pIRES2-EGFP-4-1BBL质粒的导入对减毒沙门菌生物学行为的影响

The Biological Behaviour Change of Attenuation Salmonella Imported pIRES2-EGFP-4-1BBL Plasmid

目的探讨真核表达质粒pIRES2-EGFP-4-1BBL转入对减毒鼠伤寒沙门菌SL3261生物学行为的影响。方法将真核表达质粒pIRES2-EGFP-4-1BBL通过两步电转化获得含pIRES2-EGFP质粒的SL3261,接种于LB培养基中观察其生长特性、革兰染色观察其形态和血清凝集试验检测其表面抗原变化。利用该重组菌体外感染HepG2细胞,观察其对细胞的侵袭能力的影响。结果含pIRES2-EGFP-4-1BBL质粒SL3261在体外生长繁殖较原细菌慢,革兰染色呈阴性丝状菌体,含pIRES2-EGFP-4-1BBL质粒的SL3261 A-F多价、O4菌体和H1鞭毛抗原和SL3261完全一致;体外感染HepG2能力,SL3261为201±46 CFU/200HepG2细胞,SL3261-pIRES2-EGFP为163±37,而SL3261-pIRES2-EGFP-4-1BBL为158±32,3组间的差异无统计意义(P>0.05)。结论导入pIRES2-EGFP-4-1BBL质粒对SL3261的生长和形态有部分影响,而表面抗原以及侵入HepG2细胞的功能不变,该结果为含pIRES2-EGFP-4-1BBL质粒SL3261疫苗菌的进一步研究奠定了实验依据。

Objective To explore the biological behaviour change of Attenuation Salmonella Typhimurium SL3261 imported eukaryotic plasmid pIRES2-EGFP-4-1BBL. Method Attenuation Salmonella Typhimurium SL3261 imported eukaryotic plasmid pIRES2-EGFP-4-1BBL was obtained by electrotransformation by two steps （pIRES2-EGFP4-1BBL vector was converted to LB5000 Salmonella Typhimurium for methylation then modified vector was electrotransfered to final host SL3261）, The recombined bacterial strain then was grown in Lurian broth and the characteristics of it＇s reproduction was monitored.The appearance of the recombined staining was observed by Gram stain and it＇s surface antigen was detect by serum agglutination test. The reconstructed SL3261 strain was loaded in vitro with HepG2 ceils to study it＇s potential invasive ability. Result Production of the reconstructed SL3261 strain containing pIRES2-EGFP-4-1BBL plasmid was much slower than that in its predecessor, with an appereance of negative Gram staining and trichobacteria. The recombined strain showed no difference in polyvalency A-F,thalline O4, HL antigen H1 with it＇s predecessor, and it＇s invasive ability cultured with HepG2 cells in vitro also showed no difference：SL3261：201 ±46 CFU/200HepG2 cells,SL3261-pIRES2-EGFP：163±37 and SL3261-pIRES2-EGFP-4-1BBL：158±32 （P〉0.05）. Conclusion It seems that the reconstructed SL3261 containing pIRES2-EGFP-4-1BBL plasmid shows change in it＇s reproduction and appearance,while it＇s surface antigen and invasive ability shows no difference with it＇ s predecessor, these findings might be helpful to the development of strain SL3261 vaccine containing rat 4-1BBL gene eukaryotie expression vector.