论文部分内容阅读
目的:以重组B群脑膜炎奈瑟菌(Nesseria meningitidis,Nm)外膜蛋白fHBP(recombinant factorH 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rfH-BP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。
OBJECTIVE: To prepare a conjugate vaccine by covalently binding to group C meningococcal polysaccharide (GCMP) with recombinant factor H binding protein (fHBP) of recombinant Neisseria meningitidis group B The mice were immunized and the bactericidal antibodies to B and C Nm in the sera of the mice were tested to evaluate the feasibility of the recombinant protein as a carrier for the combination vaccine and the cross protection of the combination vaccine against B group Nm. Methods: fhbp was amplified and ligated into pET30a (+) vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by isopropylthiogalactoside (IPTG). Purified rfHBP and CNBr were activated by nickel affinity chromatography The rFH-BP and GCMP were covalently combined to prepare the conjugate vaccine, and the mice were immunized. The antibody titer of GCMP and rfHBP in serum were detected by indirect ELISA. The TTC method was used to detect the bactericidal activity of the conjugate against Bm and C groups respectively Antibody level. Results: The fhbp was successfully amplified and ligated into pET30a (+) expression vector. The rfHBP was induced in E. coli and was purified and combined with GCMP. After immunization, the titer of IgG antibody against GCMP was significantly enhanced compared with that of GCMP group. After three-needle immunization, the titer of bactericidal antibody against C group Nm was higher than that of GCMP control group (P <0.05). The titer of bactericidal antibody against Nm of group B was not significantly different from that of rfHBP control group (P> 0.05). The bactericidal antibody drops against the group B Nm CMCC29361 and ATCC700111 respectively There was no significant difference between degrees (P> 0.05). CONCLUSIONS: The bactericidal antibodies to C group Nm detected after immunization with the vaccine were higher than the GCMP alone group, demonstrating that the recombinant protein can serve as a carrier protein for the conjugate vaccine and retains the protein immunogen well during the binding Sex, B group Nm have a good cross-protection.