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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SOSP-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。
OBJECTIVE: To clone cDNA of BLCAP gene and construct and identify siRNA eukaryotic expression vector targeting BLCAP gene. Methods: Total RNA was extracted from cultured human osteosarcoma cell line SOSP-9607 and the BLCAP gene was obtained by RT-PCR. The gene was cloned into pGEM-T-Easy vector, digested and sequenced. The synthesized siRNA nucleic acid fragment was annealed to form a double strand and ligated into the pSilencer4.1 eukaryotic expression vector digested with BamHI and HindIII, and named as pSilencer4.1-B1 and pSilencer4.1-B2, and digested and sequenced Identification. Liposome method transfected SOSP-9607 cell line, G418 screening and RT-PCR verified the construction of the eukaryotic expression vector on the purpose of gene interference. Results: After digestion and sequencing, the BLCAP gene of the target fragment was completely consistent. The recombinant plasmids contained inserts consistent with the theoretical values, and the sequencing results were consistent with the designed sequences. After transfection with pSilencer4.1-B2, the BLCAP expression in SOSP-9607 cells was significantly inhibited by liposome. Conclusion: The BLCAP gene was cloned successfully and its siRNA eukaryotic expression vector was constructed and verified.