2008•Zhongguo shouyi xuebaoRequires access
Construction and analysis of cDNA library of Rana catesbeiana skin tissue
Ruili Zhao, Han Junyou, Lianrui Li, Hongwei Qiao, Liancheng Lei, Hu Bo, Xie Fang, Li‐Fan Liu, Wenyu Han
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Abstract
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 contains1.42×106 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 1.35×109 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.