组。HBV全基因组序列通过聚合酶链式反应(Polymerase chain reaction,PCR)产物直接测序获得,并进行重组分析和突变分析。852名西藏HBV感染者中,HBsAg/HBsAb双阳性率为2.82%(24/852)。双阳性组在S蛋白N端和主要亲水区(Major hydrophilic region,MHR)的突变率以及PreS缺失发生率均显著高于对照组。T1753C、C1990T和C2002T等核苷酸突变;S蛋白中V224A、PreS区D103E等氨基酸突变在两组内分布存在显著差异。HBV/CD重组型的HBsAg/HBsAb双阳性发生率与中国乙肝主要流行区域接近。HBV感染者血清HBsAg和HBsAb共存可能与S蛋白,特别是MHR内的高氨基酸突变造成的免疫逃逸有关。PreS缺失、S抗原蛋白C端V224A突变和PreS区D103E突变可能对HBsAg/HBsAb双阳性的产生具有协同作用。     

乙肝患者前S1抗原与HBV-M及HBV-DNA检测的关系

目的探讨前S1抗原与乙肝标志物(HBV-M)及HBV-DNA之间的关系,进而分析前S1抗原的检测在判断乙肝病毒复制中的作用。方法检测360例乙型肝炎患者血清中前S1抗原、HBV-M、HBV-DNA,对其结果进行比较分析。结果前S1抗原能够较好地反映乙肝病毒复制情况,对病情的预后及疗效判断具有指导意义。结论前S1抗原与HBV-DNA、HBeAg阳性呈高度正相关,在防治中应引起重视。     

删除Loop区域表面不稳定氨基酸提高 (R)-ω-转氨酶热稳定性

手性胺是一类具有重要价值的医药及精细化工中间体,如何实现手性胺类化合物的不对称合成是目前人们普遍关注的一个焦点问题。ω-转氨酶(ω-Transaminase,ω-TA)是一类能直接合成对映体手性胺的天然生物催化剂。相比于(S)-ω-TA,(R)-ω-TA的研究较少,但其需求量随着手性胺类药物的发展日趋增大。提高具有潜在应用价值的(R)-ω-TA的热稳定性,将有利于手性胺的制备。本文利用Py MOL软件和YASARA软件预测来源于土曲霉Aspergillus terreus的(R)-ω-TA中具有高温度因子(B-factor)的Loop区域,通过定点突变对Loop区域表面不稳定氨基酸逐步进行删除获得突变酶。结果表明,突变酶R131del和突变酶P132-E133del半失活温度分别为41.1℃和39.4℃,比野生酶提高了2.6℃和0.9℃;在40℃下的半衰期分别为15.0 min和10.0 min,为野生酶的2.2倍和1.5倍。此外,在400 K和10 ns的分子模拟条件下,突变酶R131del在Loop区域的均方根涨落(Root mean square fluctuation,RMSF)比野生型低,突变酶P132-E133del在Loop区域增加了4个氢键。本研究通过删除(R)-ω-转氨酶Loop区域表面不稳定氨基酸提高了该蛋白的热稳定性,同时也为其他酶热稳定性的理性设计提供了方法学指导。     

乙型肝炎病毒外膜大蛋白和HBVDNA血清学诊断价值

目的探讨乙型肝炎病毒外膜大蛋白和HBV-DNA在乙肝患者中的血清学诊断价值。方法收集乙肝两对半(HBV-M)中HBsAg阳性血清标本260例作为乙肝研究组,乙肝两对半(HBV-M)阴性血清标本100例作为正常对照组;在不同乙肝模式中采用酶联免疫吸附试验(ELISA)检测血清HBV-LP和Pre-S1,荧光定量PCR检测HBV DNA;比较HBeAg血清中HBV-LP与HBV-DNA和不同HBV-DNA拷贝数的条件下HBV-LP与HBV-DNA剂量关系。结果HBsAg阳性血清中HBV-LP、HBV-DNA和Pre-S1阳性率分别为68.46%、63.08%和24.23%;HBeAg阳性标本中HBV-LP、HBV-DNA和Pre-S1阳性率分别为94.87%、94.87%和79.49%;HBeAg阴性标本中HBV-LP、HBV-DNA和Pre-S1阳性率分别为62.64%、49.45%和0.55%,HBV-LP和HBV-DNA二者检出一致率为67.03%[(50+72)/182];HBV-LP吸光度(A值)与HBV DNA呈正相关。结论HBV-LP与HBV-DNA在HBeAg阳性血清中代表病毒复制具有较高检出一致率;HBV-LP与HBV-DNA在HBeAg阴性血清中具有较大的差异性,HBV-DNA阴性血清中检测HBV-LP反应乙肝病毒复制对乙肝抗病毒治疗更有重要意义;HBV DNA拷贝数与HBV-LP含量呈正相关系。     

定向进化-易错PCR方法提高华根霉Rhizopus chinensis CCTCC M201021脂肪酶的活力

运用定向进化-易错PCR的方法,提高了华根霉Rhizopus chinensis CCTCC M201021脂肪酶的活力。经过两轮易错PCR和pNPP顶层琼脂法筛选,从第一轮和第二轮突变库中分别筛选获得最佳突变株1-11和2-28,脂肪酶酶活与野生菌株相比分别提高2倍和4倍。基因比对结果表明,突变脂肪酶2-28有4个氨基酸发生了突变:A129S、K161R、A230T、K322R。蛋白质分子空间结构模拟显示,突变A129S、K161R、A230T位于脂肪酶分子表面。突变A230T增强了α-螺旋盖结构的稳定性。突变K322R处在loop上,靠近脂肪酶底物结合区域,与邻近的Asp(带负电)形成盐桥。静电引力将该loop向底物进入酶活性中心的通道口反方向牵引,使底物分子更易进入酶活性中心。酶学性质研究表明,突变株2-28脂肪酶的Km值比出发菌株下降了10%,Kcat值提高为原来的2.75倍。     

虎纹捕鸟蛛毒素XI (HWTX-XI) 突变体的真核表达及活性鉴定

虎纹捕鸟蛛毒素-XI (HWTX-XI) 是从虎纹捕鸟蛛粗毒中分离的含55个氨基酸残基的蛋白质,兼有胰蛋白酶抑制活性和电压门控钾离子通道抑制活性。通过突变HWTX-XI上的钾离子通道抑制活性关键氨基酸残基设计了2个突变体 (分别突变以下氨基酸残基：R5I,R10T,R25A和R5I,R25A),利用pVT102U/α表达载体在酿酒酵母S78中成功表达并获得了高纯度的重组蛋白质;通过分光光度计比色法、膜片钳技术和小鼠脑室注射分别比较三者的胰蛋白酶和钾通道抑制活性以及动物毒性,结果显示：HWTX-XI突变体与     

合成乙型肝炎表面抗原肽段的结构与抗原性

本文对合成的乙型肝炎表面抗原肽段的结构与抗原性进行了研究。通过对三种亚型共9个合成肽段的抗原性测定和结构分析,我们证实了在乙型肝炎表面抗原(HBsAg)氨基酸顺序的122-137区域存在着共同决定簇“a”,且半胱氨酸残基对抗原性有很大的影响。合成的16肽P_(122-137)的两个半胱氨酸残基用叔丁基保护时,其抗原性几乎检测不到,一旦去掉保护基并氧化成分子内S—S键后,抗原性明显增加。比较各种亚型肽段的抗原性测定结果,我们发现亚型决定簇d(或y)的位置可能在122—132之间。另外我们发现P_(adw)122—132的抗原性要比P_(adr)122-137的抗原性强,结构分析结果表明adw型中的Asn_(132)可能对此有较大的贡献。     

萧山地区524例健康献血者血清中乙肝前S1蛋白调查分析

目的了解健康献血者（指表面抗原阴性、丙肝抗体阴性、艾滋抗体阴性、梅毒抗体阴性、ALT小于25赖氏单位）血清中乙肝前S1蛋白的阳性状况。方法采用ELISA法对萧山地区524例健康献血者作前S1蛋白调查检测,同时对前S1蛋白阳性标本采用放免法,作乙肝三系检测。结果有12例前S1蛋白阳性,阳性率为2．3％,其中有1例乙肝三系核心抗体IgG阳性,其余11例乙肝三系各项指标全阴性。对12例阳性者作跟踪调查,1年后,在12例阳性者中,有4例前S1蛋白仍保持阳性,对4例阳性标本再作乙肝三系检测,除1例1年前核心抗体IgG阳性外,其余3例乙肝三系各项指标仍保持阴性。     

乙肝病毒表面抗原和抗体双阳性者中病毒S区基因序列分析

为了解乙肝病毒(HBV)表面抗原和抗体双阳性患者中病毒的基因型及其HVB S区是否有变异.用放射免疫试剂检测HBsAg阳性样品中的抗-HBs抗体,用聚合酶链反应法检测双阳性样品中的HBV DNA,然后对阳性样品进行克隆和基因序列分析,并将所得序列与HBV不同基因型的代表株进行比较分析.结果显示389例HBsAg阳性样品中有10例为抗HBs抗体阳性;该10例双阳性样品中有5例为HBV DNA阳性;序列分析显示该5株HBV均为B基因型,其中4株为adw亚型,1株为adr亚型;其中有2株在S区的"a"决定簇的氨基酸发生了变异.     

Impairment of hepatitis B virus virion secretion by single-amino-acid substitutions in the small envelope protein and rescue by a novel glycosylation site

Mutations in the S region of the hepatitis B virus (HBV) envelope gene are associated with immune escape, occult infection, and resistance to therapy. We previously identified naturally occurring mutations in the S gene that alter HBV virion secretion. Here we used transcomplementation assay to confirm that the I110M, G119E, and R169P mutations in the S domain of viral envelope proteins impair virion secretion and that an M133T mutation rescues virion secretion of the I110M and G119E mutants. The G119E mutation impaired detection of secreted hepatitis B surface antigen (HBsAg), suggesting immune escape. The R169P mutant protein is defective in HBsAg secretion as well and has a dominant negative effect when it is coexpressed with wild-type envelope proteins. Although the S domain is present in all three envelope proteins, the I110M, G119E, and R169P mutations impair virion secretion through the small envelope protein. Conversely, coexpression of just the small envelope protein of the M133T mutant could rescue virion secretion. The M133T mutation could also overcome the secretion defect caused by the G145R immune-escape mutation or mutation at N146, the site of N-linked glycosylation. In fact, the M133T mutation creates a novel N-linked glycosylation site ((131)NST(133)). Destroying this site by N131Q/T mutation or preventing glycosylation by tunicamycin treatment of transfected cells abrogated the effect of the M133T mutation. Our findings demonstrate that N-linked glycosylation of HBV envelope proteins is critical for virion secretion and that the secretion defect caused by mutations in the S protein can be rescued by an extra glycosylation site.     

Patterns of circulating hepatitis B surface antigen variants among vaccinated children born to hepatitis B surface antigen carrier and non-carrier mothers

Hepatitis B virus (HBV) variants that possessed missense mutation within the neutralization epitope of the major S antigen as defined by amino acid residues (aa#) 124–147, termed the a determinant variants, were identified through a population-based serosurvey of 2,305 children of the vaccinated birth cohorts born after 1986. Data on the 678 nucleotides encoding the S antigen of HBV were available for 75 HBV strains that were collected from 63 vaccinated children and 12 unvaccinated or incompletely vaccinated children, and 21 HBV strains from 25 unvaccinated adults. Among the diverse patterns of one to three amino acid substitutions within the a determinant, 145-Arg occurred most frequently (5/14); other variants were: 126-Ala, 127-Thr, 126-Ser/131-Asn/133-Thr, 129-His, 129-Arg, 123-Asn/131-Ile, 133-Leu, 141-Glu, and 141-Arg/144-Ala. Only one of these variants occurred in the 16 hepatitis B surface antigen (HBsAg)-carrier children born to HBsAg-negative mothers, whereas 12 of these variants occurred in the 20 (50%) children born to HBsAg-positive mothers. In addition, early administration of HBV vaccine within the noenatal period increased the likelihood of the emergence of these variants to 64.7% (11/17). Five of the 21 (23.8%) unvaccinated HBsAg-carrier adults harbored the a determinant variants possessing mutations within aa# 125–136, i.e. the putative first loop formed by the cysteine disulfide bonds. Vaccinated children were likely to harbor HBV variants possessing mutations involving altered charge of side chains and/or its hydrophobicity of amino acid residues within the putative second loop between aa#140 and 146. Our data suggest that emergence of these HBV S gene mutants in the phase of HBV vaccination program would be most common among populations in whom perinatal/vertical transmission of HBV is most common, i.e. southeast Asian and the Taiwanese.     

Mutations in the <Emphasis Type="Italic">S</Emphasis> gene region of hepatitis B virus genotype <Emphasis Type="Italic">D</Emphasis> in Turkish patients

The S gene region of the hepatitis B virus (HBV) is responsible for the expression of surface antigens and includes the ‘a’-determinant region. Thus, mutation(s) in this region would afford HBV variants a distinct survival advantage, permitting the mutant virus to escape from the immune system. The aim of this study was to search for mutations of the S gene region in different patient groups infected with genotype D variants of HBV, and to analyse the biological significance of these mutations. Moreover, we investigated S gene mutation inductance among family members. Forty HBV-DNA-positive patients were determined among 132 hepatitis B surface antigen (HbsAg) carriers by the first stage of seminested PCR. Genotypes and subtypes were established by sequencing of the amplified S gene regions. Variants were compared with original sequences of these serotypes, and mutations were identified. All variants were designated as genotype D and subtype ayw3. Ten kinds of point mutations were identified within the S region. The highest rates of mutation were found in chronic hepatitis patients and their family members. The amino acid mutations 125 (M → T) and 127 (T → P) were found on the first loop of ‘a’-determinant. The other consequence was mutation inductance in a family member. We found some mutations in the S gene region known to be stable and observed that some of these mutations affected S gene expression.     

Characterization of the effect of Melanocortin 1 Receptor,a member of the hair color genetic locus,in alpaca (Lama pacos) fleece color differentiation

Little is known about the inheritance and influence of the fleece color gene Melanocortin 1 Receptor (MC1R). Melanocortin 1 Receptor (MC1R) is a well-known gene responsible for red versus black fleece pigmentation and is hypothesized to be a candidate gene for variation in alpaca coloration patterns. Inheritance of red versus black pigmentation in the context of genetic mutation is well understood in many domesticated mammals. We characterized the MC1R gene in a population of multi-colored alpacas in order to better understand its effect on coat color in the alpaca. Our characterization of the alpaca MC1R gene revealed 11 mutations. Of these one is a 4 bp deletion, four are silent mutations and six are single nucleotide polymorphisms (SNPs) that alter the amino acid sequence (T28V, M87V, S126G, T128I, S196F, R301C). No mutation correlated completely with fleece color in alpacas at the MC1R locus. This may be due to the epistatic relationship of MC1R with other coat color genes especially agouti signaling protein (ASIP).     

拉米夫定联合阿德福韦酯治疗前后乙型肝炎病毒的全基因组序列分析

目的探究拉米夫定治疗反弹后联合阿德福韦酯治疗前后乙型肝炎全基因组序列变化。方法分别提取服用拉米夫定治疗24周反弹后和阿德福韦酯辅助治疗24周后的患者2份血清病毒核酸,用聚合酶链反应扩增核酸后进行全基因组测序分析。结果测序结果显示,共计有29个氨基酸发生了突变,其中,S区突变点有5个（17．2％）,C区突变点有12个（41．3％）,P区突变点有6个（20．6％）,X区突变点有6个（20．6％）,其中P区与拉米夫定的相关位点173和204位点发生了突变翻转,但服用阿德福韦后出现了与之相关的突变位点（181、214、236和237位点）。结论核苷酸药物的使用和HBV基因耐药突变密切相关,定期检测HBV基因突变对于合理使用核苷酸药物具有重要意义。     

Genetic basis for altered pathogenesis of an immune-selected antigenic variant of reovirus type 3 (Dearing).

In this paper we provide a step by step comparison of the pathogenesis of murine infection caused by reovirus type 3 (Dearing) and an antigenic variant (K) selected by its resistance to neutralization with a monoclonal antibody (G5) directed against the T3 hemagglutinin. To show that specific changes in the biologic properties of variant K were due to mutation in the S1 double-stranded RNA segment (gene), which encodes the viral hemagglutinin, we generated a reassortant virus ("1 HA K") containing the variant K S1 gene and compared its properties to variant K and to a reassortant ("1 HA 3") containing the T3 (Dearing) S1 gene. These studies, in conjunction with our previous nucleotide sequence analysis of the S1 genes of variant K and T3 (Dearing) [R. Bassel-Duby, A. Jayasuriya, D. Chatterjee, N. Sonenberg, J. V. Maizel, Jr., and B. N. Fields, Nature (London) 315:421-423, 1985; R. Bassel-Duby, D. R. Spriggs, K. L. Tyler, and B. N. Fields, submitted for publication], indicate that a single amino acid change in the T3 hemagglutinin can alter viral growth and tropism within the central nervous system without affecting either its primary replication in the intestine or its pattern of spread to or within the central nervous system.     

拉米夫定联合阿德福韦酯治疗乙型肝炎的疗效及对HBV耐药基因突变的影响

目的探讨拉米夫定联合阿德福韦酯治疗慢性乙型肝炎的疗效,并利用反向点杂交技术检测其对HBV基因耐药突变的影响。方法156例慢性乙型肝炎患者随机分为2组：对照组70例采用拉米夫定治疗,治疗组86例采用拉米夫定联合阿德福韦酯治疗。采用实时荧光定量PCR和ELISA检测2组治疗前和治疗后48周的HBV-DNA载量和HBeAg并采用PCR-反向点杂交技术（PCR-RDB）检测2组治疗48周后的HBV耐药基因突变情况。结果对照组及治疗组在经过48周治疗后HBV-DNA载量较治疗前都明显下降（P 〈0. 05）,治疗组HBV-DNA载量明显低于对照组（P〈0.05）。治疗组经过48周治疗后HBeAg的阴转率为54.9%,明显高于对照组15.0% （P 〈0.05）。对照组44例未出现耐药突变,25例拉米夫定耐药突变中rtL180M突变6例,rtM204V/I突变11例,rtL180M ＋ rtM204V/I混合突变8例;阿德福韦酯HN236T耐药突变1例。治疗组77例未出现耐药突变;5例拉米夫定耐药突变中rtL180M突变1例,rtM204V/I突变2例,rtL180M ＋ rtM204V/I混合突变2例;阿德福韦酯耐药突变中rtN236T突变1例;拉米夫定和阿德福韦酯交叉耐药rtN236T ＋ rtM204V/I混合突变3例。对照组耐药突变率为37. 1%（26/70）明显髙于治疗组的10.5%（9/86）（P〈0.05）。结论拉米夫定联合阿德福韦酯对治疗慢性乙型肝炎方面有效并在减少HBV耐药基因突变方面具有一定的作用。     

Molecular epidemiology of drug resistance markers of Plasmodium falciparum in Yunnan Province, China

ABSTRACT: BACKGROUND: The mutations in Plasmodium falciparum chloroquine resistance transporter (pfcrt), multidrug resistance 1 (pfmdr1), dihydrofolate reductase (pfdhfr), dihydropteroate synthase (pfdhps) and ATPase (pfatp6) genes were associated with anti-malaria drug resistance. The aim of this study was to investigate the prevalence of polymorphisms in pfcrt, pfmdr1, pfdhfr, pfdhps and pfatp6 in Yunnan Province. Finger-prick blood samples were collected from malaria-positive patients from Yunnan Province in 2009-2010. Single-nucleotide polymorphisms (SNPs) in the resistance-related genes were analysed by various PCR-based methods. RESULTS: A total of 108 blood samples were collected. Although chloroquine has not been used to treat falciparum malaria for nearly 30 years, 95.3% of the parasites still carried the pfcrt K76T mutation, whereas the majority of isolates displayed the wild-type pfmdr1 N86 and D1246 sequences. The molecular level of sulphadoxine-pyrimethamine resistance in P. falciparum was high. The most prevalent mutation was pfdhfr C59R (95.9%), whereas the frequencies of the quadruple, triple and double mutants were 22.7% (N51I/C59R/S108N/I164L), 51.5% (N51I/C59R/S108N, N51I/C59R/I164L and C59R/S108N/ I164L) and 21.6% (N51I/ C59R, C59R/S108N and C59R/I164L), respectively. A437G (n=77) and K540E (n=71) were the most prevalent mutations in pfdhps, and 52.7% of the samples were double mutants, among which A437G/K540E was the most common double mutation (37/49). Quadruple mutants were found in 28.0% (26/93) of samples. A total of 8.6% of isolates (8/93) carried the S436A/A437G/A581G triple mutation. No mutations were found in pfatp6 codons 623 or 769, but another two mutations (N683K and R756K) were found in 4.6% (3/97) and 9.2% (6/97) of parasite isolates, respectively. CONCLUSIONS: This study identified a high frequency of mutations in pfcrt, pfdhfr and pfdhps associated with CQ and SP resistance in P. falciparum and no mutations linked to artemisinin resistance (pfatp6). Molecular epidemiology should be included in routine surveillance protocols and used to provide complementary information to assess the appropriateness of the current national anti-malarial drug policy.     

乙型肝炎病毒X蛋白的优势氨基酸序列与热点突变位点的生物信息学分析

乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)全长154个氨基酸,与肝癌发生密切相关.为确定HBx的优势氨基酸序列和热点突变位点,在GenBank中下载所有HBx的氨基酸序列13950条,剔除插入突变、缺失突变和起始密码子非甲硫氨酸的序列,最后保留7126条.通过分析这7126条序列,计算出HBx每个位点的氨基酸分布情况,出现频率最高的氨基酸为该位点的优势氨基酸,其他氨基酸为突变氨基酸.154个位点的优势氨基酸组成HBx优势氨基酸序列.突变率>10.0%的热点突变位点有32个.其中第36、42、44、87、88和127位氨基酸有4种(突变率>1.0%)以上突变形式,具有较高的多态性.与肝癌密切相关的K130M/V131I双突变率为34.7%.通过7126条HBx序列与优势序列的同源性比较,随机选出其中50条序列(2条与优势序列同源性<75%,48条同源性为76%~99%),与23条参考序列及优势序列共同构建系统发生树.结果显示,HBx优势氨基酸序列属于基因型C,这与基因型C为全球主要流行型一致.本研究首次系统性分析了GenBank中HBx的优势序列,确定了32个HBx热点突变位点和6个多态性较高的位点,为基于HBx突变的基础和应用研究奠定了基础.