【作者】 黄琴； 李梅； 胡春霞； 李卫芬；

【Author】 HuangQin Li Mei Hu ChunXia Li Weifen(College of Animal Science,Zhejiang University,Hangzhou 310029,China)

【机构】 浙江大学动物科学学院教育部动物分子营养学重点实验室；

【摘要】 嗜水气单胞菌是水产动物常见致病菌,其血清型众多,外膜蛋白是其共同的保护性抗原.根据已发表的嗜水气单胞菌外膜蛋白基因(omp)的核苷酸序列设计引物,扩增出嗜水气单胞菌 AS1.927 主要外膜蛋白的全长基因片段(Momp).将此基因片段插入表达载体 pNZ8048构建重组质粒,转化乳酸乳球菌(Lactococcus lactis)NZ9000,制备表达嗜水气单胞菌主要外膜蛋白乳酸乳球菌活菌疫苗,并将该活菌疫苗口服免疫 BALB/c 小鼠。重组基因工程菌培养液上清的 SDS-PAGE 结果表明,经 Nisin 诱导后,主要外膜蛋白在乳酸乳球菌(Lactococcus lactis)NZ9000获得有效表达. 免疫保护试验表明,免疫小鼠对同源菌株 AS1.927约100倍半数致死量的攻击的免疫保护率为 87.5%。经重组基因工程菌两次免疫后的小鼠血清中 IgG 水平显著高于对照组(P<0.01),说明分泌表达主要外膜蛋白的乳酸乳球菌能使小鼠产生有效的体液免疫反应.本试验结果提示,重组乳酸乳球菌能够有效地将外膜蛋白分泌到胞外并具有较好的免疫保护效果,这为解决抗嗜水气单胞菌引起的感染提供思路和嗜水气单胞菌外膜蛋白基因工程口服疫苗载体的产业化应用奠定基础.更多还原

【Abstract】 Aeromonas hydrophila is an ubiquitous pathogene in which there are many kinds of serotype and outer membrane is the common protective antigene.A pairs of primers were designed according to the published nucleotide sequence of a putative outer membrane protein gene(omp)ofAeromonase hydrophila. With the specific primers,a target fragment of the major outer membrane protein ofAeromonas hydrophila was amplified from A.hydrophila AS1.927.The recombinant plasmid was constructed and transformed into Lactococcus lactis NZ9000.SDS-PAGE demonstrated that the major outer membrane protein gene was expressed in Lactococcus lactis NZ9000 successfully.The recombinant bacteria were utilized to inoculate BALB/c mice orally.At two weeks post-vaccination,the mice were challenged by peritoneal injection with the virulent strain Aeromonase hydrophila AS1.927.IgG level in the blood serum of mice was measured by ELISA.Results showed that the genetic engineering live-vector vaccine can significantly increased the IgG level of immuned mice when compared with the control(P<0.01)and increase relative percent survival.This suggested that the engineering strain can protect mice efficiently against A. hydrophila AS1.927.The present study will provide technical support for developing an advanced gene engineering vaccine against Aeromonase hydrophila.更多还原