南昌大学学报（医学版）
南昌大學學報（醫學版）
남창대학학보（의학판）
ACTA ACADEMIAE MEDICINAE JIANGXI
2014年
9期
6-12
,共7页
邹淑慧%夏方娜%周艳%肖九长%刘金辉
鄒淑慧%夏方娜%週豔%肖九長%劉金輝
추숙혜%하방나%주염%초구장%류금휘
乙型肝炎病毒%全基因组%儿童%江西地区
乙型肝炎病毒%全基因組%兒童%江西地區
을형간염병독%전기인조%인동%강서지구
hepatitis B virus%complete genome%children%Jiangxi
目的：初步调查江西地区乙肝疫苗免疫儿童感染的乙型肝炎病毒全基因组的分子特征。方法收集江西省儿童医院11445份乙肝疫苗免疫儿童血清标本，采用酶联免疫吸附试验检测 HBV-M，抽提乙肝表面抗原（HB-sAg）阳性血清中的 HBV DNA，分3个相互重叠片段 PCR 扩增 HBV 基因并测序，采用 ContigExpress 软件对所测序列拼接成全基因组序列，分析各读码框核苷酸和氨基酸变化。对 HBV 不同基因型以及所获得的 HBV 全基因组的 S 基因序列进行系统进化树的构建以确定基因型。结果11445份血清样品中，检测出117份 HBsAg 阳性标本，进行分段重叠扩增并测序，最终拼接出58份完整的 HBV 基因组，均为 B 基因型和 adw 血清型。对各开放读码框进行序列比对发现 PreS 区存在散在分布的突变：如 A2990C→ H48P、A2999C→ N51T、C3041T→ P65L、C3097A→L84I 和 A3136G→ T97A；S 区突变集中在120～200 aa，主要突变位点为 C533A→ P127T、A541C→Q129H、G588C→G145A、T753A→F200Y；前 C 区突变位点有 A1814C／T1815C→M1P、G1896A→ W28*，C 区出现1例 C1913A→P5T 变异；P 区出现散在分布的突变位点，未发现与耐药性有关的突变位点，其 RT 区 YMDD 核序也未发生变异；X 区氨基端主要突变点为：G1437T／A→ G22C／S、G1476A→ G35R 和 G1503A／T／C／T1504G→V44I／F／L／C，羧基端突变集中在120～150 aa，出现5例 A1762T／G1764A→K130M／V131I 突变。结论了解分析儿童 HBV 全基因组的分子特征和突变情况，为疫苗免疫儿童感染乙肝的预防和治疗提供病毒学分子信息。
目的：初步調查江西地區乙肝疫苗免疫兒童感染的乙型肝炎病毒全基因組的分子特徵。方法收集江西省兒童醫院11445份乙肝疫苗免疫兒童血清標本，採用酶聯免疫吸附試驗檢測 HBV-M，抽提乙肝錶麵抗原（HB-sAg）暘性血清中的 HBV DNA，分3箇相互重疊片段 PCR 擴增 HBV 基因併測序，採用 ContigExpress 軟件對所測序列拼接成全基因組序列，分析各讀碼框覈苷痠和氨基痠變化。對 HBV 不同基因型以及所穫得的 HBV 全基因組的 S 基因序列進行繫統進化樹的構建以確定基因型。結果11445份血清樣品中，檢測齣117份 HBsAg 暘性標本，進行分段重疊擴增併測序，最終拼接齣58份完整的 HBV 基因組，均為 B 基因型和 adw 血清型。對各開放讀碼框進行序列比對髮現 PreS 區存在散在分佈的突變：如 A2990C→ H48P、A2999C→ N51T、C3041T→ P65L、C3097A→L84I 和 A3136G→ T97A；S 區突變集中在120～200 aa，主要突變位點為 C533A→ P127T、A541C→Q129H、G588C→G145A、T753A→F200Y；前 C 區突變位點有 A1814C／T1815C→M1P、G1896A→ W28*，C 區齣現1例 C1913A→P5T 變異；P 區齣現散在分佈的突變位點，未髮現與耐藥性有關的突變位點，其 RT 區 YMDD 覈序也未髮生變異；X 區氨基耑主要突變點為：G1437T／A→ G22C／S、G1476A→ G35R 和 G1503A／T／C／T1504G→V44I／F／L／C，羧基耑突變集中在120～150 aa，齣現5例 A1762T／G1764A→K130M／V131I 突變。結論瞭解分析兒童 HBV 全基因組的分子特徵和突變情況，為疫苗免疫兒童感染乙肝的預防和治療提供病毒學分子信息。
목적：초보조사강서지구을간역묘면역인동감염적을형간염병독전기인조적분자특정。방법수집강서성인동의원11445빈을간역묘면역인동혈청표본，채용매련면역흡부시험검측 HBV-M，추제을간표면항원（HB-sAg）양성혈청중적 HBV DNA，분3개상호중첩편단 PCR 확증 HBV 기인병측서，채용 ContigExpress 연건대소측서렬병접성전기인조서렬，분석각독마광핵감산화안기산변화。대 HBV 불동기인형이급소획득적 HBV 전기인조적 S 기인서렬진행계통진화수적구건이학정기인형。결과11445빈혈청양품중，검측출117빈 HBsAg 양성표본，진행분단중첩확증병측서，최종병접출58빈완정적 HBV 기인조，균위 B 기인형화 adw 혈청형。대각개방독마광진행서렬비대발현 PreS 구존재산재분포적돌변：여 A2990C→ H48P、A2999C→ N51T、C3041T→ P65L、C3097A→L84I 화 A3136G→ T97A；S 구돌변집중재120～200 aa，주요돌변위점위 C533A→ P127T、A541C→Q129H、G588C→G145A、T753A→F200Y；전 C 구돌변위점유 A1814C／T1815C→M1P、G1896A→ W28*，C 구출현1례 C1913A→P5T 변이；P 구출현산재분포적돌변위점，미발현여내약성유관적돌변위점，기 RT 구 YMDD 핵서야미발생변이；X 구안기단주요돌변점위：G1437T／A→ G22C／S、G1476A→ G35R 화 G1503A／T／C／T1504G→V44I／F／L／C，최기단돌변집중재120～150 aa，출현5례 A1762T／G1764A→K130M／V131I 돌변。결론료해분석인동 HBV 전기인조적분자특정화돌변정황，위역묘면역인동감염을간적예방화치료제공병독학분자신식。
Objective To investigate the molecular characteristics of complete genome of hepa-titis B virus (HBV)in HBV-infected children.Methods A total of 11 445 serum samples from children with HBV immunization were gathered in Jiangxi Children’s Hospital.ELISA analysis was used to determine HBV-M.PCR method with three overlapping primers was used to amplify the gene of HBV DNA extracted from HBsAg positive serum samples and the PCR products were sequenced directly.The sequences of three amplicons were spliced as a complete HBV genome by Contig Express,and the nucleotide and amino acid of complete HBV genome were divided into 4 ORFs to analyze the main and significant mutation sites.The phylogenetic tree of HBV S gene was constructed to determine the genotype of HBV isolates.Results A total of 117 HBV-M posi-tive samples were collected from the 11 445 serum samples,and complete HBV genomes from 58 samples characterized by genotype B and serotype adw were successfully amplified and sequenced. Mutations in the PreS region were found through analyzing the sequence of ORFs,including A2990C→H48P,A2999C→N51T,C3041T→P65L,C3097A→ L84I and A3136G→T97A.Muta-tions in S region mainly occurred in the domain of 120-200 aa and the mutation sites included C533A→P127T,A541C→Q129H,G588C→G145A and T753A→F200Y.Mutation sites in PreC region included A1814C/T1815C→M1P and G1896A→ W28*.Moreover,C1913A→ P5T muta-tion in C region was found in 1 case.Scattered distribution of mutations was observed in P re-gion.Furthermore,no drug resistance associated mutations were found in P region and no YMDD mutations in RT region.Mutation sites in N-terminal of X region included G1437T/A→G22C/S, G1476A→G35R and G1503A/T/C/T1504G→V44I/F/L/C.Mutations in C-terminus mainly oc-curred in the domain of 120-150 aa,including A1762T/G1764A→K130M/V131I mutation in 5 ca-ses.Conclusion In this study,we analyzed the molecular characteristics and mutations of com-plete genome of HBV in HBV-infected children and provided molecular information for the pre-vention and treatment of hepatitis B among children with HBV immunization.
