摘要
目的构建微小脲原体核糖体蛋白L23基因原核表达载体并表达蛋白,为研究微小脲原体的生物学特性提供依据。方法根据NCBI上的微小脲原体核糖体蛋白L23基因序列设计引物,以微小脲原体基因组DNA为模板,通过PCR扩增目的基因后进行纯化,利用限制性内切酶BamH I和Not I对原核表达载体pGEX-6P-2和目的基因进行双酶切,并通过T4连接酶连接,连接产物转化XL1-Blue感受态细菌后接种至含氨苄青霉素的LB固体培养基培养,对形成的菌落进行PCR筛选和质粒测序验证,利用IPTG诱导表达GST融合蛋白,表达产物超声裂解后经GST Sepharose 4B纯化,SDS-PAGE电泳鉴定。结果经PCR成功克隆出目的基因,全长345bp,使用pGEX-6P-2质粒通用引物对转化有连接产物的大肠杆菌菌落进行PCR筛选鉴定,阳性菌落的PCR条带大小为468bp,与预期相符,对筛选出的菌落扩大培养并提取质粒进行基因测序,测序结果与NCBI中的基因序列完全一致,对诱导表达的重组蛋白进行SDS-PAGE鉴定,融合蛋白分子量质约为36.6kD,与预期一致。结论成功构建了微小脲原体核糖体蛋白L23原核表达载体,并在大肠杆菌中实现融合蛋白的表达,为微小脲原体核糖体蛋白L23基因以及解脲脲原体的后续研究奠定了基础。
Objective To construct expression vector of ribosomal protein L23 gene of Ureaplasma parvum and express its fusion protein,which may lay foundation for further exploiting in its biological characteristics.Methods The primers were designed according to the published NCBI gene sequence.The gene was amplified with PCR and purified.The target gene and vector PGEX-6 P-2 were digested by BamH I and Not I,and then recombined with T4 DNA ligase.The recombinant plasmid was transformed into XL1-Blue bacteria,and the bacteria were inoculated into agar plates with LB medium and ampicillin.The colonies were identified by colony PCR and plasmid sequencing.The expression of GST fusion protein was induced by IPTG.The bacterial supersonic lysates were purified by GST Sepharose 4 Band analyzed by SDS-PAGE.Results The PCR product of ribosomal protein L23 gene of Ureaplasma parvum was about 345 bp in length.The recombinant plasmid was identified by colony PCR and the fragment of the PCR was 468 bp.The homology between sequenced results and the published sequence was 100%.The relative molecular mass of the fusion protein was about 36.6 kD which was consistent with the presumed protein.Conclusion The prokaryotic expression vector was successfully constructed and the fusion protein was expressed in E.coli,which may be helpful for further study of Ureaplasma parvum.
出处
《河北北方学院学报（自然科学版）》
2017年第12期1-4,共4页
Journal of Hebei North University:Natural Science Edition
关键词
微小脲原体
原核表达
核糖体蛋白
解脲脲原体
Ureaplasma parvum
prokaryotic expression
ribosomal protein
Ureaplasma urealyticum
