早产儿与足月儿免疫球蛋白重链可变区基因异型性

Heterogeneity of immunoglobulin heavy chain variable region gene between premature and mature neonates

目的 :分析和比较早产儿和足月儿免疫球蛋白重链可变区 (IgH)基因中的可变区基因片段 (VH)、多样化基因片段 (D)和连接区基因片段 (JH)的使用 ,VH 上的体突变 ,在VH D和D JH 连接点插入的NDN长度以及开放性阅读框 (ORF)等特征及其差异性。方法 :7例胎龄为 2 5 30周的早产儿和 8例胎龄为 37 41周的足月儿的脐带单核细胞DNA被抽提 ,使用套式PCR扩增IgH基因 ,扩增产物被克隆筛选和测序。结果 :在早产儿 ,共有 2 0个不同的VH 被使用 ,其中DP73(V3 家族成员 )被最优先使用 ,使用率为 2 1.4% ,其次是DP75 (V1家族成员 ) ,使用率为 19.0 % ,VH 上有少数体突变 ( 16 .7% )。JH6和JH3使用率分别为 42 .8%和 41.7% ,J1未被使用。IgH重排基因的VH D JH 连接点处偶有N区插入 ,DND长度为 15 .5± 7.3bp。6 4 3%的IgH基因序列具有开放性阅读框。在足月儿 ,32个不同的VH 被使用 ,DP73和DP75的使用率分别降到 11.6 %和8.3 % ,VH 上的突变为 2 9.2 %。JH6和JH3使用率分别降至 19.3 %和 34.1% ,J1仍未被使用。IgH重排基因的VH-D -JH 连接点处有少量N区插入 ,DND为 2 2 .6± 6 .8bp。 91.7%IgH基因序列的阅读框是开放的。结论 :胎龄 2 5周以上的早产儿已有IgH重排基因 ,且大部分处于活化状态 ,但由于VH 和JH 的偏倚使用 。

Objective:To analyse the features and compare differences in Ig heavy chain variable region gene (IgH) between premature and mature neonates,such as the usage of variable (V H),diversity(D) and joining (J H) segments,the mutation in V H, the insertion of N region and NDN length in V HD and DJ H points,and the opening reading frame (ORF).Methods:DNA were extracted from cord blood of 7 premature (2530 weeks)and 8 mature neonates (3741 weeks),IgH gene was amplified using nested PCR technique,then cloned and sequenced.Results:① In premature newborns,there was a preferential usgae of DP 73 (number of V 3 family),following by DP 75 (number of V 1 family) in 20 seleted V H,the use rate of DP 73 and DP 75 was 21.4% and 19.0%,respectively.A few somatic mutation (16.7%) occured in V H.J 6 and J 3 and a use rate of 42.8% and 41.7%,respectively,while J 1 was not seleted.There was a accidential sertion of N region,and DND length was 15.5±7.3bp in the V HDJ H point of rearranged IgH.64.3% of rearranged IgH and an ORF.② In mature newborns,the use rate of DP 73 and DP 75 decreased down to 11.6% and 8.3% among 32 seleted V H,respectively.A somatic mutation (point mutation)rate of 29.2% existed in V H.The use rate of J 6 and J 3 was 19.3% and 34.1%,respectively,and J 1 was not still seleted.There was some sertion of N region,and an DND length was 22.6±6.8bp in the V HDJ H point of rearranged IgH.An ORF existed in 92.4% of the rearranged IgH.Conclusion:An activated IgH rearrangement occurs early in neonates with gestational age of 25 weeks,but its diversity is limited since a preferential seletion of V H and J H segments,short NDN and few mutation.The activation and diversity rise with increasing GA.