摘要
目的构建表达绿色荧光蛋白(GFP)的重组绵羊李斯特菌,为绵羊李斯特菌的研究提供重要工具。方法利用SOEing PCR的方法将单核细胞增生性李斯特菌溶血素的启动子(phly)与GFP基因融合,连接到pCW质粒上,构建重组原核表达质粒pCW-phly-GFP。将重组质粒电转化绵羊李斯特菌,利用荧光显微镜分析荧光表达情况。分别在含红霉素和不含红霉素的BHI肉汤中连续传代培养携带重组质粒的绵羊李斯特菌,通过提取质粒和观察荧光的方法研究重组质粒pCW-phly-GFP及GFP在绵羊李斯特菌中的稳定性。结果重组质粒pCW-phly-GFP酶切验证和测序均正确。在荧光显微镜下可以见到携带重组质粒的绵羊李斯特菌发绿色荧光。在红霉素抗性压力选择下重组质粒pCW-phly-GFP能稳定存在于绵羊李斯特菌中,并高效表达GFP。结论成功构建了GFP基因原核表达质粒pCW-phly-GFP,能在绵羊李斯特菌中高效表达GFP,为进一步研究绵羊李斯特菌作为疫苗载体提供了重要的工具。
Objective Constructing the recombinant Listeria ivanovii strain expressing green fluorescent protein to provide an important tool for study of Listeria ivanovii. Methods The promoter of Listeria monocytogenes Listeriolysin O(phly)and the green fluorescent protein(GFP)gene were fused by SOEing PCR,and then ligated the fusion gene into plasmid pCW to result in recombinant plasmid pCW-phly-GFP.Recombinant plasmid was electroporated into Listeria ivanovii,and fluorescence microscope was used to analyze the expression of GFP.To observe the stability of recombinant plasmid and the stable expression of GFP in Listeria ivanovii,bacteria were cultured in the BHI broth with or without erythromycin for several generations.The stability of recombinant plasmid pCW-phly-GFPand fluorescent protein in each generation of bacteriawas studied by extracting plasmids and observing fluorescence.Results The exactness of recombinant plasmid pCW-phly-GFP was confirmed with restrictive endonuclease assay and sequence analysis.Under the fluorescence microscope,the green fluorescence was obvious in Listeria ivanovii carried with pCW-phly-GFP.The recombinant plasmid pCW-phlyGFP was stable in Listeria ivanovii and the GFP kept expressing in a high level under the pressure of erythromycin.Conclusion The prokaryotic expression plasmid pCW-phly-GFP containing GFP gene was successfully constructed.Listeria ivanovii carried with the plasmid efficiently expressed GFP.This research provides an important tool for further study of Listeria ivanovii as a vaccine carrier.
引文
[1]周梦莹,蒲启康,任晨艳,等.致病性李斯特菌毒力因子蛋白组学及基因组学研究进展.现代预防医学,2015,42(4):694-697.
[2]ZHOU MY,JIANG MJ,REN CY,et al.Listeria ivanovii infection in mice:restricted to the liver and lung with limited replication in the spleen.Front Microbiol,2016,7:790[2017-09-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4894877/pdf/fmicb-07-00790.pdf.doi:10.3389/fmicb.2016.00790.
[3]LIN QQ,ZHOU MY,XU ZK,et al.Construction of two Listeria ivanovii attenuated strains expressing Mycobacterium tuberculosis antigens for TB vaccine purposes.J Biotechnol,2015,196-197:20-26[2017-09-28].http://www.sciencedirect.com/science/article/pii/S0168165615000176?via%3Dihub.doi:10.1016/j.jbiotec.2015.01.008.
[4]ERRAMPALLI D,LEUNG K,CASSIDY MB,et al.Applications of the green fluorescent protein as a molecular marker in environmental microorganisms.J Microbiol Methods,1999,35(3):187-199.
[5]LE DT,DUBENSKY TW,Jr,BROCKSTEDT DG.Clinical development of Listeria monocytogenes-based immunotherapies.Semi Oncol,2012,39(3):311-322.
[6]李常艳,李倩,刘海涛,等.量子点荧光标记技术的研究热点及面临的挑战.生物化学与生物物理进展,2010,37(1):103-110.
[7]ZHANG C,XING XH.Fluorescent proteins as a visible molecular signal for rapid quantification of bioprocesses:potential and challenges.Chinese J Chem Eng,2010,18(5):863-869.
[8]钟俐强,杨斯皓,贾钰铭,等.pGEX-4T-2-TK原核表达质粒的构建及其表达.重庆医学,2010,39(9):1036-1038.
[9]殷幼平,李强,袁训娥,等.绿色荧光蛋白基因原核表达载体的构建及其在昆虫肠道短短芽孢杆菌和苏云金芽孢杆菌中的表达.微生物学报,2010,50(5):614-620.
[10]江伟华,刘益丽,江明锋.大肠杆菌外源蛋白表达载体稳定性的研究进展.生物技术通报,2014(5):25-31[2017-09-28].http://mall.cnki.net/magazine/Article/SWJT201405006.htm.doi:10.13560/j.cnki.biotech.bull.1985.2014.05.012.
