重组登革膜泡疫苗的构建及其免疫效能研究

Dengue diseases are transmitted to humans through the bites of the dengue viruses (DVs) infected mosquitoes, which are widely prevailing in tropical and subtropical areas. Approximately 50 million to 100 million cases of dengue fever occur annually in the world, making DV an increasingly important global health concern. Unfortunately, specific treatment measures and effective vaccines for dengue diseases are still lacking. The development of new-type of dengue vaccines, especially those effective against all serotypes of DVs, increasingly attracts the attention of biological scientists and pharmacologists. Bacterial membrane vesicles (MVs) are spherical, bilayered, nanometer-grade, membranous structures released naturally from the surfaces of bacteria during their growth. MVs can encapsulate or be embedded by specific antigens derived from bacteria to form the "virion-like" particles, which have good immunogenicities and make MVs the promising vaccine candidates. In the present proposal, the major antigen molecules that secreted by MVs secretion pathway of Gram-positive Staphylococcus aureus will be determined, and the sequence of a degenerated dengue protective peptide (DV-EDIII) will be inserted into the genes of MVs carrying staphylococcal antigens to construct recombinant bacteria. The recombinant dengue MVs are prepared by culturing of the bacteria, and purified to homogeneity by the designed strategy of affinity chromatography. The protective role of the recombinant dengue MVs will be evaluated in both cell and animal models. The antibody production and cytokine levels in animal sera will be detected post challenged with the prepared MVs, and the neutralization test and lympholeukocyte proliferation experiment will also be performed in vitro to assess the general immunopotency of the recombinant dengue MVs. The success of this proposal will lay down a good foundation for preparation of new generation of dengue vaccines.

登革病毒以蚊虫为媒介进行传播，广泛流行于热带与亚热带地区，每年有上亿人感染，严重危害人类健康。目前，对登革病毒性疾病尚缺乏特异的治疗方法，也无有效的疫苗上市，故借鉴现有的登革疫苗研究经验，设计新型的登革疫苗具有重要的现实意义。细菌膜泡是细菌在生长过程中自然向外分泌的纳米级膜性结构,可包裹或镶嵌细菌分泌的特定抗原成分，形成"病毒体"样结构，有良好的免疫原性，是一种极为理想的候选疫苗。本研究在鉴定金葡菌膜泡携带主要抗原分子的基础上，拟将自行设计的简并登革保护肽编码序列插入金葡菌膜泡携带主要靶抗原基因中，构建重组登革膜泡产生工程菌，利用金葡菌的膜泡分泌机制，制备登革膜泡；设计亲合层析等纯化方案对重组膜泡进行纯化；再用重组膜泡进行动物实验，检测抗体产生及细胞因子水平，在体观察膜泡对动物的保护能力，结合体外中和试验和淋巴细胞增殖实验评价重组登革膜泡的免疫效能，为制备新型重组登革膜泡疫苗奠定基础。

登革病毒以蚊虫为媒介进行传播，广泛流行于热带与亚热带地区，每年有上亿人感染，严重危害人类健康。目前，对登革病毒性疾病尚缺乏特异的治疗方法，也无有效的疫苗上市，故借鉴现有的登革疫苗研究经验，设计新型的登革疫苗具有重要的现实意义。细菌膜泡是细菌在生长过程中自然向外分泌的纳米级膜性结构,可包裹或镶嵌细菌分泌的特定抗原成分，形成"病毒体"样结构，有良好的免疫原性，是一种极为理想的候选疫苗。本研究对分离自北京、上海、广州、重庆、沈阳、乌鲁木齐的309株金黄色葡萄球菌进行了分子分型研究，筛选了毒力较弱的菌株进行细菌膜泡的制备，发现弱毒株膜泡携带的主要抗原分子条带与RN4220菌株相似，采用质谱技术对其中的5条蛋白条带进行鉴定，证实一37kDa的蛋白为pdhB基因编码的丙酮酸脱氢酶－β亚单位。以pdhB为靶标，采用同源重组技术，将登革病毒简并EDIII 编码序列插入pdhB的3’端，获得重组菌；采用Western-blot技术，在菌体和细菌膜泡水平都能检测到EDIII蛋白的表达。然而，利用重组金葡菌制备的膜泡对小鼠有一定毒力，故又对重组菌调控毒力表达的agr基因进行了基因敲除，发现敲除后菌株分泌的膜泡毒力大大降低。确定了重组登革热膜泡的制备工艺，产量约为23mg/L。用所制备的膜泡免疫动物，能产生效价为1:15000的抗登革病毒EDIII抗体，该抗体1:320稀释后仍对DENV-2感染的细胞具有75%的保护率。这些研究结果为后续制备新型重组登革热膜泡疫苗奠定了坚实基础。发表论著3篇，其中SCI收录期刊论文2篇，参加全国、地区性学术会议论文3篇，培养硕士研究生2名。