【作者】 汪淼；

【导师】 李一经；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2009， 硕士

【摘要】 猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)是冠状病毒家庭成员。猪流行性腹泻病毒感染猪的肠道上皮细胞而引起严重腹泻,尤其对于仔猪,感染后死亡率几乎为100%。该病是以发病猪呕吐、腹泻、严重脱水为临床特征的高度接触性肠道传染病。在世界范围内分布,呈地方流行性,尤其在东亚各国流行更为广泛,是各养猪国家导致仔猪早期死亡的重要疫病之一。针对猪流行性腹泻病毒经黏膜感染的特点,研制能有效的刺激黏膜免疫系统产生局部免疫应答,进一步引起全身性系统免疫反应的疫苗,对该病的防治具有重要意义。本研究以乳酸乳球菌Lactococcus lactis NZ9000作为递呈抗原的活菌载体,以猪流行性腹泻病毒免疫保护性抗原S蛋白部分基因片段ps420(499-638aa)为靶基因,构建了表达猪流行性腹泻病毒ps420基因的重组乳酸乳球菌表达系统。以含有猪流行性腹泻病毒ps420基因的质粒pPG612.1-ps420为模板,PCR扩增出约420bp的目的基因ps420,并克隆到pMD18-T simple载体,进行酶切、PCR鉴定,重组质粒命名为pMD18-TS-ps420。重组质粒与表达载体均经限制性内切酶双酶切,回收目的基因片段ps420与表达载体pNZ8112连接,电转化感受态NZ9000,筛选阳性克隆进行酶切、PCR和测序鉴定,重组质粒命名pNZ8112-ps420,重组乳酸乳球菌pNZ8112-ps420/NZ9000。重组菌pNZ8112-ps420在GM17(含氯霉素)培养液中培养,经Nisin诱导目的蛋白的表达。重组菌中蛋白的表达及表达蛋白的定位通过SDS-PAGE、Western blotting和间接免疫荧光进行鉴定。考马斯亮蓝染色结果显示:pNZ8112-ps420的菌体裂解物表达了15KD的融合蛋白,诱导后表达的ps420蛋白经Western-blot分析,可检测到特异性反应条带。将重组菌免疫接种家兔,每只家兔经口灌服1010CFU重组菌,每次连续灌服3d,每次间隔14d,定期采集血清样本进行PEDV细胞中和试验,结果表明,重组菌可有效诱发血清中IgG产生。血清中和抗体效价可达1:90。从而初步证明ps420蛋白具有中和活性,同时也证明了乳酸乳球菌可以作为口服免疫传递抗原的载体。更多还原

【Abstract】 Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae, was an intestinal pathogen of infectious disease of porcine which characterized by severe enteritis with anorexia, vomiting, acute diarrhea dehydration and significant mortality in swine. Histologically, the viruse cause destruction of villous enterocytes and villous atrophy within the jejunum and ileum. Morbidity and mortality in infected neonatal piglets less than 5 days old approach 100% because of severe diarrhea and dehydration. Porcine epidemic diarrhea has been widely distributed in the world, especially in the Eastern Asia and causing pig producers serious economical loss.Lactic acid bacteria (LAB), considered to be safe bacteria with a GRAS (generally regarded as safe) status, possesses many properties that has been thought to be an ideal vaccine which has a good perspective. An oral vaccine of expressing the antigen then induced efficient systemic and mucosal immune responses is very useful resulting in the inhibition of the virus.Lactococcus lactis NZ9000 was selected as an antigen delivery vehicle for the development of live mucosal vaccine. The neutralizing fragment of the spike protein ps420 was selected as the target gene. We constructed recombinant Lactococcus lactis NZ9000 systems expressing ps420. About 420bp gene fragment (ps420) was amplified by PCR using the plasmid pPG612.1-ps420. The products were linked with pMD-18-T Simple vector and digested by restriction enzyme. The target gene was digested by restriction enzyme, linked with expression vector pNZ8112 digested by restriction enzyme, giving rise to pNZ8112-ps420. The recombinant plasmids pNZ8112-ps420 was electroporated into Lactococcus lactis NZ9000 respectively, generating pNZ8112-ps420/NZ9000 followed enzyme digestion, PCR identification and sequence analysis.The recombinant strains pNZ8112-ps420 constructed was induced by Nisin in GM17 medium to express interest protein. The expression and localization of the protein from recombinant strains were detected via SDS-PAGE, Western blotting and indirect immunofluorescence. The lysates of the cells were analyzed by SDS-PAGE. Coomassie blue gel staining showed that 15KD fusion protein was expressed in lysates of pNZ8112-ps420. The localization of the ps420 protein nisin-induced was analyzed via Western blotting. Immunoreactive band (15KD) was detected, and corresponding immunoreactive bands did not display when they were not induced.Rabbits were immunized via oral route with pNZ8112-ps420/NZ9000 to identify whether the recombinant strain have the ability to induce systemic antibody responses. Each rabbit received dose of 1010 colony-forming units (c.f.u.)/mL of recombinant strains. The immune protocol was administered on three consecutive days at days 1,2 and 3. A booster immunization was given at days 15,16 and 17 and a second booster was given at days 29,30 and 31. Serum of rabbits were collected 7 days after every immunization and the results of neutralization test showed that the IgG in serum reach a comparative high level. The gene encoding PEDV ps420 protein could be certificated as a neutralizing antigen domain. All theses work established a good foundation for further study on the new and effective recombinant oral vaccine of porcine epidemic diarrhea.更多还原