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IgG1的Fd片段及κ链基因，重组型载体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VH基因片段中，存在用于克隆轻链的Sac I 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VH等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。

Analysis of abnormal recombinant clones in human phage antibody library

Aim To elucidate the importance of selecting suitable restriction enzyme digestion sites in human phage antibody library construction and to introduce the results of restriction enzyme site analysis of human germline V genes. Methods Total RNA was extracted from peripheral blood lymphocytes of normal volunteers. K chain and Fd segment genes in IgGl and IgM were amplified by RT - PCR, and cloned into p3MH to construct phage antibody library. Clone samples of the library were analyzed by restriction enzyme di- gestion. Restriction enzyme digestion sites of human germline V genes were analyzed by PCGENE software. Results Abnormal re- combinant clones with high frequency were found in the process of antibody library construction. Sequence analysis proved that there was a Sad site in the V_H~4 gene. Restriction enzyme digestion site analysis of human germline V genes revealed that all the 11 human V_H~4 germline genes contained the Sac I site. Other restriction en- zyme digestion sites often used in antibody gene cloning were also present at different frequencies in human gennline V genes. Con- clusion It is very important to choose carefully the restriction en- zyme digestion sites in antibody library construction. The widely used pCOMB series vectors are not suitable for the construction of high quality antibody libraries.