摘要
为使囊素肽(Bursin,BS)更好地发挥免疫增强作用,本研究采用EDC/NHS反应,使透明质酸(Hyaluronic acid,HA)的羧基与囊素肽的氨基发生酰胺化反应,合成囊素肽接枝透明质酸聚合物(BS@HA),进一步通过傅里叶变换红外光谱鉴定聚合物的结构,通过紫外分光光度计进行定量分析,然后以卵清蛋白OVA为模式抗原,BS@HA作为免疫增强剂添加至铝胶做成疫苗,免疫小鼠,利用ELISA和流式细胞仪评价BS@HA的免疫增强作用.结果表明:1)当透明质酸、EDC、NHS和囊素肽的反应摩尔比为1∶4∶6∶0.4,加入EDC后的反应时间为2～4 h,加入NHS后的反应时间为16 h时,合成聚合物的产率可以达到95％,透明质酸的傅里叶红外光谱特征峰从1 605 cm-1红移到1 632 cm-1,囊素肽成功接枝透明质酸.2)当透明质酸羧基取代率为35％左右时,囊素肽接枝透明质酸聚合物可以诱导小鼠产生更高水平的OVA特异性抗体.3)囊素肽接枝透明质酸聚合物能够诱导小鼠产生更高水平的滤泡辅助性T细胞(TFH)和生发中心B细胞(GC B细胞).综上,囊素肽接枝透明质酸聚合物可以使囊素肽更高效地进入引流淋巴结,诱导活化更高水平的TFH和GC B细胞,促进特异性IgG抗体的产生.本研究将丰富透明质酸在动物疫苗领域的理论研究,拓展透明质酸在动物疫苗领域的实际应用,同时为新型免疫增强剂的研制奠定基础.
Abstract
To enhance the immune effect of Bursin(BS),we attempted to graft BS to Hyaluronic acid.The amino groups of BS was utilized for direct coupling with the carboxylic groups on hyaluronic acid to obtain BS@HA by EDC and NHS.The structure of the polymer was identified by Fourier transform infrared spectroscopy(FTIR),quantitative analysis was performed using UV measurement.Then the mice were immunized with the vaccine formulated with simulated antigen OVA,BS@HA and aluminum hydroxide adjuvant and detected the immune enhancing effect by ELISA and flow cytometry.The results showed that:1)The yield of the synthesized polymer could reach 95％when the molar ratio of hyaluronic acid,EDC,NHS,and BS was 1∶4∶6∶0.4,the reaction time was 2-4 hours and 16 hours after adding EDC and NHS respectively.The FTIR characteristic peaks of HA shifting from 1 605 cm-1to 1 632 cm-1 showed BS@HA was successfully prepared.2)BS@HA can induce higher expression of OVA-specific antibodies when the degrees of substitution(DS)was about 35％.3)BS@HA also can induce higher percentages of Follicular helper T cells and germinal center B cells.Therefore,it is spected BS@HA could enhance targeted delivery of immunogens to draining lymph nodes,induce more TFH and GC B cells and evoke high antigen-specific IgG titers.This study will enrich the theoretical research of hyaluronic acid in the field of animal vaccines,expand its practical application in the animal vaccines and lay the foundation for the development of novel immune enhancers.
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