不同胎龄新生儿免疫球蛋白重链基因CDR3序列特征

Feature of immunoglobulin heavy chain complementarity determining region 3 gene in neonates with different gestational age

目的 分析和比较不同胎龄新生儿免疫球蛋白重链 (IgH)基因CDR3序列特征 ,探讨新生儿成熟度对CDR3序列多样性的影响。方法 从 10例胎龄 2 5～ 30周的极不成熟儿、12例胎龄 31～ 36周的不成熟儿和 11例胎龄 37～ 41周的成熟儿脐血B细胞中抽提模板DNA ,使用巢式PCR技术扩增IgH基因、然后对扩增物进行克隆和CDR3序列测定。结果 ①在极不成熟儿、不成熟儿和成熟儿 ,CDR3长度分别为 2 9.4± 7.8、32 .4± 9.2和 40 .8± 10 .7bp ,其中的N区 D基因片段 N区 (NDN)长度分别为 13.5± 5 .6、16 .1± 7.8和 2 2 .0± 8.5bp。②极不成熟儿和不成熟儿优先使用DP73和DP75 ,成熟儿优先使用VH5、DP73和DP75 ;随着胎龄的增加 ,DP73和DP75的使用率下降 ,而VH5的使用率上升。③对于D基因片段的使用 ,极不成熟儿主要是DN、DQ5 2和DXP ,不成熟儿和成熟儿主要是DXP、DLR和DN。④所有新生儿中 ,JH4的使用率最高 ,其次是JH6 ,但随着胎龄增加 ,JH4和JH6的使用率下降。⑤极不成熟儿、不成熟儿和成熟儿中 ,分别 6 3.3%、6 8.8%和 92 .0 %的克隆有开放性阅读框 ,其长度为 30 3bp ,编码 10 1个氨基酸残基。结论 新生儿的早期生长发育阶段 ,IgH基因的VH D JH 重排机制已处于活化状态 ,但其多样性有限 ;新生儿体液免疫系统的发育?

Objective To study the feature of immunoglobulin complementarity determining region 3 (CDR3) gene in neonates of different gestational age (GA), and the effect of neonatal maturity on the diversity of CDR3 nucleotide sequence. Methods DNA were extracted from cord blood of 10 neonates of very immature (25-30 weeks), 12 immature (31-36 weeks), and 11 mature (37-41 weeks). CDR3 sequence was amplified suing nested PCR technique then cloned and sequenced. Results 1. There was a CDR3 length of 29.4±7.8, 32.4±9.2 and 40.8±10.7bp, including NDN length of 13.5±5.6, 16.1±7.8 and 22.0±8.5bp respectively in very immature, immature and mature neonates. 2. DP73 and DP75 were preferentially used in very immature and immature neonates, V H5, DP73 and DP75 were used in mature neonates. The usage rate of DP73 and DP75 reduced, whereas V H5 was raised with GA increasing. 3. For D gene segment, there was a frequent use of DN, DQ52 and DXP in very immature neonates, whereas DXP, DLR and DN in immature and mature neonates. 4. J H4 usage was preferential, followed by J H6 in all neonates, and their usage rate was decreased with GA increasing. 5. 63.3%, 68.8% and 92.0% of CDR3 sequence respectively in very immature, immature and mature neonates have an open reading frame with 303bp long encoding 101 residues. Conclusion During the early life of neonates, V H-D-J H rearrangement of IgH gene is in an active condition, but the diversity is limited. Humoral immunity is a gradual development with increasing GA. Both heterogeneity and similarity of CDR3 sequence exists in neonates with different GA.