【作者】 姚全良；

【导师】 刘金辉；

【作者基本信息】 南昌大学 ， 病原生物学， 2010， 硕士

【摘要】 人感染乙型肝炎病毒(hepatitis b vius,HBV)后可导致急性自限性肝炎,或者转为慢性乙型肝炎(chronic hepatitis B,CHB)。尽管只有3~10%个体感染HBV后成为CHB患者,但据世界卫生组织统计全世界超过20亿人感染过HBV,CHB人口仍巨大。我国多年来一直是HBV感染的高流行区,全国约有1.2亿CHB患者。乙型肝炎慢性化的机制比较复杂,仍然有待进一步阐述。HBV感染人体后,特异性T、B淋巴细胞可清除病毒。然而CHB患者在感染急性期HBV DNA水平较高,进入慢性期后HBV DNA水平维持在较低水平,免疫系统未能成功清除病毒,导致了HBV持续存在。目前较多的研究认为HBV结构基因选择性突变逃避宿主免疫监测,但HBV调控序列中增强子的变异对HBV基因组的转录与复制水平的下调作用导致感染慢性化的机制却较少报道。HBV基因组上有二个增强子序列。研究表明HBV增强子Ⅰ(enhancer I,Enh I)对HBV基因组的转录与复制起重要调控作用,其序列上的突变与HBV感染慢性化的相关性未见报道。本研究拟选取CHB患者,采用PCR技术对HBV Enh I区域DNA进行扩增并直接测序,采用Genotyping软件分析序列并鉴定其基因型(genotype),利用DNAMAN软件将获得的HBV序列与genebank上相应基因型HBV参考株序列比对以分析变异,同时检测血清中HBV DNA水平、免疫学指标HBsAg、HBeAg滴度,分析Enh I基因变异与HBV DNA水平、HBsAg和HBeAg滴度的关联性,以分析Enh I基因变异对乙肝慢性化的影响。目的(1)比较Taq PCR MasterMix和Taq Plus PCR MasterMix扩增HBV靶DNA保真度影响。(2)研究江西地区CHB患者体内HBV Enh I区域基因变异。(3)研究CHB患者HBV Enh I基因变异对血清HBV DNA水平及HBsAg、HBeAg滴度影响。方法(1)取CHB患者血清与阴性对照血清标本各一份,抽提血清中DNA,采用Taq PCR MasterMix和Taq Plus PCR MasterMix分别扩增HBV DNA nt835-nt1396片段,PCR产物测序并DNAMAN软件进行比对。(2)收集来我院就诊的CHB患者70名,其中“大三阳”35例,“小三阳”35例,健康体检者2名血清,PCR扩增HBV Enh I基因,产物测序后利用Genbank中Genotyping对HBV进行分型,利用DNAMAN软件与标准株比对分析DNA序列中的变异位点。荧光定量PCR(Fluorescence quantitative PCR,FQ-PCR)法定量检测血清HBV DNA拷贝数,倍比稀释血清后用ELISA法检测HBsAg、HBeAg滴度。采用SPSS 13.0统计软件分析,HBV DNA拷贝数以其对数值计算x±s,滴度均值采用几何均数表示。阳性率比较采用χ2检验,组间比较HBV DNA拷贝数阳性对数均值差异采用多应变量方差分析,两两比较均采用不假定方差相等Dunnett’s T3检验, P<0.05为差异有统计学意义。结果(1)二种PCR试剂均能特异地扩增出HBV靶基因片段,PCR产物测序比对结果显示,Taq PCR MasterMix扩增产物与Taq Plus PCR MasterMix扩增产物相比,产生了nt1390缺失和nt1389T→C及nt1392G→T二个突变位点。(2)70例乙肝患者HBV DNA测序后经Genotyping比对,均属于B型,与参考序列AF100309同源性最高,CHB患者HBV Enh I主要变异位点为: nt1010A→T,nt1013G→A,nt1031T→C,nt1034A→G,nt1053C→T,nt1082A→T,nt1133G→A,nt1167C→T,nt1223C→A,nt1227G→C,nt1230A→C,nt1241T→A,nt1244G→A,nt1246C→A,nt1251C→G,nt1289A→C,nt1349A→C。(4)CHB患者HBV Enh I区域nt1133G→A变异对应的HBV DNA拷贝数与HBsAg、HBeAg滴度明显降低。结论(1)两种不同PCR试剂中Taq和Pfu混合DNA聚合酶的Taq Plus PCR MasterMix试剂催化PCR反应中HBV靶DNA片段扩增的保真度高于Taq DNA聚合酶。(2)江西地区乙肝患者感染HBV基因型主要为B型,HBV Enh I区域nt1133G→A可降低Enh I功能,下调HBV基因表达,Enh I区域基因变异可能是导致乙肝慢性化的原因之一。更多还原

【Abstract】 Human infection with the hepatitis B virus(HBV) will lead either to acute,self-limiting disease,or to chronic hepatitis B(CHB).Despite only 3 to 10% individuals who infected HBV will become chronic carriers. However, more than 2 billion people worldwide had been infected with hepatitis B virus, population of CHB is still huge.After infection with HBV, specific T and B lymphocytes response against viral proteins leads to clearance of the virus. However in CHB patients,HBV DNA levels were high during the acute phase after infection.Later,in the chronic phase,there were a strong decrease in serum HBV DNA levels,but failed to eliminate the virus,leading to a persistent infection with HBV.The switch mechanism between a high to a low HBV DNA levels during chronic HBV infection was very complex. One was selective mutation of HBV structural genes to evade the host immune surveillance. The relationship between HBV enhancer I (Enh I) DNA mutation with chronic HBV infection mechanism remained unclear. There are two enhancers in HBV genome. Studies showed that the HBV Enh I play an important regulatory role in the HBV genome transcription and replication, Site mutations at HBV Enh Iassociated with HBV chronic infection has not been addressed so far.In this paper, HBV Enh I region of CHB patients were amplified by PCR technology and sequenced, then typed using Genotyping software in Genbank , and analyzed of variations compared with the reference HBV sequence in Genebank with DNAMAN software, while serum marker such as HBV DNA, HBsAg and HBeAg titer were detected to analyze the relationship between the Enh I gene mutations.Objective:(1)To investigate the influence of Taq PCR MasterMix and Taq plus PCR MasterMix on the fidelity of PCR products of HBV DNA. (2)To investigate DNA Variations in HBV genome Enh I region of CHB patients in jiangxi province.(3)To study the influence of serum HBV DNA levels and HBsAg, HBeAg titer with HBV Enh I DNA mutations in CHB patients.Methods: (1)Two serum samples from patients infected with or without hepatitis B virus were collected for specific amplification of the gene of nt835 to nt1396 in HBV genome using two different PCR MasterMix respectively. The PCR products were sequenced and matched with DNAMAN software.(2)Serum samples were collected from 70 patients with CHB including 35 cases with seroconversion HBsAg(+)HBeAg(+)HBcAb(+), other 35 cases with seroconversion HBsAg(+)HBeAg(-)HBcAb(+) and 2 cases of healthy people. Enh I region DNA of HBV genome were amplified by PCR and sequenced, then HBV genotype were determined by Genotyping software in Genbank, and matched the standard strains by DNAMAN software to determine the relevant DNA mutations. Fluorescence quantitative PCR(FQ-PCR) was used to measure serum HBV DNA copies. Enzymer-linked immunosorbent assay (ELISA) was used to detect serum HBsAg, HBeAg titer by coubling dilution. The data were analyzed by SPSS 13.0 statistical software. HBV DNA copy number logarithmic mean show as ( x±s), antigen titers show as geometric mean, and positive rate was compared by the chi-square test , HBV DNA copy number of different groups was compared with multiple comparisons ANOVA and equal variance Not Vssumed Dunnett’s T3 , and a P value less than 0.05 was regarded as significant.Results: (1) Two PCR Matermix could amplify the target gene in HBV genome specifically. Compared with the sequence of PCR product using Taq Plus PCR MasterMix, the amplified gene product using Taq PCR MasterMix showed deletion of nt1390 and two mutation sites of nt1389T→C and nt1392G→T. (2)The results with Sequence Alignment with Genotyping showed that all 70 sequenced HBV DNA belong to type B and were homologous to the reference sequence AF100309. Seventeen mutation sites were found in Enh I region and they were nt1010A→T,nt1013G→A,nt1031T→C,nt1034A→G,nt1053C→T,nt1082A→T,nt1133G→A,nt1167C→T,nt1223C→A,nt1227G→C,nt1230A→C,nt1241T→A,nt1244G→A,nt1246C→A,nt1251C→G,nt1289A→C,nt1349A→C.(3)Serum HBV DNA levels and HBsAg, HBeAg titer decreased obviously in CHB patients with HBV Enh I region nt1133G→A mutation.Conclusions: (1) The fidelity of the target DNA fragment amplification catalyzed by Taq and Pfu mixed DNA polymerase was higher than by Taq DNA polymerase. (2) HBV genotype B is the major genotype in jiangxi province. The mutation of nt1133G→A at HBV Enh I region could decrease Enh I function, down regulate HBV gene transcription and replication, mutations at HBV Enh I region DNA may be a possible mechanism for the progression to chronic hepatitis.更多还原