Cpn0147基因真核表达载体的构建及在HeLa细胞中的表达
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摘要
目的构建肺炎衣原体Cpn 0147基因真核表达重组质粒,为进一步研究其与宿主相互作用的分子机制打下基础。方法以Cpn AR39株基因组为模板,以Cpn0147全长编码特异性引物进行PCR扩增;将Cpn0147基因插入至pcDNA3.1+/His/Myc载体,构建pcDNA3.1+/His/Myc-Cpn0147重组质粒,经双酶切及测序鉴定后转染至HeLa细胞中,采用RT-PCR检测重组质粒转染情况,采用免疫荧光法及Western blot检测Cpn0147蛋白在细胞中的表达。试验设pcDNA3.1+/His/Myc载体为阴性对照。结果从CpnAR39株基因组DNA中扩增出Cpn0147基因片段,大小466bp,经双酶切、连接、转化、筛选,得到pcDNA3.1+/His/Myc-Cpn0147重组质粒,序列测定证实与GenBank Cpn AR39株Cpn0147基因序列一致;RT-PCR扩增出Cpn0147基因,与理论大小一致;免疫荧光检测重组质粒转染后HeLa细胞胞浆观察到红色荧光,对照组无荧光;Western blot检测到特异反应条带位于16kd,对照组无此条带。结论成功构建pcDNA3.1+/His/Myc-Cpn0147重组质粒并在真核细胞内表达Cpn0147蛋白,为进一步研究其分子生物学功能奠定了基础。
Objective To construct a eukaryotic expression vector for Cpn0147 in order to provide a basis for further study of the molecular mechanism of its interaction with the host. Methods Primers for the full-length sequence of Cpn0147 were designed with the genome from the AR 39 strain of Chlamydia pneumoniae serving as a template,and PCR was used to amplify Cpn0147.Cpn0147 was then inserted into a pcDNA3.1 +/His/Myc vector to construct a recombinant plasmid,pcDNA3.1+/His/Myc-Cpn0147.The recombinant plasmid was transformed into E.coli.Colony PCR was used to screen for positive colonies.The recombinant plasmid was then extracted and identified using restriction enzyme digestion and DNA sequencing.The recombinant plasmid was transfected into a HeLa cell line using Lipofectamine 2000.Total RNA was extracted from transfected HeLa cells and the level ofβ-actin and Cpn0147 mRNA was detected with RT-PCR.An indirect immunofluorescence assay and Western blotting were used to analyze the level of Cpn0147 expression.HeLa cells transfected with an empty plasmid(pcDNA3.1+/His/Myc)served as negative controls. Results The Cpn0147 gene was cloned from the genome of the AR 39 strain of C.pneumoniae and the gene fragment was 466 bps in length.The recombinant plasmid pcDNA3.1+/His/Myc-Cpn0147 was obtained through double endoenzyme digestion,ligation,transformation,and screening.Sequencing indicated that the recombinant plasmid was identical to the Cpn0147 gene from the AR 39 strain of C.pneumoniaein GenBank(Gene ID:894891).RT-PCR indicated that the transfected plasmid produced an amplified band at 466 bp for Cpn0147 and a band at 613 bp for theβ-actin gene,both of which were consistent with expectations.An indirect immunofluorescence assay indicated the presence of red fluorescence in the cytoplasm of HeLa cells transfected with the recombined plasmid and the absence of this fluorescence in the control group.Western blotting revealed a specific band with a molecular weight about 16 kd in the transfected cells but not in the control group. Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1+/His/Myc-Cpn0147 was successfully constructed,and Cpn0147 protein was expressed in HeLa cells.This work has laid thefoundation for further study of the biological function of Cpn0147.
Objective To construct a eukaryotic expression vector for Cpn0147 in order to provide a basis for further study of the molecular mechanism of its interaction with the host. Methods Primers for the full-length sequence of Cpn0147 were designed with the genome from the AR 39 strain of Chlamydia pneumoniae serving as a template,and PCR was used to amplify Cpn0147.Cpn0147 was then inserted into a pcDNA3.1 +/His/Myc vector to construct a recombinant plasmid,pcDNA3.1+/His/Myc-Cpn0147.The recombinant plasmid was transformed into E.coli.Colony PCR was used to screen for positive colonies.The recombinant plasmid was then extracted and identified using restriction enzyme digestion and DNA sequencing.The recombinant plasmid was transfected into a HeLa cell line using Lipofectamine 2000.Total RNA was extracted from transfected HeLa cells and the level ofβ-actin and Cpn0147 mRNA was detected with RT-PCR.An indirect immunofluorescence assay and Western blotting were used to analyze the level of Cpn0147 expression.HeLa cells transfected with an empty plasmid(pcDNA3.1+/His/Myc)served as negative controls. Results The Cpn0147 gene was cloned from the genome of the AR 39 strain of C.pneumoniae and the gene fragment was 466 bps in length.The recombinant plasmid pcDNA3.1+/His/Myc-Cpn0147 was obtained through double endoenzyme digestion,ligation,transformation,and screening.Sequencing indicated that the recombinant plasmid was identical to the Cpn0147 gene from the AR 39 strain of C.pneumoniaein GenBank(Gene ID:894891).RT-PCR indicated that the transfected plasmid produced an amplified band at 466 bp for Cpn0147 and a band at 613 bp for theβ-actin gene,both of which were consistent with expectations.An indirect immunofluorescence assay indicated the presence of red fluorescence in the cytoplasm of HeLa cells transfected with the recombined plasmid and the absence of this fluorescence in the control group.Western blotting revealed a specific band with a molecular weight about 16 kd in the transfected cells but not in the control group. Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1+/His/Myc-Cpn0147 was successfully constructed,and Cpn0147 protein was expressed in HeLa cells.This work has laid thefoundation for further study of the biological function of Cpn0147.
引文
[1]魏俊燕,王蓓蓓,张利军,等.肺炎衣原体在血管平滑肌细胞中的生长状态及细胞超微结构的观察[J].中国病原生物学杂志,2013,8(1):5-8,34.
[2]粟盛梅,李忠玉,黄秋林,等.沙眼衣原体pORF8质粒蛋白的表达及其单克隆抗体的制备与鉴定[J].中国病原生物学杂志,2013,8(2):102-5.
[3]Grayston JT,Kuo CC,Wang SP,et al.A new Chlamydia psittaci strain,TWAR,isolated in acute respiratory tract infections[J].New Engl J Med,1986,315(3):161-8.
[4]Izadi M,Fazel M,Akrami M,et al.Chlamydia pneumoniae in the atherosclerotic plaques of coronary artery disease patients[J].Acta Medica Iranica,2013,51(12):864-70.
[5]Sansoni J,Anderson KH,Varona LM,et al.Caregivers of Alzheimer's patients and factors influencing institutionalization of loved ones:some considerations on existing literature[J].Ann Ig,2013,25(3):235-46.
[6]Subtil A,Dautry-Varsat A.Chlamydia:five years A.G.(after genome)[J].Curr Opin Microbiol,2004,7(1):85-92.
[7]童伟,李峥,贾天军.肺炎衣原体包涵体膜蛋白Cpn0147抗原性分析[J].湖北医药学院学报,2011,4(6):569-72.
[8]童伟,周淑芬,贾天军.肺炎衣原体感染儿童血清中抗Cpn0147抗体的检测[J].临床检验杂志,2012,2(8):258.
[9]童伟,张战军,贾天军.肺炎衣原体Cpn0147基因的克隆、表达与定位及其重组蛋白的生物信息学分析[J].中国病原生物学杂志,2009,4(8):567-9.
[10]Luo J,Liu G,Zhong Y,et al.Characterization of hypothetical proteins Cpn0146,0147,0284&0285that are predicted to be in the Chlamydia pneumoniae inclusion membrane[J].BMC Microbiol,2007(7):38-50.
[11]代国知,马忠夏,周安文,等.肺炎嗜衣原体Cpn0147重组蛋白的免疫学活性研究[J].国际检验医学杂志,2012,33(14):1671-2.
[2]粟盛梅,李忠玉,黄秋林,等.沙眼衣原体pORF8质粒蛋白的表达及其单克隆抗体的制备与鉴定[J].中国病原生物学杂志,2013,8(2):102-5.
[3]Grayston JT,Kuo CC,Wang SP,et al.A new Chlamydia psittaci strain,TWAR,isolated in acute respiratory tract infections[J].New Engl J Med,1986,315(3):161-8.
[4]Izadi M,Fazel M,Akrami M,et al.Chlamydia pneumoniae in the atherosclerotic plaques of coronary artery disease patients[J].Acta Medica Iranica,2013,51(12):864-70.
[5]Sansoni J,Anderson KH,Varona LM,et al.Caregivers of Alzheimer's patients and factors influencing institutionalization of loved ones:some considerations on existing literature[J].Ann Ig,2013,25(3):235-46.
[6]Subtil A,Dautry-Varsat A.Chlamydia:five years A.G.(after genome)[J].Curr Opin Microbiol,2004,7(1):85-92.
[7]童伟,李峥,贾天军.肺炎衣原体包涵体膜蛋白Cpn0147抗原性分析[J].湖北医药学院学报,2011,4(6):569-72.
[8]童伟,周淑芬,贾天军.肺炎衣原体感染儿童血清中抗Cpn0147抗体的检测[J].临床检验杂志,2012,2(8):258.
[9]童伟,张战军,贾天军.肺炎衣原体Cpn0147基因的克隆、表达与定位及其重组蛋白的生物信息学分析[J].中国病原生物学杂志,2009,4(8):567-9.
[10]Luo J,Liu G,Zhong Y,et al.Characterization of hypothetical proteins Cpn0146,0147,0284&0285that are predicted to be in the Chlamydia pneumoniae inclusion membrane[J].BMC Microbiol,2007(7):38-50.
[11]代国知,马忠夏,周安文,等.肺炎嗜衣原体Cpn0147重组蛋白的免疫学活性研究[J].国际检验医学杂志,2012,33(14):1671-2.