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

【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30.5kDa的外源保护性抗原rPmtC。C501(pYA-PmtC)腹腔感染BALB/c小鼠的LD50为8.5×106CFU,毒力稍低于C500(LD50为4.4×106CFU);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。

[ 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 LDs0 of 8.5 ×10^6CFU was a little lower than C500 with LDs0 of 4.4 ×10^6CFU 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.