重组百日咳杆菌粘附素C端蛋白的免疫原性

Immunogenicity of C Terminal Recombinant of Pertactin

分别将支气管败血波氏杆菌百日咳杆菌粘附素(PRN)基因prn的全长编码区2040bp及3’端867bp的片段(prnC)克隆到原核表达载体pGEX-KG,经IPTG诱导后在大肠杆菌中实现了表达。Western blot检测证实两种表达产物GST-PC和GST-PRN均具有良好的免疫学反应活性。在主动免疫保护试验中,GST-PC和GST-PRN免疫组小鼠均能产生较高的PRN抗体水平;当使用3LD50的Bb强毒株HH0809进行鼻腔攻毒后,其保护率均为100%(9/9);当使用10LD50HH0809攻毒时,其保护率分别为66.7%(6/9)和77.8%(7/9)。在被动免疫保护试验中,腹腔免疫GST-PC和GST-PRN的兔抗血清均能100%(10/10)保护小鼠抵抗10LD50的HH0809的腹腔攻击,但经PRN吸附后的2种兔抗血清均失去了保护力(0/5)。研究结果表明:重组PRNC端多肽具有良好的免疫原性,可考虑作为新型亚单位疫苗或活载体疫苗的抗原成分。

Pertactin(PRN)is identified as one of the most important protective immunogen of Bordetella bronchiseptica(Bb).To study the immunogenicity of the C terminal region of PRN,the complete coding sequence(2040bp)and its 3'-terminal 867 bp fragment of the prn gene were separately cloned into the prokaryotic expression vector pGEX-KG,and expressed in the Escherichia coli BL21(DE3) with induction by IPTG,named with GST-PRN and GST-PC.The GST-PC and GST-PRN showed the similar immunological reactivity in Western-blot analysis.Mice,immunized subcutaneously with two doses of purified GST-PC or GST-PRN proteins mixed with an equal volume of Freund's adjuvant,produced robust PRN-specific IgG antibody levels.In these two groups,all 9 mice survived intranasal challenge with three times the 50% lethal dose(LD50) of virulent Bb HH0809,whereas 6 of 9 and 7 of 9 mice survived challenge with ten times LD50,respectively.Furthermore,complete protection(10/10)against intraperitoneal(i.p.)challenge with ten times the LD50 of virulent HH0809 strain was seen in mice that were injected i.p.with 0.5m L rabbit anti-GST-PC or anti-GST-PRN serum,whilst there were no survivors in group of mice that received PRN-absorbed rabbit anti-GST-PC(0/5) or anti-GST-PRN serum(0/5).The results show that the recombinant GST-PC protein has strong immunogenicity against Bb infection,which provides a good foundation for the further research of highly efficient vaccine against bordetellosis.