摘要
[目的]对单增李斯特菌新疆绵羊脑炎临床分离株LM90SB2的lmo2192基因进行克隆及原核表达。[方法]利用PCR方法扩增lmo2192基因,连接p MD19-T载体进行克隆,筛选阳性菌进行测序比对。构建重组表达质粒p ET32a-2192,将其转入大肠杆菌感受态细胞,经诱导表达,利用SDS-PAGE与Western Blotting鉴定重组蛋白。[结果]扩增lmo2192基因序列长度为969 bp,与预期一致。该基因在大肠杆菌中大量表达,经SDS-PAGE和Western Blotting鉴定分析该产物为56 k Da左右的融合重组蛋白,与预期大小一致。[结论]成功克隆lmo2192基因,并获得大量表达,为进一步研究该基因功能奠定理论基础。
[Objective] The lmo2192 gene of Listeria monocytogenes Xinjiang sheep encephalitis clinical isolate LM90 SB2 was cloned and prokaryotic expression.[Method]The lmo2192 gene was amplified by PCR,ligated with p MD19-T vector,and the positive bacteria were screened for sequencing. The recombinant expression plasmid p ET32 a-2192 was constructed and transformed into E. coli competent cells. After induction,the recombinant protein was identified by SDS-PAGE and Western Blotting.[Result] The amplified lmo2192 gene sequence was 969 bp in length,which was consistent with expectations. The gene was expressed in Escherichia coli in a large amount,and the fusion protein of 56 k Da was identified by SDS-PAGE and Western Blotting,which was consistent with the expected sizes. [Conclusion] The lmo2192 gene was successfully cloned and obtained a large amount of expression,it provided a theoretical basis for further study on the function of the gene.
引文
[1]Danielle R Balay,Ramana V Dangeti,Kamaljit Kaur,et al.Purification of leucocin A for use on wieners to inhibit Listeria monocytogenes,in the presence of spoilage organisms[J].International Journal of Food Microbiology,2017,255:25-31.
[2]孙静娟,邱景璇,曾海娟,等.单增李斯特菌prs A1基因的截短表达、纯化及多克隆抗体的制备[J].生物技术进展,2018,8(03):254-260,278.
[3]任静静,杨铭伟,剡根强,等.环境因子及接种量对单增李斯特菌生长的影响[J].中国畜牧兽医,2017,44(05):1491-1497.
[4]张伟欣,李春阳,陈秀兰.细菌肽转运蛋白的研究进展[J].微生物学通报,2014,41(9):1856-1863.
[5]何庆芳,王晴.病原菌寡肽转运蛋白免疫原性的研究进展[J].内蒙古中医药,2011,30(08):110.
[6]何庆芳.哈维氏弧菌寡肽透性酶原核表达、突变株构建及其性质研究[D].青岛:中国海洋大学,2011.
[7]Mylène M Maury,Yu-Huan Tsai,Caroline Charlier,et al.Uncovering Listeria monocytogenes hypervirulence by harnessing its biodiversity[J].Nature Genetics,2016,48(3):308-313.
[8]钱凌霄,康立超,李红欢,等.食源性单增李斯特菌14种潜在毒力基因的检测[J].中国人兽共患病学报,2018,34(10):878-882.
[9]Ana Camejo,Filipe Carvalho,Olga Reis,et al.The arsenal of virulence factors deployed by Listeria monocytogenes to promote its cell infection cycle[J].Virulence,2011,2(5):379-394.
[10]Ferreira V,Wiedmann M,Teixeira P,et al.Listeria monocytogenes persistence in food-associated environments:epidemiology,strain characteristics,and implications for public health[J].Journal of Food Protection,2014,77(1):150.
[11]Wolfgang Eisenreich,Thomas Dandekar,Jürgen Heesemann,et al.Carbon metabolism of intracellular bacterial pathogens and possible links to virulence[J].Nature Reviews Microbiology,2010,8(6):401-412.
[12]Grischa Y Chen,Daniel A Pensinger,John-Demian Sauer.Listeria monocytogenes cytosolic metabolism promotes replication,survival,and evasion of innate immunity[J].Cellular Microbiology,2017,19(10).DOI:10.1111/cmi.12762.
[13]Wolfgang Eisenreich,Jürgen Heesemann,Thomas Rudel,et al.Metabolicadaptations of intracellullar bacterial pathogens and their mammalian host cells during infection("Pathometabolism")[J].Microbiology Spectrum,2015,3(3).DOI:10.1128/microloiolspec.MBP-0002-2014.
[14]Lior Lobel,Anat A Herskovits.Systemslevel analyses reveal multiple regulatory activities of CodY controlling metabolism,motility and virulence in Listeria monocytogenes[J].PLo S Genetics,2016,12(2):e1005870.DOI:10.1371/journal.pgen.1005870.
[15]Kevin E.Bruce,Britta E.Rued,Ho-ching Tiffany Tsui,et al.The Opp(Ami ACDEF)oligopeptide transporter mediates resistance of Serotype 2 Streptococcus pneumoniae D39 to killing by Chemokine CXCL10 and other antimicrobial peptides[J].Journal of Bacteriology,2018,DOI:10.1128/JB.00745-17.
[16]王真,王根立,吴同垒,等.寡肽转运酶Opp C对羊种布鲁氏菌致病性的影响[C].中国畜牧兽医学会家畜传染病学分会全国会员代表大会暨第十五次学术研讨会,2013.
[17]吴学林,曹树珠,马勋.单核细胞增生性李斯特菌srt A基因的克隆与原核表达[J].石河子大学学报:自然科学版,2015,33(2):196-200.
[18]杜冬冬,张奇文,李红欢,等.单增李斯特菌lmo0331基因的克隆、序列分析及原核表达[J].中国畜牧兽医,2018,45(5):1170-1176.
[19]Elise Borezee,Elisabeth Pellegrini,Pellegrini Berche.OppA of Listeria monocytogenes,an oligopeptide-binding protein required for bacterial growth at low temperature and involved in intracellular survival[J].Infection&Immunity,2000,68(12):7069-7077.
