调控内切酶表达重塑猪霍乱沙门氏菌载体免疫特性的研究

To develop the new technologies of improving immune response in Salmonella Choleraesuis vector, the advantage of the regulating arabinose promoter technology in Salmonella virulence genes and ACA-specific endoribonuclease MazF would be combined. First, the regulated delayed attenuated system (RDAS) derived from the regulating arabinose promoters technology will be introduced into Salmonella Choleraesuis chromosome by the methods of gene clone and suicide vector for homologous recombination, to obtain the balance of attenuation and immunogenecity when Salmonella Choleraesuis immunize the piglet; Second, the MazEF system will be cloned fron E.coli and connected with the genes of regulating arabinose promoter, araC PBAD, and introduced into Salmonella Choleraesuis with RDAS, then the expression of MazF would be indirectly regulated by arabinose in Salmonella Choleraesuis, which should synthesis higher concentration of the heterologous antigen, and decreased Salmonella Choleraesuis protein. The Salmonella Choleraesuis strain with RDAS and MazF will produce more and more heterologous antigens and present a long lasting stimulation of the immune system regulted by arabinose. After that, the strains will programmed die to release antigens into the extracellular environment of the host lymphoid tissue as opposed to being sequestered in the bacterial cytoplasm, and obtained significantly enhance immune response and balanced humoral and cellular immune responses. The Cap protein of PCV2 will be cloned and selected to become the heterologous antigen delivered by the Salmonella Choleraesuis strain. The feature of immune response stimulated by the Salmonella Choleraesuis strain with new system carrying Cap protein will be studied in weaned pigs by flow cytometry，cytokine detection and quantitative PCR to explore the immune reponse mechanism from Salmonella Choleraesuis vector with regulating endoribonuclease expression.

本项目拟将MazF特异性酶切ACA-序列特性与阿拉伯糖启动子调控技术相结合，以重塑猪霍乱沙门氏菌载体的免疫特性，提高载体诱导优化的免疫应答。通过基因克隆和自杀载体同源重组技术，在猪霍乱沙门氏菌中引入源于阿拉伯糖启动子调控的延迟减毒系统，使载体菌获得平衡的减毒和免疫原性；从大肠杆菌中克隆MazEF系统，与阿拉伯糖启动子串联，插入猪霍乱沙门氏菌染色体，由阿拉伯糖调控MazF内切酶的表达，进而调控载体菌合成更高浓度的外源抗原，相对降低载体背景蛋白，调控载体菌持续刺激宿主免疫应答的时间，最后程序性死亡释放抗原到宿主淋巴组织外环境，获得显著提升的免疫应答和均衡的体液和细胞免疫应答特性；以PCV2核衣壳蛋白Cap作为猪霍乱沙门氏菌递送的外源抗原模型。通过流式细胞术、细胞因子检测和定量PCR等，在断奶仔猪中检测猪霍乱沙门氏菌载体诱导免疫应答，探讨调控MazF内切酶表达提升猪霍乱沙门氏菌载体免疫效力的机制

沙门氏菌对宿主具备较强的侵袭性，其表达的异源蛋白可对宿主持续刺激，诱导良好的黏膜免疫、细胞免疫和体液免疫应答，因此减毒沙门氏菌作为疫苗载体有着无比的优越性，但是存在绝对的安全性和较高的免疫原性之间的矛盾，递送病毒抗原蛋白表达低效率以及载体菌排毒环境安全的问题。本研究通过阿拉伯糖启动子调控技术对强毒株猪霍乱沙门氏菌的染色体进行修饰，创建了延迟减毒系统、延迟表达外源抗原系统和mazEF延迟裂解系统，构建了安全高免疫原性，并显著提高外源抗原蛋白表达的猪霍乱沙门氏菌载体。研究结果表明，随着阿拉伯糖的减少，载体菌逐渐减毒，外源抗原表达量增加，载体菌呈现程序性死亡，同时诱导强大的黏膜免疫，细胞免疫和体液免疫应答，并提供猪圆环病毒攻毒很好的保护。本研究是国内外首次将阿拉伯糖启动子调控基因技术与mazEF系统中mazF核糖核酸内切酶特异性酶切特性相结合进行重塑猪霍乱沙门菌载体免疫特性的研究，为开发安全高效猪霍乱沙门氏菌载体递送猪源病原的多价疫苗打下坚实基础。