CAJ | 학술논문

目的探讨乙型肝炎患者和健康携带者乙型肝炎病毒(hepatitis B virus,HBV)C区基因变异规律及其临床意义。方法采用实时荧光定量PCR检测乙肝患者和HBV健康携带者外周血HBV DNA载量；PCR结合DNA测序对HBV C区基因进行多态性检测，并应用DNAstar软件进行比对和分析。结果①与标准株相比，所有标本C区基因序列均出现变异。在≥2例标本中出现的突变31处，包括PreC基因3处，C基因28处；其中9处为错义突变，1处为终止突变，其余21处均为同义突变。nt 1827 c→a和nt 2221 c→t出现在所有标本，有6处同义突变出现于较多标本中。4例乙肝患者标本在nt1896位发生g→a突变，另有4例乙肝患者在HBcAgCTL识别表位84～101处发生2处突变即S87G和I97F或197L。②DNA扩增及测序的成功与否与HBV的DNA拷贝数密切相关，本次研究DNA扩增及测序成功的标本其DNA拷贝数多大于40 193/ml,DNA拷贝数较低的标本不易获得成功。结论HBV基因组极易发生突变，C区基因有2个位点的突变见于所有扩增成功标本，为此研究标本所特有，提示这些突变热点可能具有地域特异性。C区基因变异可引起HBeAg和HBcAg的结构及功能改变，进而导致病毒逃逸机体对病毒的免疫清除作用，或影响HBeAg的检出。
목적 탐토을형간염환자화건강휴대자을형간염병독(hepatitis B virus,HBV)C구기인변이규률급기림상의의。방법채용실시형광정량PCR검측을간환자화HBV건강휴대자외주혈HBV DNA재량；PCR결합DNA측서대HBV C구기인진행다태성검측，병응용DNAstar연건진행비대화분석。결과①여표준주상비，소유표본C구기인서렬균출현변이。재≥2례표본중출현적돌변31처，포괄PreC기인3처，C기인28처；기중9처위착의돌변，1처위종지돌변，기여21처균위동의돌변。nt 1827 c→a화nt 2221 c→t출현재소유표본，유6처동의돌변출현우교다표본중。4례을간환자표본재nt1896위발생g→a돌변，령유4례을간환자재HBcAgCTL식별표위84～101처발생2처돌변즉S87G화I97F혹197L。②DNA확증급측서적성공여부여HBV적DNA고패수밀절상관，본차연구DNA확증급측서성공적표본기DNA고패수다대우40 193/ml,DNA고패수교저적표본불역획득성공。결론HBV기인조겁역발생돌변，C구기인유2개위점적돌변견우소유확증성공표본，위차연구표본소특유，제시저사돌변열점가능구유지역특이성。C구기인변이가인기HBeAg화HBcAg적결구급공능개변，진이도치병독도일궤체대병독적면역청제작용，혹영향HBeAg적검출。
Objective To explore the variations of gene C in hepatitis B viruses between hepatitis B patients and healthy carriers, and provide experimental evidences for analysis of virus gene mutations acting on the virus material science and response of the body to the virus. MethodsThe virus DNA load in hepatitis B patients and healthy blood donors was investigated by real-time polymerase chain reaction (PCR). Gene sequence analysis was taken to detect gene polymorphism, and all the success samples were compaired with standard strain by DNAstar. Results(①)Compared with standard strain, C region in all samples had mutations, there were 31 mutations in at least 2 samples (3 mutations in gene PreC and 28 mutations in gene C), including 9 missense mutations, 1 chain termination mutation and 21 synonymous mutation. Mutations nt 1827 c→a and nt 2221 c→t existed in all the samples, and most samples had 6synonymous mutations. Four hepatitis B patients had mutation nt1896 g→a, and another 4 patients had 2mutations, namely, S87G and I97F (or I97L) in HBcAg CTL recognition episome. ②The success ratio of amplification and sequencing of HBV DNA was closely associated with its copy numbers. In the present study, copy numbers of HBV DNA which were successfully amplified and sequenced were almost more than 40 193/ml. ConclusionsHBV genome were easily affected by nucleotide mutations, 2 residues had mutations in gene of C region, which is firstly reported, suggesting these mutations may be geographical restricted. Mutations in gene of C region may either change the structure and function of HBeAg and HBcAg,which may further induce the escape of immune clearance for HBV or influence the detection of HBsAg or HBeAg, which may creat new problems for the prevention, diagnosis and treatment of hepatitis B.