干酪乳杆菌作为减蛋综合症口服基因工程疫苗载体关键技术的研究

Egg-drop syndrome (EDS) is now an economic problem for the poultry industry world-wide，A conventional vaccination against EDS was developed.in 1977, consisting of inactivated virus in oil adjuvant.Vaccination with the recombinant proteins provided the production of antibodies that recognized the whole virus，but the neutralizing antibody will become to be low after 50 days.Lactobacillus casei is suitable to be used as vaccine carrier because of its safety and stimulating immunity.We will modify E.coli coli Red system in order to use it to improve the recomdination efficiency of exogenous genes into Lactobacillus casei genome, at the same time, we will construct an suicide recombination carrier in which the expression of mazF gene is under control by LacI protein. The GFP gene is used as selected marker gene by recombining it into lactobacillus casei genome,and then the fusion gene F-LTB which is under control of appropriate promoter and signal peptide is recombined into lactobacillus casei genome too. The recombination of the two gene deponds on the help of both modified E.coli Red system and the suicide recombination carrier.The flourescence microscope is used to identify the expression of GFP, and the stable and continuous secretion expression of the fussion proteins of F-LTB can been identify by Werstern blotting.The animal experiments are performed to analyze and assess the immunogenicity,distribution in intestinal tracts and security of the obtained restructuring lactobacillus casei.The technology platform ,modified E.coli Red system and the suicide type recombination carrier, will help to develop oral feasibility of developing new oral genetic engineering vaccine.

减蛋综合症-76（EDS-76）是使禽产蛋率下降的传染性病毒，研究其口服基因工程疫苗对预防该病有重要意义。本研究在前期克隆并验证EDS-76主要抗原基因的基础上开展干酪乳杆菌作为口服疫苗载体的研究。方法：对E.coli Red重组系统进行改造使其能用于干酪乳杆菌的基因修饰，提高外源基因在基因组中的重组效率；构建自杀型重组载体以提高无抗性标记重组菌株的筛选效率；用所开发的载体将GFP基因整合到干酪乳杆菌基因组作为基因标记，再将构建的融合基因（启动子+信号肽+EDS-76的抗原基因+LTB）整合到干酪乳酸菌基因组中，并使融合基因编码的蛋白能稳定而持续的分泌表达。对所获得重组干酪乳杆菌分别在裸鼠与SPF鸡中进行免疫原性、肠植能力以及安全性评价，检验其作为口服疫苗的可行性，为开发口服、持续免疫、安全性高的新型疫苗打下基础。所改造的重组系统与自杀重组载体还可用于其它疫苗的开发与研究。

减蛋综合症-76（EDS-76）是使禽产蛋率下降的传染性病毒，研究其口服基因工程疫苗对预防该病有重要意义。本研究在前期克隆并验证EDS-76主要抗原基因的基础上开展干酪乳杆菌作为口服疫苗载体的研究。方法：制备干酪乳杆菌的原生质体，以提高外源基因的转化效率；构建自杀型重组载体以提高无抗性标记重组菌株的筛选效率；用所开发的载体将mCherry基因整合到干酪乳杆菌基因组作为基因标记，再将构建的融合基因（启动子+信号肽+EDS-76的抗原基因+LTB）整合到干酪乳酸菌基因组中，验证融合基因分泌表达。并将重组干酪乳杆菌分别在裸鼠中进行免疫原性、肠植能力以及安全性评价，检验其作为口服疫苗的可行性，结果表明，整合效率在3－5%，所整合的外源基因mCherry与EDS－76的F蛋白均能有效分泌表达，在裸鼠中能有效激活体液免疫与细胞免疫。表明这一系统有开发成口服疫苗的价值。