pcDNA3.1(+)/ChM-Ⅰ表达质粒的构建及鉴定  

Construction and Identification of pcDNA3.1(+)/ChM-ⅠExpression Plasmid

作　　者：周连仲[1] 刘冲 苏玢 王明辉[1] ZHOU Lianzhong;LIU Chong;SU Bin(Department of Otolaryngology Head and Neck Surgery,Tianjin 4th Centre Hospital&4th Central Hospital Affiliated to Nankai University&Otorhinolaryngology Head and Neck surgery Hospital of Tian Jin,Tianjin City 300140;不详)

机构地区：[1]天津市第四中心医院,南开大学附属第四中心医院,天津市耳鼻咽喉头颈外科医院耳鼻咽喉头颈外科,300140 [2]天津市第四中心医院,南开大学附属第四中心医院,天津市耳鼻咽喉头颈外科医院麻醉科,300140

出　　处：《医学理论与实践》2022年第4期544-547,共4页The Journal of Medical Theory and Practice

基　　金：天津市卫生健康委、天津市中医药管理局中医中西医结合科研课题(2019129)。

摘　　要：目的:为了研究软骨调节素Ⅰ的生物学作用,构建pcDNA3.1(+)/ChM-Ⅰ表达质粒。方法:用Trizol法提取大鼠软骨组织总RNA,根据ChM-Ⅰ基因序列(序列号:AF051425)设计特异引物,通过PCR获取ChM-Ⅰ目的基因,分别将ChM-Ⅰ目的基因的PCR产物和pcDNA3.1(+)质粒双酶切,并将两者定向连接,构建pcDNA3.1(+)/ChM-Ⅰ质粒,转化感受态细菌,挑取阳性单克隆并扩增,酶切鉴定阳性克隆,并对插入的ChM-Ⅰ片段进行测序。结果:结果显示与ChM-Ⅰ基因长度相符,序列无误,且读码框正确。结论:成功构建pcDNA3.1(+)/ChM-Ⅰ表达质粒,为进一步研究ChM-Ⅰ的生物学作用打下基础。Objective:Construct pcDNA3.1(+)/ChM-Ⅰexpression plasmid to study the biological effect of chondromodulinⅠ.Methods:Extract the total RNA of rats cartilage by Trizol.Our group designed the specific primers for the ChM-Ⅰgene and amplified the ChM-Ⅰgene(NO.AF051425)by PCR.The product acquired by PCR and the pcDNA3.1(+)were digested by the enzymes,and then connected directionally with each other by the T4 DNA ligase.The ligation product was used to transform E.coli TOP 10 and the positive clones were identified by being digested,sequencing and comparing with the sequence according to the Gene Bank.Results:The results showed that the positive clones were consistent with the length of ChM-Ⅰgene,the sequence was correct,and the reading frame was correct.Conclusion:The pcDNA3.1(+)/ChM-Ⅰexpression plasmid was successfully constructed,which laid a foundation for further study on the biological function of ChM-Ⅰ.

关 键 词：软骨调节素Ⅰ pcDNA3.1(+)质粒 肿瘤治疗 

分 类 号：R73-3[医药卫生—肿瘤]