江西地区免疫儿童感染的乙型肝炎病毒S基因突变分析

发布时间：2018-01-27 04:02

本文关键词： 乙型肝炎病毒变异基因型血清型免疫儿童　出处：《南昌大学》2012年硕士论文　论文类型：学位论文

【摘要】：研究背景：自从1992年病毒性乙型肝炎疫苗在我国被纳入儿童计划免疫，低年龄组儿童人群HBV阳性检出率逐年下降，但与此同时，由疫苗免疫压力引发的HBV “a”抗原决定簇变异株的出现和流行，越来越受到了人们的广泛关注，但是对江西地区免疫儿童感染的HBV “a”抗原决定簇变异情况的研究还未见报道。研究目的：本研究拟调查1996年后出生的乙肝疫苗免疫儿童HBV的感染，分析所感染的HBV S基因“a”抗原决定簇的变异，寻找可能与免疫逃避相关的变异位点，并鉴定HBV基因型和血清型，为诊疗和预防儿童HBV感染提供重要参考。研究方法： 1.临床标本筛选：收集在江西省儿童医院就医的全程乙肝疫苗免疫儿童血清标本，ELISA方法检测HBV-M，筛选出HBV感染的阳性标本。 2. HBＶ感染的统计学分析：采用x2检验分析不同年龄、性别组间免疫儿童的HBsAg阳性率的差异。 3. HBV S基因的PCR扩增与测序：采用高保真PCR方法对HBsAg阳性血清进行HBV S区基因DNA扩增，PCR产物直接测序。 4.“a”抗原决定簇变异位点分析：采用DNAman软件对所测HBV S基因的核苷酸和氨基酸序列与参考序列进行比对以分析血清型和“a”决定簇的变异。 5. HBV基因型的鉴定：采用Editseq软件、Clustalx(1.83)软件和Mega4软件绘制S基因遗传进化树和使用在线Genotyping软件与参考序列比对以鉴定基因型。研究结果： 1.标本采集：研究者于2010年6月-2011年5月间在江西省儿童医院采集就医的乙肝疫苗全程免疫儿童(7.39±3.66岁)血清标本13117份，男童7139人(54.42%)，女童5978人(45.58%)。 2. HBsAg感染分析：从13117份血清标本中共筛选出230份HBsAg阳性标本，HBsAg阳性率为1.75%，男女儿童HBsAg阳性率分别为1.78%和1.72%，两者间的HBsAg阳性率差异不明显（P0.05）；每5岁设置的不同年龄段儿童HBsAg阳性率分别为1.80%、1.53%和2.00%，三者之间没有明显差异（P0.05）；同一年龄段不同性别间免疫儿童HBsAg阳性率差异无统计学意义（P0.05）。 3.“a”抗原决定簇突变位点的检测：从230份HBsAg阳性标本成功扩增并测序了118份HBV S区基因，从中检测出24份标本有“a”抗原决定簇变异，变异率20.34%。在“a”决定簇中共发现了7个位点8种错义突变，分别为I126T/S，P127T，Q129H，S132T，P142T，S143T和G145A。HBV “a”抗原决定簇两茎环间的变异率和男女儿童感染的“a”抗原决定簇的变异率无显著性差异（P0.05）。 4.血清型分析：氨基酸序列比对分析发现，118标本中，105份为adw血清型，均为adw2亚型；7份为ayw血清型，均为ayw1亚型；6份为adr血清型，adrq+4份，adrq-2份。 5.基因型分析：进化树分析与在线Genotyping软件检测结果显示，118标本中，112份为HBV B基因型，占94.92%，都是B2亚型；6份为HBV C基因型，占5.08%，其中4份为C2亚型，2份为C3亚型。基因型分布在不同性别间无明显差异（P0.05），基因型和血清型之间未反映出异质性，存在一定的关联。研究结论： 1.从1992年起将乙肝疫苗纳入儿童计划免疫管理后，江西地区儿童HBsAg阳性率有了明显的下降，，低于乙肝疫苗纳入计划免疫前的儿童HBsAg阳性率。 2.在江西地区乙肝疫苗免疫儿童人群检测出“a”抗原决定簇变异的HBV感染，但并未发现有明显优势的变异株类型，提示S基因中“a”决定簇的某些位点变异可能是逃避乙肝疫苗免疫导致HBV感染的机制之一。 3.江西地区乙肝疫苗免疫儿童感染的HBV以adw血清型为主，并有ayw血清型和adr血清型存在。 4.江西地区乙肝疫苗免疫儿童感染的HBV以B基因型中B2基因亚型为主，有少量的C基因型（C2基因亚型和C3基因亚型）；该地区儿童感染的HBV遗传距离近。
[Abstract]:Research background:
Since 1992 hepatitis B vaccine was brought into EPI in China, children in low age groups. The positive rate of HBV decreased year by year, but at the same time, the pressure caused by immune vaccine HBV in "a" determinant variants of emergence and popularity of cluster, more and more people's attention, but for children infection in Jiangxi area HBV in "a" determinant cluster mutation has not been reported.
The purpose of the study is:
Born after 1996 this study intended to investigate the children's hepatitis B immune vaccine against HBV infection, infected by the HBV mutation analysis of S gene "a" epitope, and find possible immune escape mutation related, and identification of HBV genotypes and serotypes, provides an important reference for the treatment and prevention of HBV infection in children.
Research methods:
1. clinical screening: serum samples collected from children in Jiangxi provincial hospital for children, hepatitis B vaccine immunized children, HBV-M detected by ELISA method, and positive specimens of HBV infection were screened out.
2. HBV infection statistical analysis: x2 test was used to analyze the difference of HBsAg positive rate between different age groups and sex groups.
PCR amplification and sequencing of 3. HBV S gene: a high fidelity PCR method was used to amplify the HBV S region gene of HBsAg positive serum, and the PCR product was directly sequenced.
4., "a" epitope mutation point analysis: DNAman software was used to compare the nucleotide sequence and amino acid sequence of HBV S gene with reference sequence, so as to analyze the variation of serum type and "a" determinant.
5. identification of HBV genotypes: Editseq gene, Clustalx (1.83) software and Mega4 software were used to draw S genetic evolution tree and online Genotyping software and reference sequence alignment to identify genotypes.
The results of the study:
1. specimen collection: in June 2010 -2011 May, a total of 13117 children (7.39 + 3.66 years old) were immunized with hepatitis B vaccine in Jiangxi children's Hospital, 13117 boys, 7139 boys (54.42%), 5978 girls (45.58%).
Analysis of the infection of 2. HBsAg from 13117 serum samples were screened out of 230 HBsAg positive samples, the positive rate of HBsAg was 1.75%, the positive rate of HBsAg of male and female children were 1.78% and 1.72%, the positive rate of HBsAg, the difference between the two is not significant (P0.05); the positive rate of children of different ages HBsAg every 5 years set were 1.80% 1.53%, and 2%, there was no significant difference between the three (P0.05); no significant positive rate between HBsAg don't immunized children the same age sex difference (P0.05).
Detection of 3. "a" epitope mutations: from 230 HBsAg positive samples were successfully amplified and sequenced the gene 118 HBV S region, detected from 24 samples of "a" antigen determinant mutation, mutation rate of 20.34%. in the "a" determinant were found in 7 loci of 8 missense the mutation, respectively I126T/S, P127T, Q129H, S132T, P142T, S143T and G145A.HBV "a" antigen determinant two stem loop between the mutation rate and children infected with the "a" epitope mutation rate had no significant difference (P0.05).
4. serotype analysis: amino acid sequence alignment analysis showed that 105 of the 118 samples were ADW serotypes, all of them were adw2 subtypes, 7 were ayw serotypes, all were ayw1 subtypes, 6 were ADR serotypes, adrq+4 parts, adrq-2 parts.
Analysis of 5. genotypes: analysis of test results with Genotyping software online phylogenetic tree showed that 118 samples, 112 samples were HBV genotype B, accounted for 94.92% of all B2 subtypes; 6 were HBV genotype C, accounting for 5.08%, of which 4 were C2 subtype, 2 were C3 subtype gene. Type distribution in different genders, no significant difference (P0.05) between genotype and serotype does not reflect the heterogeneity, there is a certain correlation.
The conclusions are as follows:
1., since 1992, when the hepatitis B vaccine was incorporated into the planned immunization management of children, the HBsAg positive rate of children in Jiangxi has decreased significantly, which is lower than the HBsAg positive rate of hepatitis B vaccine before vaccination.
In 2. hepatitis B immune vaccines for children population in Jiangxi detected "a" antigen determinant mutation of HBV infection, but did not find the obvious advantage of the mutant type, some mutations suggest that S gene "a" determinant may be one of the mechanisms leading to immune escape of hepatitis B vaccine for HBV infection.
3. the HBV of children infected with hepatitis B vaccine in Jiangxi area was mainly ADW serotype, and there were ayw serotypes and ADR serotypes.
4. in Jiangxi area, the HBV infection of hepatitis B vaccine children was mainly B genotype B2 gene subtype. There were a small number of C genotype (C2 gene subtype and C3 gene subtype). The HBV genetic distance of children infected by hepatitis B vaccine was near.

【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.1

【参考文献】