BLCAP基因cDNA的克隆及siRNA表达载体的构建

目的：克隆BLCAP基因cDNA并构建和鉴定针对于BLCAP基因的siRNA真核表达载体.方法：从培养的人骨肉瘤细胞系SOSP-9607细胞中提取总RNA,经RT-PCR获得BLCAP基因.将该基因克隆到pGEM-T-Easy载体中,酶切及测序鉴定.将合成的siRNA核酸片段退火形成双链后连接到经BamHI和HindⅢ双酶切后的pSilencer4.1真核表达载体,命名为pSilencer4.1-B1以及pSilencer4.1-B2,并进行酶切及测序鉴定.脂质体法转染SOSP-9607细胞系,G418筛选以及RT-PCR验证构建的真核表达载体对目的基因的干涉情况.结果：经酶切及测序鉴定,所获得的目的片段序列BLCAP基因完全相符.重组质粒中含有与理论值相符的插入片段,其测序结果与设计序列一致.经脂质体法转染后pSilencer4.1-B2可明显抑制SOAP-9607细胞的BLCAP表达.结论：成功克隆了BLCAP基因并cDNA构建和验证了其siRNA真核表达载体.

Clone BLCAP gene and construct siRNA vector intercept its expression

Objective: To clone BLCAP gene ,construct and identify the siRNA eukaryotic vector intercept the expression of BLCAP in SOSP-9607 cell line. Methods:Total RNA was extracted from human osteosarcoma cell line SOSP-9607, and the BLCAP was obtained by RT-PCR. Then it was cloned into pGEM-T-Easy vector and sequenced. According to the BLCAP gene sequence BLACP siRNA cDNAs were synthesized and cloned into the vector pSilencer4.1-CMV neo and named pSilencer4.1-BLACP,which were further identified by restriction endonuclease digestion analysis and DNA sequencing. Then it was transfected into SOSP-9607 cell line by Lipofectamin and screened by G418.The intercepting effect was detected by RT-PCR. Results:Restriction endonuclease digestion analysis and DNA sequencing results showed that BLCAP gene was exactly consistent with the sequence reported in genebank, the target segment was cloned into pSilencer4.1-CMV neo eukaryotic vector successfully and it could intercept the expression of BLCAP effectively. Conclusion:BLACP gene was successfully cloned and constructed siRNA eukaryotic vector.