花狭口蛙海南亚种皮肤cDNA文库的构建及鉴定

Construction and Identification of the Skin cDNA Library from Kaloula pulchra hianana

在两栖动物,尤其是蛙皮肤中已分离得到多种生物学活性物质.而构建皮肤c DNA文库,有助于筛选活性组分,并保存其功能基因.本试验以花狭口蛙皮肤组织为材料,根据Trizol法提取皮肤总RNA后,使用Creator?SMART?c DNA文库构建试剂盒,运用SMART技术构建文库,经反转录反应合成c DNA第一链,LD PCR法将第一链产物扩增再合成双链c DNA;经蛋白酶K和Sfi I消化后,用CHROMA APIN-400分离双链c DNA;将合适大小的ds c DNA通过T4DNA连接酶16℃过夜孵育连接到经过相同酶切的p DNR-LIB载体中,体外包装后转染DH5α宿主菌,构建形成原始文库,进行滴度测试.研究表明,构建的花狭口蛙皮肤c DNA文库原始文库滴度为1.18×108 PFU/m L,插入片段大小不同,没有特异性,构建的文库不符合要求,不可用于大规模的功能基因分析,但可为花狭口蛙皮肤抗菌肽基因的前期研究提供借鉴.

To construct a c DNA library of the frog,Kaloula pulchra hianana skin,in order to facilitate the screening of active components,and maintaining its functional gene. The c DNA library of Kaloula pulchra hianana skin tissue was constructed using the Creator？ SMART？ c DNA Library Construction Kit. Total RNA was extracted by Trizol,followed by constructing c DNA library by SMART（switching mechanism at 5′end of RNA transcript）technology：the first strand c DNA was synthesized by reverse transcript,followed by producing the second strand and double strand c DNA by LD-PCR. The double strand c DNA was digested by proteinase K and Sfi I and then the different sizes c DNA were isolated by CHROMA SPIN-400. Some ds c DNA was transferred into the p DNR-LIB vector at16 ℃ overnight and then packaged into DH5α in vitro to construct the c DNA library.The titer of the c DNA library was estimated as 1.18×108 PFU/m L. PCR results showed that the inserted fragments varied in size with no specificity. This c DNA library gives an unsatisfactory base for further study of genes. c DNA library constructed with this method is unsuitable for functional genomic analysis..