Construction of a Fluorescent-labeled Localization Reporter Strain of Salmonella typhimurium and Its Tracer Application in Porcine Alveolar Macrophages

doi:10.7668/hbnxb.20193402

Abstract

Abstract:

The enhanced green fluorescent protein gene(EGFP)was used to characterize Salmonella typhimurium strain 14028(Sal-14028)to study its tracer role in porcine alveolar macrophages(3D4/21),and firstly,the EGFP was transferred into Sal-14028 by electroshock method and obtained green fluorescent positive and stable strain,named Sal-pPpagC-EGFP.Then,the growth characteristics of the fluorescent strain and the wild strain(WT)were compared under the same conditions,and the virulence of the fluorescent strain was examined by mouse infection test to determine whether the insertion of the EGFP gene had any significant effect on the virulence of the fluorescent strain,so as to examine the biological characteristics of the fluorescent strain,and analysis the localization of fluorescent strains in the phagocytic lysosomes of porcine alveolar macrophages(3D4/21)using fluorescence microscopy and indirect immunofluorescence.The results showed that EGFP-labeled Salmonella typhimurium emitted strong green fluorescence under fluorescence microscopy at different time points,and the fluorescence carried was not affected by resistance selection and could be stably inherited.The trend of the growth curve of the fluorescent strain Sal-pPpagC-EGFP was basically the same as that of the wild strain under the same culture conditions.In addition,the LD₅₀ calculated by the modified Kou method was not significantly different from that of the wild strain(P>0.05),indicating that the insertion of EGFP gene had no significant effect on the virulence of the fluorescent strain.After the fluorescent strains infected porcine alveolar macrophages,labeled strains were observed in the phagolysosomes,and the number of strains showed an increasing trend with the increase of time.It showed that the fluorescent strains had no significant changes in biological traits other than having luminescent properties.These results suggested that the construction of Sal-pPpagC-EGFP provided an effective tool to further reveal its infestation pathogenic process.

Key words: Salmonella typhimurium, EGFP, Electrical transformation, Biological properties, Intracellular tracing

XIAO Hong, HE Zhen, TIAN Qi, HUANG Xinmiao, LIAO Weidong, HUANG Tinghua, YAO Min. Construction of a Fluorescent-labeled Localization Reporter Strain of Salmonella typhimurium and Its Tracer Application in Porcine Alveolar Macrophages[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(2): 232-238. doi: 10.7668/hbnxb.20193402.

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Figures/Tables 7

Fig.1 Structural mapping of pPpagC-EGFP plasmid

Fig.2 PCR Amplification of PpagC fragment and validation of pMD18T-PpagC and pPpagC-EGFP by enzymatic cleavage A.PCR amplification of PpagC promoter fragment:M.DNA Marker DL2000;B.pMD18T-PpagC,pET28a-EGFP double digestion 1.5% agarose gel electrophoresis map:1.pET28a-EGFP,2.pMD18T-PpagC,M.DNA Marker DL2000;C.1.5% agarose gel electrophoresis plot of recombinant plasmid pPpagC-EGFP double digested(Fsp Ⅰ and Xba Ⅰ)product:1.The original plasmid,2-3.The digested plasmid,M.DNA Marker DL2000.

Fig.3 pPpagC-EGFP plasmid sequencing peak map(part 1) The sequencing primer is 5'-GCTGCCCATGGTATATCTCCT-3'.

Fig.4 Fluorescence map of salmonella labeled with EGFP A.Picture of Sal-pPpagC-EGFP Salmonella typhimurium under fluorescence microscope (excitation 488 nm/emission 507 nm,100×);B.Blank control under fluorescence microscope(excitation 488 nm/emission 507 nm,100×).

Fig.5 Comparison of the growth curves of Sal-14028 and Sal-pPpagC-EGFP

Tab.1 Determination of LD₅₀ after intraperitoneal injection of Sal-14028 and Sal-pPpagC-EGFP strains in 8-week-old mice

菌株 Strains	菌落总数/(cfu/mL) Total number of bacterial colonies	小鼠 Mice	小鼠死亡数 Mouse death	半数致死量/(cfu/mL) LD₅₀
Sal-14028	1.0×10⁴	12	0	1.45×10⁶
	1.0×10⁵	12	2
	1.0×10⁶	12	5
	1.0×10⁷	12	9
	1.0×10⁸	12	12
Sal-pPpagC-EGFP	1.0×10⁴	12	0	1.20×10⁶
	1.0×10⁵	12	2
	1.0×10⁶	12	5
	1.0×10⁷	12	10
	1.0×10⁸	12	12
对照Control	生理盐水	10	0	-

Fig.6 Co-localization of Salmonella fluorescens with phagocytic lysosomes in 3D4/21(100×)

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