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目的:将淋球菌脂寡糖的2C7表位插入乙型肝炎病毒核心抗原(HBc)的MIR区,通过原核表达系统进行融合蛋白的表达,以期获得高免疫原性的淋球菌亚单位疫苗的候选靶位。方法:通过间接ELISA法,以CMCC29403菌株的LOS为包被抗原,对7个表位进行筛选;并通过杀菌试验检测肽段免疫动物产生血清的保护力。对已筛选好的肽段基因进行人工合成,通过重叠PCR将肽段基因嵌入HBc基因中,以增强表位的免疫原性,并进行原核表达。结果:初步研究表明了表位PEP1,PEP2,PEP7具有较强的脂寡糖(LOS)的免疫原性,能够模拟脂寡糖的抗原性。这3个肽段可能成为候选疫苗亚单位。通过重叠PCR方法:成功地将PEP1,PEP2,PEP7三个表位基因序列插入乙型肝炎病毒核心抗原(HBc)的刺突(MIR)部位,构建了HBc-PEP1,HBc-PEP2,HBc-PEP7融合基因,通过PET22b(+)载体进行原核表达,为下一步疫苗的研究奠定了基础。结论:2C7表位PEP1,PEP2,PEP7具有较强的脂寡糖(LOS)的免疫原性,能够模拟脂寡糖的抗原性。表位与HBc的融合蛋白可以通过原核表达系统进行可溶性表达,为下一步疫苗的研究奠定基础。
OBJECTIVE: To insert the 2C7 epitope of gonococcal lipooligosaccharides into the MIR region of hepatitis B virus (HBc) core antigen and express the fusion protein by prokaryotic expression system in order to obtain high immunogenicity of Neisseria gonorrhoeae subunit vaccine candidates Target. Methods: Seven epitopes were screened by indirect ELISA with LOS of CMCC29403 as the antigen, and the protective effect of the immunized animals on serum was tested by bactericidal test. The screened peptide gene was synthesized, and the peptide gene was inserted into HBc gene by overlap PCR to enhance the immunogenicity of the epitope and prokaryotic expression. Results: Preliminary studies showed that the epitopes PEP1, PEP2 and PEP7 have strong lipogenicity (LOS) immunogenicity and can simulate the antigenicity of lipooligosaccharides. These three peptides may become candidate vaccine subunits. By overlapping PCR, the three epitopes of PEP1, PEP2 and PEP7 were successfully inserted into the spike region (MIR) of HBc and HBc-PEP1, HBc-PEP2 and HBc-PEP7 Fusion gene, prokaryotic expression through PET22b (+) vector, laid the foundation for the next vaccine research. CONCLUSION: The 2C7 epitopes PEP1, PEP2 and PEP7 have strong immunogenicity against lipooligosaccharides (LOS), which can simulate the antigenicity of lipooligosaccharides. The fusion protein of epitope and HBc can be expressed by the prokaryotic expression system, which lays the foundation for further vaccine research.