用suc2信号肽捕获系统筛选小鼠胚胎cDNA文库基因

Screening of cDNA Library from Mouse Fetus Using suc2 Signal Sequence Trap

PCR扩增 1 1d小鼠胚胎cDNA文库插入片段 ,将 0 .5～ 2 0kb的扩增产物插入筛选载体的多克隆位点 ,转化suc2基因缺陷酵母宿主菌 .然后将约 1 0 5个酵母菌落接种于选择性平板上进行筛选 ,得到了 1 82个可在选择性培养基上生长的菌落 .PCR扩增显示 ,插入片段大小分布于 0 1～ 1 5kb之间 .对其中 1 4个阳性菌落的重组子进行序列测定 ,分别代表 6种不同的基因序列 ,与报告基因都有正确的读框内融合 .其中两种基因序列反复被筛到 ,分别命名为spt1、spt2 .spt1 [gi:2 772 876 6 ],可能以非编码RNA的身份参与蛋白质向细胞外分泌的过程 ,而spt2编码多个连续的赖氨酸 。

The cDNA fragments were amplified from an 11 days old mouse embryonic cDNA library. And the 0 5～2 0 kb products were inserted into the cloning site of a screening vector aimed at secreted peptides. The generated plasmids were transformed into yeasts with mutated suc 2 gene. 182 out of 10 5 colonies were shown to be positive after screening using suc 2 signal sequence trap system. PCR analysis of all the positive colonies showed that inserted fragments were among 0.1～1 5 kb. Fourteen colonies corresponding to six different genes showed in frame fusion junction with the reporter gene( suc 2) as indicated by sequence analysis. Among them, two kinds of sequence named spt 1 and spt 2 were repeatedly obtained. It was suggested that spt 1 could be part of a novel non coding RNA gene which was involved in protein secretion, and spt 2 mediated protein secretion by a non classical pathway.