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目的 对 10 6例免疫失败儿童研究分析HBVS变异株的感染和意义。方法 生于携带HBV母亲的 132 4名新生儿按 0、1、6程序接种乙型肝炎疫苗 ,联合应用HBIG ,定期临床随访和检测HBV血清学标志。其中 10 6例免疫失败者为研究对象 ,从血清中提取DNA ,经PCR扩增HBVS区基因片段 ,Southernblot转移至尼龙膜 ,与S区标准序列探针杂交 ,选择PCR阳性、与寡核苷酸探针不杂交的样本作序列分析。结果 免疫失败儿童中HBVDNA阳性 99例 (93.4 % ) ,99例阳性样本有 30份 (30 .3% )与探针不杂交。选择 11份作序列分析 ,10份存在核苷酸变异伴氨基酸改变 ,分别位于氨基酸残基 113(ST、TP)、12 6 (IT)、12 7(PT)、131(NT)、143(ST)、16 1(YF)和 175(LF)。同时对一对母婴测序结果显示其序列完全一致 ,均存在 131和 16 1位氨基酸被取代。结论 乙型肝炎主被动联合免疫失败儿童中 30 %存在S区基因变异。HBV变异株可经母婴传播。由于基因序列分析受试剂和仪器限制 ,难以常规在大样本中开展 ,应用寡核苷酸探针杂交是一种简便实用的方法。
Objective To study the infection and significance of HBV variant in 106 children with immune failure. Methods 132 4 newborns born to HBV mothers were vaccinated with hepatitis B vaccine according to 0, 1, 6 procedures. HBIG was used in combination with regular clinical follow-up and HBV serological markers. One hundred and sixty cases of immune failure were studied. DNA was extracted from serum. The HBVS gene fragment was amplified by PCR. Southern blot was transferred to nylon membrane and hybridized with the standard sequence probe of S region. The PCR was positive and matched with the oligonucleotide Samples that did not hybridize to the probe were sequenced. Results 99 cases (93.4%) were positive for HBVDNA in immunocompromised children and 30 cases (30.3%) in 99 cases were not hybridized with the probe. Eleven were selected for sequence analysis, and 10 were found to have nucleotide variation with amino acid changes, which were located at amino acid residues 113 (ST, TP), 126 (IT), 127 (PT) 131 (NT), 143 (ST), 16 1 (YF) and 175 (LF). At the same time a pair of maternal and child sequencing results showed that its sequence is exactly the same, there are 131 and 16 amino acids are replaced. Conclusion S-region gene variation exists in 30% of children with primary and passive failure of hepatitis B infection. HBV variants can be transmitted by mother to child. Due to the limitations of reagents and instruments for gene sequence analysis, it is difficult to routinely carry out large samples. The application of oligonucleotide probe hybridization is a simple and practical method.