【摘要】 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。更多还原

【Abstract】 To construct a live attenuated Streptococcus pneumoniae vaccine with a Ca Pi shell, and further to explore its self-stability and thermal stability, the gene of lyt A-ins was amplified by PCR with specific primers from S. pn D39 and then cloned into the expression vector p EVP3, the recombinant p EVP3-lyt A-ins was transformed into SPY1 to construct SPY1-△lyt A mutant strain; then the SPY1-△lyt A mutant strain was cultured in the calcium-rich environment, and then titrated with different concentrations of phosphate, coated itself with a layer of Ca Pi mineralization shell; scanning electron microscopy and direct fluorescent reaction was used to detecte the Ca Pi shell; the thermal stability of mineralized and non-mineralized strains was assessed by calculated the survival rate of these strains while placed them at 37℃ in different times. PCR results and the growth characteristics of the SPY1- △lyt A mutant strain showed that the mutant strain was successfully constructed;Scanning electron microscopy and direct fluorescent reaction showed the surface of the mutant strain was mineralized with Ca Pi; and the survival rate showed that the mineralized strains had well thermal stability than non-mineralized strains. This study would provide theoretical and experimental basis for further development of highly efficient and stable Streptococcus pneumoniae vaccines.更多还原