副猪格拉瑟菌<i>Lon</i>基因缺失株的构建及生物学特性研究

doi:10.7668/hbnxb.20195705

摘要/Abstract

摘要：

为探索副猪格拉瑟菌Lon基因的生物学功能及其对该菌致病力的影响,利用重叠 PCR法扩增得到包含Lon基因上下游同源臂及卡那霉素抗性基因筛选标记的融合基因片段,构建包含该基因片段的重组自杀质粒pToPo-LR-Kana;采用自然转化法将 pToPo-LR-Kana转化至亲本菌株ZJ1208中,通过卡那霉素抗性筛选、PCR和测序鉴定Lon基因缺失菌株ZJ1208-ΔLon;通过生长速率测定、透射电镜观察、抗应激试验、紫外抗性试验、血清抗性试验、生物膜试验、荚膜多糖含量测定以及小鼠毒力试验等对比了野毒株ZJ1208与基因缺失株ZJ1208-ΔLon之间的差异。结果显示,成功获得了Lon基因缺失株 ZJ1208-ΔLon,该基因缺失株菌体明显变长,但与 ZJ1208野毒株生长速度和外膜囊泡数量相似;ZJ1208-ΔLon对渗透压应激、氧化应激及热应激耐受能力明显下降,且对紫外线的抗逆性明显下降;和亲本菌株 ZJ1208相比,ZJ1208-ΔLon的生物膜形成能力相似,荚膜多糖含量升高,但血清抗性显著降低,对小鼠的致死能力显著降低。这表明Lon基因在调节副猪格拉瑟菌多种关键生物功能方面起着极为关键的作用,为进一步解析 Lon基因在副猪格拉瑟菌中的生物学功能提供新的信息。

关键词: 副猪格拉瑟菌, ΔLon缺失株, 自然转化, 致病性, 生物学特性

Abstract:

The aim is to explore the biological function of the Lon gene in Glaesserella parasuis and its impact on the pathogenicity of the bacterium.A fusion gene fragment containing the homologous arms of the Lon gene and the kanamycin resistance gene selection marker was amplified using overlapping PCR,and a recombinant suicide plasmid pToPo-LR-Kana containing this gene fragment was constructed.The pToPo-LR-Kana was transformed into the parent strain ZJ1208 using natural transformation.The Lon gene-deleted strain ZJ1208-ΔLon was identified by kanamycin resistance screening,PCR,and sequencing.The differences between the wild-type strain ZJ1208 and the gene-deleted strain ZJ1208-ΔLon were compared through determination of growth rate,observation by transmission electron microscopy,stress resistance test,ultraviolet resistance test,serum resistance test,the biofilm formation assay,determination of capsule polysaccharide content and virulence test.The results showed that the Lon gene deletion strain ZJ1208-ΔLon was successfully obtained.The deletion strain had a significantly longer cell size,but its growth rate and number of outer membrane vesicles were similar to those of the wild-type strain ZJ1208.ZJ1208-ΔLon had significantly decreased tolerance to osmotic stress,oxidative stress,and heat stress,as well as significantly decreased resistance to ultraviolet radiation. Compared with the parent strain ZJ1208,the biofilm production capacity of ZJ1208-ΔLon and ZJ1208 was similar,but the capsular polysaccharide content of ZJ1208-ΔLon increased significantly,and the serum resistance and virulence in mice decreased significantly.The above results indicate that the deletion of Lon gene has an impact on multiple biological characteristics of G.parasuis,providing new information for further analysis of the biological function of Lon gene in G.parasuis.

Key words: Glaeserella parasuis, ΔLon deficient strain, Natural transformation, Pathogenicity, Biological characteristics

中图分类号:

S858.28

黄令, 孙洪超, 叶十一, 苏菲, 袁秀芳, 徐丽华, 余斌, 张晖, 李军星. 副猪格拉瑟菌Lon基因缺失株的构建及生物学特性研究[J]. 华北农学报, 2026, 41(1): 222-229. doi: 10.7668/hbnxb.20195705.

HUANG Ling, SUN Hongchao, YE Shiyi, SU Fei, YUAN Xiufang, XU Lihua, YU Bin, ZHANG Hui, LI Junxing. Construction and Biological Characteristics of Lon Gene Deletion Strain of Glaesserella parasuis[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(1): 222-229. doi: 10.7668/hbnxb.20195705.

http://www.hbnxb.net/CN/Y2026/V41/I1/222

图/表 11

表1 试验中所用的引物

Tab.1 Primers used in this study

引物名称 Name of the primers	引物序列(5'—3') Primer sequence(5'—3')	目的片段/bp Product size
Lon-U-F-BamHⅠ	GAG GGATCCTAAGATAGGTATAGTTACCAAAGTG	93
Lon-U-R	CCGTTGAATATGGCTCATTGGATTGCTCTCCTAAAATAGAATT
Lon-Kana-F	TTAGGAGAGCAATCCAATGAGCCATATTCAACGGGAAACGTCT	846
Lon-Kana-R	CTAATATCAACTATTTAGAAAAACTCATCGAGCATCAAATGAAAC
Lon-D-F	TCGATGAGTTTTTCTAAATAGTTGATATTAGCTAAGGTGAC	858
Lon-D-R-Hind Ⅲ	GGG CTCGAGCGCAACATTCCGCCATTTTGGAAGG

图1 缺失株的构建与验证 A.同源臂及抗性基因PCR产物;B.重组融合片段重叠PCR产物;B.重组融合片段重叠PCR产物;C.双酶切鉴定pToPo-LR-Kana;D.ZJ1208-ΔLon的PCR鉴定。1.Lon-UP(893 bp);2.Kana(846 bp);3.Lon-Down(858 bp);4.Lon-UP+Kana+Lon-Down(2 597 bp);5.双酶切后的载体3 000 bp,目的基因片段2 597 bp;6.未酶切的重组质粒5 597 bp;7.ZJ1208-ΔLon Kana PCR扩增片段(846 bp);8.ZJ1208-ΔLon Lon PCR扩增片段;M1.1 000 bp Marker;M2、M3.5 000 bp Marker;M4.2 000 bp Marker.

Fig.1 Construction and verification of deletion strain A.PCR product of homologous arms and drug resistance gene; B.Overlap PCR product of fusion gene fragment; C.Double enzyme digestion identification of pToPo-LR-Kana; D.ZJ1208-ΔLon PCR identification.1.Lon-UP (893 bp); 2.Kana (846 bp); 3.Lon-Down(858 bp); 4.Lon-UP+Kana+Lon-Down(2 597 bp); 5.The vector after double enzyme digestion is 3 000 bp, and the target gene fragment is 2 597 bp; 6.Untreated recombinant plasmid 5 597 bp; 7.ZJ1208-ΔLon Kana PCR amplification fragment (846 bp); 8.ZJ1208-ΔLon Lon PCR amplification fragment; M1.1 000 bp Marker; M2, M3.5 000 bp Marker; M4.2 000 bp Marker.

图2 缺失株与野毒株的菌落形态和菌体革兰氏染色 A、B.ZJ1208-ΔLon缺失株;C、D.ZJ1208野毒株。

Fig.2 Colony morphology and bacterial Gram staining of deletion strain and wild strain A,B.ZJ1208-ΔLon deficient strain;C,D.ZJ1208 wild-type strain.

图3 缺失株与野毒株的菌体透射电镜观察 A.ZJ1208-ΔLon基因缺失株;B.ZJ1208野毒株。

Fig.3 Transmission electron microscopic observation of the deletion strain and wild strain A.ZJ1208-ΔLon gene deletion strain;B.ZJ1208 wild-type strain.

图4 ZJ1208-ΔLon缺失株和ZJ1208野毒株生长曲线

Fig.4 The growth rate curves of ZJ1208-ΔLon deficient strain and ZJ1208 wild-type strain

图5 ZJ1208-ΔLon缺失株和ZJ1208野毒株的生物膜产量不同小写字母表示不同处理间在P<0.05水平差异显著。图6-8同。

Fig.5 The biofilm formation of ZJ1208-ΔLon deficient strain and ZJ1208 wild-type strain Different lowercase letters indicate significant differences between different treatments at P<0.05 level.The same as Fig.6-8.

图6 Lon基因对ZJ1208菌株抗逆性的影响 A.渗透压应激;B.氧化应激;C.热应激。

Fig.6 Effect of Lon gene on stress resistance of ZJ1208 strain A.Osmotic stress;B.Oxidation stress;C.Heat shock stress.

图7 荚膜多糖SDS-PAGE电泳及定量分析 A.ZJ1208野毒株与ZJ1208-ΔLon缺失株的SDS-PAGE;B.荚膜多糖表达量。

Fig.7 SDS-PAGE electrophoresis and quantification of capsule polysaccharides A.SDS-PAGE of ZJ1208 wild-type strain and ZJ1208-ΔLon deletion strain;B.Capsular polysaccharide expression between ZJ1208 wild strain and ZJ1208-ΔLon deletion strain.

图8 副猪格拉瑟菌在猪血清中的存活率

Fig.8 Detection of survival rate of G.parasuis in porcine serum

图9 ZJ1208与ZJ1208-ΔLon紫外光抗性检测

Fig.9 UV resistance detection of ZJ1208 and ZJ1208-ΔLon

图10 小鼠内脏病变与生存曲线 A.ZJ1208-ΔLon组;B.ZJ1208组;C.正常小鼠内脏;D.3组小鼠的生存曲线。

Fig.10 Visceral lesions and survival curves in mice A.Group of ZJ1208-ΔLon;B.Group of ZJ1208;C.Normal mice viscera;D.Survival curves of three groups of mice.

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副猪格拉瑟菌Lon基因缺失株的构建及生物学特性研究

Construction and Biological Characteristics of Lon Gene Deletion Strain of Glaesserella parasuis

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