摘要
目的克隆幽门螺杆菌(Helicobacter pylori,Hp)D-丙氨酸-D-丙氨酸连接酶A(D-alanine-D-alanine ligase A, DdlA)基因ddlA编码区,并对其进行测序和生物信息学分析。方法以MEL-Hp27菌株基因组DNA为模版,PCR扩增ddlA基因编码区;将ddlA基因与真核表达载体pcDNA3.1(+)连接,构建重组表达载体;测序获得ddlA基因序列,利用生物信息学软件对编码DdlA蛋白的理化性质、结构特点进行分析。结果 MEL-Hp27 ddlA基因的编码区长为1 044 bp,编码347个氨基酸;编码的DdlA蛋白是一种性质稳定的亲水蛋白,相对分子质量为39.61×10~3,属于D-ala-D-ala连接酶N端家族;DdlA蛋白无跨膜区和信号肽,具有磷酸化位点和O-糖基化位点;二级结构中α-螺旋占36.02%,无规卷曲Cc占42.36%,延伸链Ee占18.73%,β-转角Tt占2.88%。DdlA蛋白含有7个B淋巴细胞和15个CTL细胞相关抗原表位。结论成功克隆了Hp27 ddlA基因,构建了该基因的真核表达载体,表达的DdlA蛋白含有B、T淋巴细胞抗原表位,为研究Hp DdlA蛋白基因在Hp感染致病及防治中的作用提供了理论依据。
Objectives To clone and sequence the nucleotide sequence of the D-alanine-D-alanine ligase A(ddlA) gene of Helicobacter pylori(Hp) and to bioinformatically analyze that sequence. Methods Genomic DNA of the MEL-Hp27 strain was used as a template to amplify the ddlA gene via a polymerase chain reaction. The ddlA gene and the eukaryotic expression vector pcDNA3.1(+) were ligated to construct a recombinant expression vector. The sequence of the ddlA gene was obtained via sequencing, the homology of nucleotide sequences in the ddlA coding region of different H. pylori strains was analyzed using the BLAST tool from the NCBI, and the physicochemical properties, structural characteristics, and epitopes of the DdlA protein were analyzed using bioinformatic software. Results The coding region of the ddlA gene of MEL-Hp27 was 1,044 bp in length. It encoded a protein consisting of 347 amino acids. The protein included 19 amino acids, the most prevalent of which was leucine(13.5%), followed by lysine(Lys)(10.7%). The remaining amino acids all accounted for less than 10%. The encoded DdlA protein is a stable hydrophilic protein with a molecular weight of 39.61×10~3 and an isoelectric point of 7.10, belonging to the D-ala-D-ala ligase N-terminal family. The protein has no transmembrane regions or signal peptides, 1 O-glycosylation site, and 23 potential phosphorylation sites. Alpha helices account for 36.02% of the protein’s secondary structure, random coils account for 42.36 %, extended strands account for 18.73%, and β-turns account for 2.88%. This protein has 7 B lymphocyte-and 15 CTL-associated epitopes. Conclusion This study successfully cloned the ddlA gene of MEL-Hp27, and it constructed a eukaryotic expression vector for the ddlA gene. The expressed DdlA has both B-and T-lymphocyte epitopes, providing a theoretical basis for the study of the role of the ddlA gene in the pathogenesis and prevention of H. pylori infection.
作者
何梦雅
王海燕
张荣光
余飞艳
陈帅印
郗园林
段广才
HE Meng-ya;WANG Hai-yan;ZHANG Rong-guang;YU Fei-yan;CHEN Shuai-yin;XI Yuan-lin;DUAN Guang-cai(Department of Epidemiology and Statistics,College of Public Health,Zhengzhou University,Zhengzhou,Henan,China450001)
出处
《中国病原生物学杂志》
CSCD
北大核心
2019年第10期1119-1124,1134,共7页
Journal of Pathogen Biology
基金
国家自然科学基金面上项目(No.81773495)
国家科技重大专项(No.2018ZX10301407)
