海豚链球菌兼职蛋白FBA的克隆表达、抗原性检测及免疫效果评价

为检测斑点叉尾鮰源海豚链球菌兼职蛋白(fructose-1,6-bisphosphate aldolases,FBA)的抗原性和潜在的疫苗价值,本实验克隆得到斑点叉尾鮰源海豚链球菌DX09(基因组登陆号LXQF01)的fba基因序列(基因登录号A7N10_RS06935),对克隆序列进行生物信息学分析,并通过原核表达得到重组FBA蛋白(r FBA),制备了兔抗r FBA血清用于FBA蛋白抗原性检测,同时通过免疫保护实验评估重组蛋白的免疫保护效果。结果显示,海豚链球菌DX09 fba基因有1个882 bp的开放阅读框(ORF),编码293个氨基酸。生物信息学分析显示,其分子式为C_(1378)H_(2172)N_(368)O_(422)S_8,分子质量为30.9 ku,理论等电点为5.01,不具有信号肽和跨膜区域;具有保守的裂解酶结构域,且与其他来源的FBA蛋白同源性达100%;具有较高的抗原指数,表明其可形成多个抗原表位。SDS-PAGE检测发现,诱导表达的重组蛋白以包涵体的形式出现在沉淀中,大小约为47 ku。Western blot分析表明,兔抗r FBA血清能特异性结合菌体蛋白。同时免疫保护实验显示,重组蛋白对斑点叉尾鮰的相对保护率可达55%,免疫后鱼体抗体水平相对对照组显著升高。本研究表明,原核表达的斑点叉尾鮰源海豚链球菌DX09 rFBA具备较好的抗原性和免疫保护作用,具有研发斑点叉尾鮰海豚链球菌亚单位疫苗的潜在价值。

Cloning, prokaryotic expression, antigenicity detection and immunization efficacy of moonlighting protein FBA of Streptococcus iniae

In order to detect the antigenicity of moonlighting protein FBA (fructose-1,6-bisphosphate aldolases), fba gene from Streptococcus iniae DX09 isolated from Ictalurus punctatus was cloned. The related properties of FBA protein were predicted and its immunization efficacy assay was conducted. rFBA protein was obtained by prokaryotic expression systems and purified by Ni-NTA-Sefinose Column. The purified rFBA protein was used to immunize rabbits (Oryctolagus cuniculus) to obtain the polyclonal rabbit anti-rFBA sera for antigenicity detection. The results showed that fba gene had an ORF with 882 bases, encoding 293 amino acids with a C₁₃₇₈H₂₁₇₂N₃₆₈O₄₂₂S₈ formula, 30.9 ku molecular mass, and a 5.01 theoretical isoelectric point. Furthermore, the deduced amino acids comprised phosphorylation sites, not containing the transmembrane domain and signal peptide sequence. The conserved domains, namely aldolase were predicted via NCBI conseverd domains tool. The comparative analysis revealed an exaggerated degree of homology with other S. iniae FBA protein in amino sequences. Additionally, high antigen index of the deduced amino acids was predicted using DNAstar-Protean, which means it can form numerous epitopes. rFBA proteins formed into inclusion bodies were found in the pellet and a band about 47 ku was observed by SDS-PAGE. Moreover, western blot analysis showed that rabbit anti-rFBA sera can combine with the mycoprotein specifically. Immunization efficacy assay suggested that rFBA prevented S. iniae DX09 infecting I. punctatus with a 55% protective rate. In this study, our results showed that rFBA possesses nice antigenicity and optimal immune protection, implying that the FBA protein can be a subunit vaccine candidate against S. iniae in I. punctatus.