表达T+Pm保护性抗原的重组猪霍乱沙门氏菌C500株的构建及其生物学特性

Construction and characterization of recombinant Salmonella enterica serovar choleraesuis vaccine strain expressing heterologous antigen of Pasteurella multocida toxin

摘要:

摘要：【目的】本研究利用Asd+平衡致死系统构建表达巴氏杆菌毒素(Pasteurella multocida toxin, PMT)的重组猪霍乱沙门氏菌株，并对重组菌株的生物学特性进行比较研究。【方法和结果】通过基因克隆的方法构建表达PMT的重组质粒pYA-PmtC，再将其电转化减毒猪霍乱沙门氏菌C500的asd基因缺失株C501，构建口服活疫苗菌株C501(pYA-PmtC)。研究结果表明重组菌株C501(pYA-PmtC)的生化特性、血清型和生长速度与亲本菌株C500一致；在没有选择压力的条件下，C501(pYA-PmtC)能够稳定遗传重组质粒及其外源基因片段，并能稳定、高效、分泌性表达30.5 kDa的外源保护性抗原rPmtC。C501(pYA-PmtC)腹腔感染BALB/c小鼠的LD50为8.5×106 CFU，毒力稍低于C500（LD50为4.4×106 CFU）；口服接种C501(pYA-PmtC)和C500的所有仔猪未见任何发病症状，两者没有显著差别。【结论】本研究利用Asd+平衡致死系统的原理构建表达T+Pm保护性抗原重组猪霍乱沙门氏菌弱毒菌株C501(pYA-PmtC)，为进一步开发猪萎缩性鼻炎-副伤寒的双价基因工程疫苗奠定基础。

Abstract:

Abstract: [Objective] This study was aimed to construct and characterize Salmonella vaccine strain C500 expressing the recombinant Pasteurella multocida toxin (PMT) antigen by the Asd+ balanced-lethal host-vector system. [Methods and results] The DNA fragment encoding C terminal of PMT was cloned downstream from the beta-lactamase signal sequence in the multicopy Asd+ pYA3493 vector to create pYA-PmtC. Fermentation patterns, serotype, and mean generation time of the vaccine strain C500 harboring pYA-PmtC (named with C501(pYA-PmtC)) were identical to those of the parent strain C500. The recombinant pYA-PmtC plasmid was very stable in C501(pYA-F1P2), which expressed secretorily a large amount of the recombinant PMT antigen (named with rPmtC). The virulence of C501(pYA-PmtC) with LD50 of 8.5×106 CFU was a little lower than C500 with LD50 of 4.4×106 CFU based on the method of Reed and Muench. All piglets inoculated with C501(pYA-PmtC) or C500 survived, and had no signs of disease during the entire experimental period. No significant differences were found between these two groups. [Conclusion] The recombinant vaccine strain C501(pYA-PmtC) had a series of biological characteristics silimar to the parent vaccine strain C500. It is likely that C501(pYA-PmtC) could be adapted to develop multivalent recombinant Salmonella vaccine against both infections with S. enterica serovar Choleraesuis and toxigenic P. multocida.