支气管败血波氏杆菌PRN黏附素R1区蛋白的免疫原性

Immunogenicity of Recombinant R1 Region Antigen of Pertactin and Its Efficacy against Bordetella bronchiseptica Challenge

百日咳杆菌黏附素(PRN)是支气管败血波氏杆菌最重要的保护性抗原。为研究PRN的R1区多肽的免疫原性,分别将prn基因的全长编码区2 040 bp及5′端1 173 bp的片段(prnR1)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。SDS-PAGE和Western blot检测证实2种表达产物GST-R1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-R1和GST-PRN免疫组小鼠均能产生较高的PRN抗体水平;当使用3 LD50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10 LD50HH0809攻毒时,其保护率分别为33.3%(3/9)和77.8%(7/9)。在被动免疫保护试验中,腹腔免疫GST-R1和GST-PRN兔抗血清均能100%(10/10)保护小鼠抵抗10 LD50HH0809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)。这些研究结果表明,重组PRN的N端R1区多肽具有良好的免疫原性。

Pertactin（PRN） is identified as one of the most important protective immunogen of Bordetella bronchiseptica（Bb）.To study the immunogenicity of region 1（R1） of PRN,the complete coding sequence（2 040 bp） and its 5′-terminal 1 173 bp fragment of the prn gene were separately cloned into the prokaryotic expression vector pGEX-KG,and expressed in the E.coli BL21（DE3） with induction by IPTG.The concentration of purified expression products,glutathione S-transferase（GST）-R1 and GST-PRN were 312.6 and 207.3 μg·mL-1,and the purity of them were 95.2% and 92.4%,respectively.The GST-R1 and GST-PRN showed the similar immunological reactivity in Western-blot analysis.Mice,immunized subcutaneously with two doses of purified GST-R1 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 3 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 0.5 mL rabbit anti-GST-R1 or anti-GST-PRN serum,whilst there were no survivors in group of mice that received PRN-absorbed rabbit anti-GST-R1（0/5） or anti-GST-PRN serum（0/5）.In this study,we have shown that the recombinant GST-R1 protein had strong immunogenicity against Bb infection,which built a good foundation for the further research of highly efficient vaccine against bordetellosis.