常氏肝癌细胞cDNA文库的构建及ADAMs相关基因的免疫筛选与序列分析

抽提常氏肝癌细胞的总RNA，以oligo(dT)为引物，通过两次转换模板，采用LD-PCR合成全长cDNA，在cDNA两端引入SfiI的酶切位点。以λTriplEX2为载体经过包装后构建了常氏肝癌细胞cDNA表达文库。以ADAMs通用抗体，用免疫筛选技术从常氏肝癌cDNA文库中筛选出22个阳性克隆，经测序分析和BLAST检索，证实其中一个为新基因，部分区域具有蛋白酶的功能，对该基因进行了GenBank登录，获注册号为AY078070。该基因的克隆为研究ADAMs相关基因的功能奠定了基础。

Construction of Chang Liver cDNA Library and Immunoscreening of ADAMS Related Genes

Total RNA was isolated from Chang Liver cell. The full-length cDNA was synthesized using LD PCR by RNA 5' end switching mechanism, oligo(dT) as primer. SfiI digestion sites were introduced at both 3' and 5' end of cDNA. The cDNA was ligated with gammaTriplEX2 vector and packaged. Twenty-two positive clones were obtained by immunoscreening to the library, they were sequenced and blast in NCBI, one of them was identified a novel gene. It was submitted to GenBank and obtained accession number AY078070. It may lay down a foundation for studying on functions of ADAMs related gene.