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为构建维氏气单胞菌OmpAⅠ基因重组植物乳杆菌并检测其表达产物的免疫原性,将目的基因克隆至大肠杆菌-乳杆菌穿梭表达载体p SIP409中,并电转至植物乳杆菌中,经诱导后对表达产物进行Western-blot分析。结果显示,LP/p SIP409-OmpAⅠ重组植物乳杆菌出现一条约42 ku的印迹,与预期大小相符,表明重组蛋白具有反应原性。LP/p SIP409-OmpAⅠ重组植物乳杆菌免疫鲫鱼后,在第25天血清中Ig M水平达到高峰;RT-PCR检测发现免疫后鲫鱼肝、脾、肾及心组织中的细胞因子IL-8、IFN-γ与IL-1β表达水平均有不同程度提升;攻毒后LP/p SIP409-OmpAⅠ免疫组的保护率在65%以上,高于对照组的存活率。结果表明,应用重组植物乳杆菌LP/p SIP409-OmpAⅠ免疫鲫鱼能刺激机体产生免疫应答反应,为预防鱼类维氏气单胞菌感染的重组植物乳杆菌口服制剂的研发奠定了基础。
To construct the Lactobacillus plantarum OmpAⅠ gene recombinant Lactobacillus plantarum and to detect the immunogenicity of the expressed product, the target gene was cloned into the E. coli-Lactobacillus shuttle expression vector p SIP409 and electroporated into Lactobacillus plantarum, After induction, the expression products were analyzed by Western-blot. The results showed that an approximate 42 ku blot of LP / p SIP409-OmpA I recombinant Lactobacillus plantarum, in line with the expected size, showed that the recombinant protein was reactive. Serum IgM levels peaked on day 25 after LP / p SIP409-OmpAI recombinant Lactobacillus plantarum was immunized. RT-PCR results showed that cytokines IL-8 in liver, spleen, The levels of IFN-γ and IL-1βwere increased to some extent. The protection rate of LP / p SIP409-OmpAⅠimmunohistochemistry group was above 65% after challenge, which was higher than the control group. The results showed that the immunization of crucian carp with Lactobacillus plantarum LP / p SIP409-OmpAⅠ could stimulate the immune response and lay a foundation for the research and development of recombinant Lactobacillus plantarum oral preparations for prevention of Aeromonas sobria infection.