人子宫颈癌基因HCCR-2真核表达载体的构建及鉴定

目的:克隆人子宫癌基因HCCR-2并构建其真核表达载体,为研究其在人胃癌细胞中与P53的相互作用关系奠定基础. 方法:从人肝癌细胞株HepG2提取RNA,利用RT-PCR方法,先克隆出一包括HCCR-2编码区的长约1 kb的片段,并将其与T载体连接并转化,测序证实无误后,以HCCR-2/T载体为模板,再通过PCR克隆出HCCR-2的编码区序列,将其连接到pIRES2-EGFP真核表达载体,并通过测序获得证实. 结果:RT-PCR扩增出一长约1 kb的HCCR-2片段,PCR扩增出约0.9 kb大小的目的片段,HCCR-2/T,pIRES2-EGFP/HCCR-2( ) DNA测序均表明编码序列及读框完全正确. 结论:成功构建HCCR-2真核表达载体pIRES2-EGFP/HCCR-2( ),为进一步研究其致癌机制打下基础.

Construction and identification of recombinant eukaryotic expression vector of human cervical cancer oncogene HCCR-2

AIM: To clone and construct the recombinant eukarotic plasmid of human cervical cancer oncogene HCCR-2 and provide the premise for its application to study the mutual relation between HCCR-2 and P53 in gastric cancer. METHODS: Total RNA was isolated from hepatocellular carcinoma cell line HepG2. Subsequently, a 1kb fragment of HCCR-2 encoding region was amplified by RT-PCR and cloned into Promega TEasy vector. After confirmed by DNA sequencing, the full-length encoding region of HCCR-2 was amplified by PCR and cloned into pIRES2-EGFP. The recombinant eukaryotic expression vector was confimed by DNA sequencing. RESULTS: A 1 kb fragment and a 0.9 kb fragment of HCCR-2 was successfully amplified by RT-PCR and PCR respectively. DNA sequencing suggested that both HCCR-2/T and pIRES2-EGFP/HCCR-2(+) possesses correct read frame and nucleotide sequence of HCCR-2. CONCLUSION: The pIRES2-EGFP/HCCR-2(+) was successfully constructed and this laid a solid foundation for uncovering the carcinogenic mechanism of HCCR-2.