以重组B群脑膜炎奈瑟菌fHBP为载体的结合疫苗制备及免疫原性研究

摘要

To express and purify the meningococcal outer membrane protein factor H binding protein (fHBP) , and conjugate this recombinant protein with group C meningococcal polysaccharide (GCMP) u-sing chemical method. Then to immunize mice with this conjugate, and to detect the IgG and serum bactericidal antibody (SBA) against GCMP ( GCMP-IgG) and recombinant fHBP ( rfHBP-IgG) in order to evaluate its application as a protein carrier in conjugates vaccine. Methods: The gene encoding fHBP was amplified from the genomic DNA of meningococcal using PCR. The PCR product was then cloned into the prokaryotic expression vector pET30a ( + ) and the recombinant was transformed into the host cell E. coli BL21 (DE3) . The rfHBP was induced by IPTG and purified by Ni-NTA. The rfHBP was successfully conjugated with GCMP. Mice were immunized with subcutaneous injection of the conjugates and the immune responses against GCMP and rfHBP were detected by ELISA. The SBA was detected with the TTC method. Results: The inserted fhbp gene sequence was confirmed by DNA sequencing and the rfHBP was successfully induced and conjugated with GCMP. High levels of rfHBP-IgG and GCMP-IgG were significantly boosted in the mice immunized with conjugate vaccine in comparison with those immunized with GCMP alone (P <0.05). There was no statistical significance in rfHBP-IgG and SBA between the conjugate vaccine group and the rfHBP group ( P > 0. 05 ). There was no statistical significance between the SBA against ATCC700111 and CMCC29361 (P>0.05). Conclusion: High level of antibody and SBA against meningococcal group C have been detected in mice immunized with the conjugate vaccine, suggesting that the rfHBP is a promising protein carrier. At the same time, high level of antibody and SBA against meningococcal group B have also been detected , showing that this kind of conjugate vaccine may provide universal protection against meningococcal infection.%目的:以重组B群脑膜炎奈瑟菌(Nesseria meningitidis,Nm)外膜蛋白fHBP(recombinant factor H binding protein,rfHBP)为载体与C群多糖(group C meningococcal polysaccharide,GCMP)共价结合制备结合疫苗并免疫小鼠,检测小鼠血清中针对B群和C群Nm的杀菌抗体,评价该重组蛋白作为结合疫苗载体的可行性及该结合疫苗对B群Nm的交叉保护性.方法:扩增fhbp并连接到pET30a(+)表达载体上,转化至大肠杆菌BL21(DE3),异丙基硫代半乳糖苷(IPTG)诱导表达,镍亲和层析柱纯化rfHBP,CNBr活化法将rfHBP与GCMP共价结合,制备结合疫苗,免疫小鼠,间接ELISA法检测血清中GCMP抗体和rfHBP抗体滴度,TTC法检测结合物诱导的分别针对B群和C群Nm的杀菌抗体水平.结果:成功扩增fhbp并连接至pET30a(+)表达载体上,大肠杆菌中诱导表达rfHBP,纯化后与GCMP结合.结合物免疫小鼠后,检测到的针对GCMP的IgG抗体滴度与GCMP组比较有明显加强效应；三针免疫后,针对C群Nm的杀菌抗体滴度高于GCMP对照组,有统计学意义(P＜0.05)；针对B群Nm的杀菌抗体滴度与rfHBP对照组间无统计学意义(P＞0.05)；两组结合物诱导的分别针对B群Nm CMCC29361株和ATCC700111株的杀菌抗体滴度间无统计学意义(P＞0.05).结论:结合疫苗免疫小鼠后检测到的针对C群Nm的杀菌抗体高于单独GCMP组,证明该重组蛋白可作为结合疫苗的载体蛋白；且在结合过程中较好地保留了蛋白的免疫原性,对B群Nm有良好的交叉保护作用.