[1]陈俊红,蔡煜,戴薇,等.禽致病性大肠杆菌P型菌毛papA基因克隆与基因分型分析[J].江苏农业学报,2022,38(06):1594-1602.[doi:doi:10.3969/j.issn.1000-4440.2022.06.018]
　CHEN Jun-hong,CAI Yu,DAI Wei,et al.Cloning and genotyping of papA gene of type P pili of avian pathogenic Escherichia coli[J].,2022,38(06):1594-1602.[doi:doi:10.3969/j.issn.1000-4440.2022.06.018]
点击复制

禽致病性大肠杆菌P型菌毛papA基因克隆与基因分型分析()
分享到：

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
38
期数:
2022年06期
页码:
1594-1602
栏目:
畜牧兽医·水产养殖
出版日期:
2022-12-31

文章信息/Info

Title:
Cloning and genotyping of papA gene of type P pili of avian pathogenic Escherichia coli
作者:
陈俊红; 蔡煜; 戴薇; 雷卫强; 戴鼎震
（金陵科技学院动物科学与食品工程学院，江苏南京210046)
Author(s):
CHEN Jun-hong; CAI Yu; DAI Wei; LEI Wei-qiang; DAI Ding-zhen
(School of Animal Science and Food Engineering, Jinling Institute of Technology, Nanjing 210046, China)
关键词:
禽大肠杆菌; P型菌毛; papA基因
Keywords:
avian pathogenic Escherichia coli; type P pili; papA gene; variation
分类号:
Q785
DOI:
doi:10.3969/j.issn.1000-4440.2022.06.018
文献标志码:
A
摘要:
为克隆禽致病性大肠杆菌P型菌毛papA基因，确定其基因分型并分析其变异性，从GenBank数据库中查找大肠杆菌P型菌毛papA基因，并运用Snap Gene 3.2.1软件设计合成3对引物。其中，第1对引物为扩增不同基因分型通用引物，预计扩增片段大小为281 bp；第2对和第3对引物是为了扩增完整papA基因并分析有无变异而设计的，预计扩增片段大小分别为522 bp和943 bp。以禽大肠杆菌HJ2、TK3菌株的基因组为模板进行PCR扩增；将所有扩增片段回收、酶切，并分别与pTG19-T载体进行连接，转化进入DH5α受体菌，提取获得含扩增片段的重组质粒，测序并进行序列分析。结果表明，用引物1从HJ2、TK3菌株的基因组中均扩增获得接近 281 bp大小的片段，用引物2则只从HJ2菌株基因组中扩增获得接近522 bp大小的片段，而未能从TK3菌株基因组中扩增出片段；用引物3从TK3菌株基因组中扩增出接近943 bp大小的片段，而未能从HJ2菌株基因组中扩增出产物。经过基因碱基序列分析比对，完整的papA基因则位于943 bp的片段之中，确定HJ2和TK3两个菌株P型菌毛papA基因均属基因11型，但发现二者papA基因存在一定的碱基差异。PapA蛋白氨基酸序列比对发现，同属于基因11型的papA基因编码的PapA蛋白氨基酸序列也存在位点差异。
Abstract:
In order to clone the papA gene of type P pili of avian pathogenic Escherichia coli, determine its genotyping and analyze its variability, the papA gene of Escherichia coli P-type pili was searched from the GenBank database, and three pairs of primers were designed and synthesized using Snap Gene 3.2.1 software. Among them, the first pair of primers was a universal primer for amplifying different genotypes, and the expected amplified fragment size was 281 bp. The second and third pairs of primers were designed to amplify the complete papA gene and analyze the variation, and the expected amplified fragments were 522 bp and 943 bp, respectively. The genes of avian Escherichia coli HJ2 and TK3 strains were used as templates for PCR amplification. All amplified fragments were recovered, digested and connected with the pTG19-T vector, respectively, and transformed into DH5α receptor bacteria. Recombinant plasmids containing amplified fragments were extracted, sequenced and analyzed. The results showed that a fragment of 281 bp was amplified from the genome of HJ2 and TK3 strains by primer 1, and a fragment of 522 bp was amplified from the genome of HJ2 strain by primer 2 but not from the genome of TK3 strain. A fragment of 943 bp was amplified from the genome of TK3 strain by primer 3 but not from the genome of HJ2 strain. After sequencing analysis, the complete papA gene was located in the fragment of 943 bp. It was confirmed that the papA genes of P-type pili of the two strains belonged to genotype 11, but there were some base differences in their papA genes. Amino acid sequence alignment of PapA protein showed that there were site differences in the amino acid sequence of PapA protein encoded by papA gene, which belonged to genotype 11.

参考文献/References:

[1]RAMREZ RM, ALMANZA Y, GONZLEZ R, et al. Avian pathogenic Escherichia coli bind fibronectin and laminin[J]. Vet Res Commun, 2009, 33(4):379-386.
[2]DOZOIS C M, POURBAKHSH S A, FAIRBROTHER J M. Expression of P and type 1 (F1) fimbriae in pathogenic Escherichia coli from poultry[J]. Vet Microbiol, 1995, 45(4):297-309.
[3]SMYTH C J, MARRON M B, TWOHIG J M, et al. Fimbrial adhesins: similarities and variations in structure and biogenesis[J]. FEMS Immunol Med Microbiol, 1996, 16(2):127-139.
[4]GYIMAH J E, PANIGRAHY B. Adhesin-receptor interactions mediating the attachment of pathogenic Escherichia coli to chicken tracheal epithelium[J]. Avian Dis, 1988, 32(1):74-78.
[5]KLEMM P, ROOS V, ULETT GC, et al. Molecular characterization of the Escherichia coli asymptomatic bacteriuria strain 83972: the taming of a pathogen[J]. Infect Immun, 2006, 74(1):781-785.
[6]POURBAKHSH S A, DHO-MOULIN M, BRE A, et al. Localization of the in vivo expression of P and F1 fimbriae in chickens experimentally inoculated with pathogenic Escherichia coli [J]. Microb Pathog, 1997, 22(6):331-341.
[7]SAUER M M, JAKOB R P, LUBER T, et al. Binding of the bacterial adhesin FimH to its natural, multivalent high-mannose type glycan targets [J]. J Am Chem Soc, 2019, 141(2):936-944.
[8]EPLER BARBERCHECK C R, BULLITT, ANDERSSON M.Bacterial adhesion pili [J]. PubMed-NCBI, 2018, (87):1-18.
[9]KARIYAWASAM S, NOLAN LK. papA gene of avian pathogenic Escherichia coli [J]. Avian Dis, 2011, 55(4):532-538.
[10]MORTEZAEI N, SINGH B, BULLITT E, et al. P-fimbriae in the presence of anti-PapA antibodies: new insight of antibodies action against pathogens[J]. Scientific Reports，2013(12):1-9.
[11]JOHNSON J R, STELL A L, SCHEUTZ F, et al. Analysis of the F antigen-specific papA alleles of extraintestinal pathogenic Escherichia coli using a novel multiplex PCR-based assay[J]. Infect Immun, 2000, 68(3):1587-1599.
[12]戴鼎震,郑明球,蔡宝祥,等. 鸡大肠杆菌P型菌毛结构基因(papA)的克隆与序列测定[J]. 中国兽医学报, 2000(5):455-458.
[13]LILLINGTON J, GEIBEL S, WAKSMAN G. Biogenesis and adhesion of type 1 and P pili[J]. Biochim Biophys Acta, 2014, 1840(9):2783-2793.
[14]MAITI S N, DESGROSEILLERS L, FAIRBROTHER J M, et al. Analysis of genes coding for the major and minor fimbrial subunits of the Prs-like fimbriae F165(1) of porcine septicemic Escherichia coli strain 4787[J]. Microb Pathog, 1994, 16(1):15-25.

备注/Memo

备注/Memo:
收稿日期：2022-09-27基金项目：江苏省高校自然科学研究面上项目（19KJB230006）；江苏省自然科学基金青年基金项目（BK20210005）；金陵科技学院高层次人才科研启动项目（jit-b-201909）；金陵科技学院“创客”虚拟班项目（2017008、2021001）；大学生创新训练计划项目（202113573090M）作者简介：陈俊红（1990-），女，安徽宿州人，博士研究生，主要从事预防兽医、中兽医学和比较医学研究。（E-mail）chenjunhong@jit.edu.cn通讯作者：戴鼎震,（E-mail）dzdai@163.com
更新日期/Last Update: 2023-01-13