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为构建肺炎链球菌减毒活疫苗SPY1矿化菌,并进一步探究其自身稳定性、热稳定性等生物学特性的改变,本研究以肺炎链球菌D39基因组DNA为模板设计合成构建突变体lyt A基因所需引物,采用插入失活的方法构建SPY1-△lyt A突变株,并以缺陷菌的生长特性以及PCR的方法进行鉴定;将SPY1-△lyt A突变株于富钙环境中培养,用不同浓度的磷酸盐进行滴定,使其形成磷酸钙矿化壳,通过扫描电镜以及直接荧光反应来检测疫苗菌株矿化情况;并将矿化菌株与未矿化菌株同时置于37℃不同时间后通过比较其存活率来评价矿化菌的热稳定性。PCR结果以及细菌长时间不溶解的生长特性表明成功构建了SPY1-△lyt A突变株;扫描电镜以及直接荧光反应结果显示细菌表面形成Ca Pi矿化外壳,并且SPY1-△lyt A-Ca Pi矿化菌具有较好的热稳定性。本研究为肺炎链球菌活菌疫苗的进一步研发奠定了基础。
In order to construct the SPY1 mineralized bacterium of Streptococcus pneumoniae attenuated live vaccine and to further explore the biological characteristics of its own stability, thermostability and so on. In this study, Streptococcus pneumoniae D39 genomic DNA was used as a template to construct the mutant lyt A SPY1- △ lyt A mutant was constructed by insertion inactivation method and identified by the growth characteristics of the defective bacteria and PCR method. The SPY1- △ lyt A mutant strain was cultured in a calcium-rich environment with Different concentrations of phosphate were titrated to form calcium phosphate mineralized shell, and the mineralization of the vaccine strain was detected by scanning electron microscopy and direct fluorescence reaction. At the same time, the mineralized strains and non-mineralized strains were incubated at 37 ℃ for different time The thermal stability of mineralized bacteria was evaluated by comparing their survival rates. PCR results and long-time bacterial undissolved growth characteristics showed that the SPY1-Δ lyt A mutant was successfully constructed. Scanning electron microscopy and direct fluorescence reaction showed that Ca Pi mineralized shell was formed on the surface of bacteria, and SPY1- △ lyt A-Ca Pi mine Bacteria have good thermal stability. This study laid the foundation for the further development of Streptococcus pneumoniae viable vaccine.