牛蛙(Rana catesbeiana)皮肤cDNA文库的构建与鉴定

Construction and analysis of cDNA library of Rana catesbeiana skin tissue

以牛蛙皮肤组织为材料获得总RNA,用偶联Oligo（dT）的磁珠纯化mRNA后,利用含SfiⅠ酶切位点的CDSⅢ引物在逆转录酶的作用下合成cDNA的第1链,以含SfiⅠ酶切位点的CDSⅢ引物和SMART引物,利用LD-PCR合成双链cDNA,双链cDNA经SfiⅠ酶切,通过T4DNA连接酶连接到经过相同酶切的λTripIEx2载体,体外包装后转染E.coliXLI-Blue宿主菌,进行滴度测试和文库扩增。结果构建的牛蛙皮肤cDNA文库原始库容量为1.42×10^6PFU/mL,插入片段长度在0.5～1.5kb,重组率为89.5%,扩增后的文库滴度为1.35×10^9PFU/mL,构建的文库质量符合要求,可用于大规模的功能基因分析。

In order to clone and express the antibacterial peptides genes of Rana catesbeiana, the cDNA library of skin tissue was constructed by SMART cDNA Library Construction Kit. Total RNA from Rana catesbeiana was isolated,and mRNA were separated and purified through the magnetic substance combined with Oligo（dT）. The CDS primer with Sfi I were induced into the first cDNA strand with the help of reverse transcriptase, then double strand cDNA with the restriction site of Sfi I were synthesized through LD-PCR（Long-distance-PCR） and ligated into TripIEx2 vector, packaged in vitro. The obtained cDNA library contains 1.42 × 10^6 recombinants, the percentage of vectors with inserts was 89.5 % and the average inserts size were between 0.5-1.5 kb. After amplification, the titer of amplified library was 1.35 × 10^9 PFU/mL. This cDNA library gives an ideal base for further study of genes of antibacterial peptides from Rana catesbeiana, cDNA library constructed with this method is suitable for functional genomic analyzing.