人LDH-B基因克隆及其真核表达载体的构建

摘要

目的：克隆人乳酸脱氢酶B（LDH-B）基因并构建其真核表达载体。方法提取人乳腺癌细胞系MDA-MB-231的总RNA，逆转录生成cDNA，以cDNA为模板，采用PCR 扩增获得LDH-B基因编码区序列片段，限制性核酸内切酶Bam HⅠ和KpnⅠ双酶切LDH-B基因片段及pUC19质粒，连接反应构建pUC19-LDH-B克隆载体，并将其转入大肠埃希菌DH5α感受态细胞，蓝白筛选挑取白色菌落，酶切验证后测序鉴定。将序列正确的LDH-B基因亚克隆到pcDNA3.1（-）载体上，构建LDH-B真核表达载体pcDNA3.1（-）-LDH-B。结果 PCR扩增后可见约1.0 kb的特异性条带，与预期的LDH-B片段长度相符合；Bam HⅠ和KpnⅠ双酶切pUC19-LDH-B后可见约2.6 kb 和1.0 kb 的条带，分别与pUC19和LDH-B的片段长度相符合，LDH-B 测序结果与 DNA 序列数据库（Genebank）中的参考序列相一致；Bam HⅠ和 KpnⅠ双酶切pcDNA3.1（-）-LDH-B后可见约5.5 kb和1.0 kb的条带，分别与pcDNA3.1（-）和LDH-B的片段长度相符合。结论成功克隆了人LDH-B基因，并构建了其真核表达载体，为进一步探究LDH-B的生物学功能奠定了基础。%Objective To clone human lactate dehydrogenase B(LDH-B) gene and to construct its eukaryotic expression vector. Methods Total RNA was extracted from human breast cancer cell line MDA-MB-231,cDNA was generated by reverse transcription,cDNA was used as the template and the sequence segment of LDH-B gene coding region was amplified by poly merase chain reaction(PCR),both LDH-B gene segment and pUC19 plasmid were digested with two restriction endonucleases of BamHⅠand KpnⅠ,pUC19-LDH-B cloning vector was constructed by ligation reaction and then transformed into E. coli DH5αcompetence cells,the single white bacterial colony was selected in the blue-white selection plates,positive recombinant plasmid was identified by double restriction endonucleases digestion and DNA sequencing. Eukaryotic expression vector of pcDNA3.1 (-)-LDH-B was constructed by subcloning LDH-B gene segment with the right sequence into pcDNA3.1 (-) vector. Results A spe-cific DNA band about 1.0 kb was found by PCR amplification , which agreed with the expected fragment length of LDH-B;two DNA bands about 2.6 kb and 1.0 kb were seen after digestion of pUC19-LDH-B with BamHⅠand KpnⅠ,which agreed with the fragment length of pUC19 and LDH-B respectively,the LDH-B sequencing result was consistent with its reference sequence in Genebank;two DNA bands about 5.5 kb and 1.0 kb were seen after digestion of pcDNA3.1 (-)-LDH-B with BamHⅠand KpnⅠ, which agreed with the fragment length of pcDNA3.1 (-) and LDH-B respectively. Conclusion Human LDH-B gene is successfully cloned in this experimental study and its eukaryotic expression vector is successfully constructed ,which lays an essential founda-tion for further exploring LDH-B biological functions.