兔肾上皮细胞cDNA表达文库的建立

CONSTRUCTION OF THE LENGTH cDNA LIBRARY OF RK13 CELLS

为筛选兔瘟病毒在兔肾上皮细胞(rabbit kidney epithelial cells,RK13)上的受体,从RK13细胞中提取基因组总RNA,然后用链霉亲和素磁珠(SA-PMP)纯化mRNA,再用SMART(Switching methanism at 5 end ofRNA transcript)技术合成cDNA第1链。采用长距离PCR(long-distance-PCR,LD-PCR)方法扩增双链cDNA,经纯化后克隆到真核表达载体pEXP-Lib中。最后,电转化宿主菌(DH5α),构建了RK13细胞的全长cDNA真核表达文库。鉴定结果表明,该文库插入的cDNA片段平均长度达到1.0 kb,库容量达到2.55×105CFU。

The full length cDNA library of rabbit kidney epithelial （RK-13） cells was constructed to identify RHDV receptors. Total mRNAs were isolated and purified from RK-13 cell and then the first strand cDNAs were synthesized by reverse transcription （RT） with RT primer （CDSIII） and the double-stranded cDNAs were amplified by long-distance-PCR. Subsequently, the ds cDNAs were inserted into the eukaryotic expression vector （pEXP-Lib） and transformed into E. coli cells （DH5ct）. The results showed that the cDNA library contained 2.55 × 10^5 clone with averaged inserts about 1.0 Kb.