百日咳杆菌粘附素中针对支气管败血波氏杆菌的保护性抗原肽的筛选

Immunogenicity and protective efficacy of pertactin recombinants against Bordetella bronchiseptica challenge

【目的】本研究通过百日咳杆菌黏附素（PRN）基因的分段克隆表达及其在BALB/c小鼠的主动和被动免疫保护试验筛选PRN中的保护性抗原肽。【方法和结果】利用大肠杆菌进行PRN的完整蛋白、N端和C端多肽及其RⅠ和RⅡ区域多肽（双拷贝）的表达,命名为GST-PRN、GST-PN、GST-PC、GST-2PRⅠ和GST-2PRⅡ。Westernblot检测证实5种表达产物均具有良好的反应原性。在主动免疫保护试验中,5种表达产物均能诱导小鼠产生较高的PRN抗体水平;当使用3LD50的支气管败血波氏杆菌（Bb）强毒株HH0809进行鼻腔攻击后,GST-2PRⅠ的保护率为66.7%（6/9）,其余4者的保护率均为100%（9/9）;当使用10LD50的HH0809攻击时,其保护率分别为77.8%（7/9）、33.3%（3/9）、66.7%（6/9）、10%（1/10）和60%（6/10）。在被动免疫保护试验中,当使用10LD50的HH0809进行腹腔攻击时,GST-2PRⅠ抗血清对小鼠的保护率为20%（2/10）,其余4者的保护率均为100%（10/10）;但经天然PRN吸附后的5种兔抗血清均无任何保护力（0/5）。【结论】本研究表明PRN的N端和C端表达多肽均具有较强的针对Bb的保护力,且C端强于N端;而C端RⅡ区域的保护力也显著强于N端RⅠ区域。

[Objective]In this study we showed the immunogenicity and protective efficacy of five pertactin recombinants against Bordetella bronchiseptica（Bb） challenge.[Methods and results]The complete coding sequence（2040 bp） of the prn gene（PRN） and its fragments,5＇-terminal 1173 bp fragment（PN）,3＇-terminal 867 bp fragment（PC）,two copies of region Ⅰ（654 bp;PRⅠ） in PN,and 2 copies of region Ⅱ（678 bp;PRⅡ） in PC,were separately cloned into the prokaryotic expression vector pGEX-KG,and expressed in the Eschierichia coli BL21（DE3） using induction by isopropylβ-D-thiogalactopyranoside.The recombinant proteins were named GST-PRN,GST-PN,GST-PC,GST-2PRⅠ and GST2PRⅡ.All five recombinant proteins showed immunological reactivity in the Western-blot analysis.Mice,immunized subcutaneously with two doses of the purified proteins mixed with an equal volume of Freund＇s adjuvant,produced robust PRN-specific IgG antibody levels.When challenged,6 of 9 mice in GST-2PRⅠgroup and all 9 mice in the other groups survived intranasal challenge with three times the 50% lethal dose（LD50） of virulent Bb HH0809.After challenge with 10 LD50 7/9,3/9,6/9,1/10 and 6/10 of the mice survived.Furthermore,complete protection against intraperitoneal（i.p.） challenge with 10 LD50 of HH0809 was observed in mice that were injected i.p.with 0.5 ml rabbit anti-GST-PRN,GST-PN,GST-PC or GST-2PRⅡ serum.Only 1 of 10 mice survived in the group of mice that received anti-GST-2PRⅠ,and no survivors were noted in the group of mice that received PRN-absorbed rabbit antiserum（0 /5）.[Conclusion]In this study,we showed that all of five pertactin recombinants had differential immunogenicity and protective efficacy against Bb challenge.Mice immunized with GST-PC had better survival against fatal Bb challenge than did those immunized with GST-PN.In addition,GST-2PRⅡ and GST-2PRⅠprovided the similar results These data may have implications for the development of safe and efficacious subunit vaccines for the prevention of bordetellosis on the basis of these five pertactin recombinants.