人噬菌体抗体库中异常重组子的分析

目的 阐明在噬菌体抗体库技术中，选择适当限制性内切酶酶切位点的重要性，并介绍人抗体可变区胚系基因的限制酶谱。方法 从正常人外周血提取淋巴细胞总ＲＮＡ，用ＲＴ－ＰＣＲ扩增 ＩｇＭ和哪 的 Ｆｄ片段及ｋ链基因，重组到载体ｐ３ＭＨ中构建噬菌体抗体库。以限制性内切酶消化及电泳分析所获重组克隆；用ＰＣＧＥＮＥ软件分析人抗体可变区胚系基因的限制性内切酶谱。结果 在构建抗体库的过程中，发现高频率的异常重组子克隆。经序列分析证实，在人Ｖ＿Ｈ基因片段中，存在用于克隆轻链的ＳａｃＩ位点。对人全部功能性可变区胚系基因进行限制性内切酶谱分析，发现人 Ｖ＿Ｈ第Ⅳ家族的 １１个成员均含有ＳａｃＩ位点，其它限制酶切位点在抗体可变区胚系基因中具有不同的出现率。结论 构建抗体库时，用于重组可变区基因的酶切位点，对库的构建具有重要的影响，因此，应对表达载体所用的限制性内切酶进行精心选择。现在比较广泛使用的ｐＣＯＭＢ系统载体不利于良好性能抗体库的构建。

Cloning, sequencing and analyzing of the heavy chain V region genes of human polyreactive antibodies

The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction (PCR) technique. We found that 2 and 3 mAbs utilized genes of the VHIV and VHIII families, respectively. The former 2 VH segments were in germline configuration. A common VH segment, with the best similarity of 90.1 % to the published VHIII germline genes, was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs. This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHIII gene. All these polyreactive mAbs displayed a large NDN region (VH-D-JH junction). The entire H chain V regions of these polyreactive mAbs are unusually basic. The analysis of the charge properties of these mAbs as well as those of other poly- and mono- reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites (CDRs), may be an important structural feature involved in antibody polyreactivity.