Xian'gan Han, E-mail:
Wei Jiang, Tel/Fax: +86-21-34293397, E-mail:
以副溶血弧菌VP2918为研究对象,研究其对副溶血弧菌的生物学特性和致病性的影响。
利用同源重组技术构建了
缺失
By using comparative proteomics, we identified several potential T6SS-2 effectors, including VP2918. The aim of this study is to elucidate the role of VP2918 in biological characteristics and pathogenicity of
The
The biological characteristics analysis
The VP2918 was not associated with the motility and biofilm formation ability, but it was a potential virulence factor and associated with the pathogenic of
副溶血性弧菌(
副溶血性弧菌感染宿主细胞需要多种毒力因子,包括粘附素、溶血素和分泌系统[
本研究首先通过构建
副溶血弧菌临床分离株SH112 (
缺失株、互补株引物设计是根据GenBank上副溶血性弧菌RIMD2210633标准株的
本试验所用引物
The primers used for gene mutant strain and complement strain in this study
Primers | Sequences (5ʹ→3ʹ) | PCR product/bp |
The underline indicates the enzyme digestion site. | ||
CGC |
666 | |
ACCAAGCTAGTCTAGCAACAGTGATGT | ||
CTAGACTAGCTTGGTCACGATGAAGAAAACG | 547 | |
TCC |
||
TGTTGTTGCAGGAAGTGA | Wide type: 1959 |
|
GTGTAGCAACCTCTTTCG | ||
sacB-F | ACGGCACTGTCGCAAACTATA | 600 |
sacB-R | TTCCGTCACCGTCAAAGAT | |
CGCGGATCCATGACGTTCTGTATTGATGG | 537 | |
AAAACTGCAGTTAGTTCCATGCCTGCTTAAG |
参照文献[
以SH112基因组为模板,以VP2918-pMMB-F/R为引物,PCR扩增含
将野生株、缺失株和互补株在含有3% NaCl的LB培养基(pH 7.2)中培养,待菌液培养至对数生长期后,转接至100 mL含有3% NaCl的LB中,每隔1 h取各菌液200 μL利用分光光度仪测定各自的吸光度,并绘制各菌株在含3% NaCl的LB培养基中的生长曲线。
取1 μL上述菌液于运动培养基(0.3%琼脂、3% NaCl-LB半固体培养基)上,37 ℃恒温正置培养4–5 h,观察细菌从中央向周围的泳动情况,并进行测量、拍照。
为评价上述菌株生物被膜形成情况,将200 μL (
用含10%胎牛血清的DMEM培养基培养HeLa细胞于24孔板内,待单层细胞布满孔底部90%时,用DMEM洗涤2次后,待用。将野生株、缺失株和互补菌株培养至对数生长期,再用DMEM培养基洗2次并重悬,均以感染率(multiplicity of infection,MOI)为10:1 (菌数: 细胞数)感染HeLa细胞,每孔加200 μL,重复3个孔,置于37 ℃、5% CO2细胞培养箱中孵育1 h。用PBS洗1次,每孔加100 μL预冷的0.5% (体积分数)TritonX-100裂解细胞,作用10 min。最后用无菌的PBS倍比稀释裂解液,并涂布于含有3% NaCl的LB琼脂平板,37 ℃培养过夜,记录单菌落数,计算各细菌株的相对黏附率。
将HeLa细胞在96孔细胞培养板上培养,用PBS洗涤3遍,每孔加入50 μL不含酚红的DMEM。将野生株、缺失株和互补菌株培养至对数生长期,用PBS洗涤重悬后,加入50 μL的菌液(MOI分别为1:1、1:10和1:100)至上述细胞孔中作为实验孔,重复6个孔。同时设立100 μL的DMEM作为细胞自发对照孔,取90 μL DMEM加10 μL裂解液作为最大释放孔,于37 ℃、5% CO2的细胞培养箱中分别孵育1.5 h。利用CytoTox96试剂盒检测细胞上清中乳酸脱氢酶(LDH)的释放,参照说明书计算各细菌感染HeLa细胞后LDH释放的百分比。
为了评价
将4周龄ICR小鼠分成4组(野生株SH112,Δ
实验数据采用GraphPad Prism8软件进行统计分析。单因素方差分析(one-way ANOVA)用于分析运动性和生物膜形成、细胞黏附、组织载菌量测定的数据,双因素方差分析(two-way ANOVA)分析细胞毒性测定数据,用平均数±标准差(
对野生株SH112、
Identification of
野生株SH112、缺失株
各菌株生长曲线测定
Growth curve detection of SH112, Δ
运动性分析
Motility analysis of SH112, Δ
各菌株生物被膜形成能力比较
Biofilm formation ability of SH112, Δ
对HeLa细胞的黏附结果表明,与野生株比较,缺失株Δ
各菌株对HeLa细胞的黏附作用
Adhesion to HeLa cell monolayers of the strains. ns:
如
各菌株对HeLa细胞的细胞毒性影响
Cytotoxic effects of the SH112, △
将各菌株以每只5×107 CFU的量腹腔注射试验组小鼠,结果表明(
各菌株攻毒ICR小鼠后的存活率
The survival rate of ICR mice infected with the
以每只1×107 CFU的量感染ICR小鼠15 h时检测细菌组织载量,结果显示(
各菌株在小鼠心脏(A)、肝(B)和脾组织(C)中的细菌载量
Bacterial loads in heart (A), liver (B) and spleen (C) of infected mice.
副溶血性弧菌不仅严重影响水产养殖业的健康发展,还是人类健康的重大威胁,对该菌致病机制的研究非常重要。副溶血弧菌能够在全球范围内流行,与毒力因子的作用密切相关[
本实验室前期通过差异蛋白质组学技术比较分析野生株SH112和T6SS-2主要结构基因缺失株
黏附、定殖或入侵宿主细胞是细菌感染宿主的重要步骤,与细菌的致病机制密切相关[
本研究通过对
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