半套式PCR扩增人抗HFRS病毒抗体基因及Fd段基因的克隆和序列分析

Amplification of genes of human antibodies against HFRS virus using semi nested polymerase chain reaction and cloning, sequencing of Fd fragments

从肾综合征出血热恢复期患者外周血淋巴细胞（ＰＢＬ） 中提取细胞总ＲＮＡ， 反转录成ｃＤＮＡ 。以之为模板，用半套式聚合酶链反应（ＰＣＲ） 扩增人Ｉｇ Ｇ 抗体Ｆｄ 段及轻链基因，并将Ｆｄ 段基因克隆入噬菌体载体ｐｃｏ ｍ ｂ３ ；经酶切鉴定后，再亚克隆入ｐＧＥＭ７ｚｆ ，筛选阳性克隆测序。经计算机分析，Ｆｄ 段基因的全长为６３９ｂｐ ，编码２１３ 个氨基酸，属于人Ｉｇ Ｇ２ 亚类。

The total cell RNA extracted from peripheral lymphocytes of HFRS patient was transcribed reversely into cDNA. The Fd fragments and light chain genes of human antibodies were amplified from cDNA using semi nested polymerase chain reaction. Fd fragments genes were cloned into vector pcomb3 and pGEM7zf. One of positive clones selected randomly was sequenced.The results indicated that the Fd fragment gene consisted of 639bp, encoded 213 amino acid residues,and belonged to human IgG2.