本文關(guān)鍵詞： 乙型肝炎病毒 變異 基因型 血清型 免疫 兒童　出處：《南昌大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景： 自從1992年病毒性乙型肝炎疫苗在我國被納入兒童計劃免疫，低年齡組兒童人群HBV陽性檢出率逐年下降，但與此同時，由疫苗免疫壓力引發(fā)的HBV “a”抗原決定簇變異株的出現(xiàn)和流行，越來越受到了人們的廣泛關(guān)注，但是對江西地區(qū)免疫兒童感染的HBV “a”抗原決定簇變異情況的研究還未見報道。 研究目的： 本研究擬調(diào)查1996年后出生的乙肝疫苗免疫兒童HBV的感染，分析所感染的HBV S基因“a”抗原決定簇的變異，尋找可能與免疫逃避相關(guān)的變異位點(diǎn)，并鑒定HBV基因型和血清型，為診療和預(yù)防兒童HBV感染提供重要參考。 研究方法： 1.臨床標(biāo)本篩選：收集在江西省兒童醫(yī)院就醫(yī)的全程乙肝疫苗免疫兒童血清標(biāo)本，ELISA方法檢測HBV-M，篩選出HBV感染的陽性標(biāo)本。 2. HBＶ感染的統(tǒng)計學(xué)分析：采用x2檢驗(yàn)分析不同年齡、性別組間免疫兒童的HBsAg陽性率的差異。 3. HBV S基因的PCR擴(kuò)增與測序：采用高保真PCR方法對HBsAg陽性血清進(jìn)行HBV S區(qū)基因DNA擴(kuò)增，PCR產(chǎn)物直接測序。 4.“a”抗原決定簇變異位點(diǎn)分析：采用DNAman軟件對所測HBV S基因的核苷酸和氨基酸序列與參考序列進(jìn)行比對以分析血清型和“a”決定簇的變異。 5. HBV基因型的鑒定：采用Editseq軟件、Clustalx(1.83)軟件和Mega4軟件繪制S基因遺傳進(jìn)化樹和使用在線Genotyping軟件與參考序列比對以鑒定基因型。 研究結(jié)果： 1.標(biāo)本采集：研究者于2010年6月-2011年5月間在江西省兒童醫(yī)院采集就醫(yī)的乙肝疫苗全程免疫兒童(7.39±3.66歲)血清標(biāo)本13117份，男童7139人(54.42%)，女童5978人(45.58%)。 2. HBsAg感染分析：從13117份血清標(biāo)本中共篩選出230份HBsAg陽性標(biāo)本，HBsAg陽性率為1.75%，男女兒童HBsAg陽性率分別為1.78%和1.72%，兩者間的HBsAg陽性率差異不明顯（P0.05）；每5歲設(shè)置的不同年齡段兒童HBsAg陽性率分別為1.80%、1.53%和2.00%，三者之間沒有明顯差異（P0.05）；同一年齡段不同性別間免疫兒童HBsAg陽性率差異無統(tǒng)計學(xué)意義（P0.05）。 3.“a”抗原決定簇突變位點(diǎn)的檢測：從230份HBsAg陽性標(biāo)本成功擴(kuò)增并測序了118份HBV S區(qū)基因，從中檢測出24份標(biāo)本有“a”抗原決定簇變異，變異率20.34%。在“a”決定簇中共發(fā)現(xiàn)了7個位點(diǎn)8種錯義突變，分別為I126T/S，P127T，Q129H，S132T，P142T，S143T和G145A。HBV “a”抗原決定簇兩莖環(huán)間的變異率和男女兒童感染的“a”抗原決定簇的變異率無顯著性差異（P0.05）。 4.血清型分析：氨基酸序列比對分析發(fā)現(xiàn)，118標(biāo)本中，105份為adw血清型，均為adw2亞型；7份為ayw血清型，均為ayw1亞型；6份為adr血清型，adrq+4份，adrq-2份。 5.基因型分析：進(jìn)化樹分析與在線Genotyping軟件檢測結(jié)果顯示，118標(biāo)本中，112份為HBV B基因型，占94.92%，都是B2亞型；6份為HBV C基因型，占5.08%，其中4份為C2亞型，2份為C3亞型�；蛐头植荚诓煌詣e間無明顯差異（P0.05），基因型和血清型之間未反映出異質(zhì)性，存在一定的關(guān)聯(lián)。 研究結(jié)論： 1.從1992年起將乙肝疫苗納入兒童計劃免疫管理后，江西地區(qū)兒童HBsAg陽性率有了明顯的下降，，低于乙肝疫苗納入計劃免疫前的兒童HBsAg陽性率。 2.在江西地區(qū)乙肝疫苗免疫兒童人群檢測出“a”抗原決定簇變異的HBV感染，但并未發(fā)現(xiàn)有明顯優(yōu)勢的變異株類型，提示S基因中“a”決定簇的某些位點(diǎn)變異可能是逃避乙肝疫苗免疫導(dǎo)致HBV感染的機(jī)制之一。 3.江西地區(qū)乙肝疫苗免疫兒童感染的HBV以adw血清型為主，并有ayw血清型和adr血清型存在。 4.江西地區(qū)乙肝疫苗免疫兒童感染的HBV以B基因型中B2基因亞型為主，有少量的C基因型（C2基因亞型和C3基因亞型）；該地區(qū)兒童感染的HBV遺傳距離近。
[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.

【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R725.1

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