宫颈癌相关新基因的筛选、克隆及鉴定

Screening,Cloning and Identification of a Novel Cervical Carcinoma Associated Gene

目的:筛选、克隆及鉴定宫颈癌相关基因———BLCAP。方法:利用基因芯片技术对1例原发性宫颈癌患者的癌组织及其癌旁正常组织进行分析;将在宫颈癌组织中表达显著降低的BLCAP基因克隆到pET-28(a)表达载体上,以构建原核表达重组质粒pET-28(a)-BLCAP,通过PCR、限制性内切酶酶切和DNA测序等技术鉴定阳性重组子。结果:通过基因芯片分析发现存在表达差异的原癌和抑癌基因共11条,其中表达降低最明显的是BLCAP基因。构建的重组表达质粒经PCR、酶切和DNA测序鉴定与预期的结果一致,即BLCAP基因共264 bp,基因序列无改变,基因插入后阅读框(ORF)正确。结论:筛选出了在宫颈癌中表达显著降低的BLCAP基因并成功构建了pET-28(a)-BLCAP原核表达质粒,为表达BLCAP目的蛋白及研究该蛋白的性质奠定了基础。

To screen, clone and identify cervical carcinoma associated BLCAP gene. Methods： A proto-oncogene and suppressor-tumor gene DNA microarray was utilized to analyze gene expression patterns from cervical carcinoma tissues and their adjacent tissues in a primary cervical carcinoma. Then BLCAP gene, which was down-regulated greatly, was cloned into pET- 28（a） vector to construct the prokaryotic expression plasmid pET-28（a）-BLCAP. The positive clone was identified by PCR method, restriction endoenzyme analysis and DNA sequencing. Results： All 11 genes were consistently upregulated or downregulated in DNA microarray, and the BLCAP gene was downregulated greatly. The recombinant expression plasimid was identified by PCR method, digested with restricted endoenzymes and DNA sequencing, and was in coincidence with the predicted result. Conclusion： It was very successful to find the BLCAP gene which is down-regulated greatly in cervical carcinoma and construct the prokaryotic expression plasmid pET-28（a）-BLCAP. It was the foundation to express and study the quality of BLCAP protein.