【作者】 董丽娜；

【导师】 胡桂学；

【作者基本信息】 吉林农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 猪流行性腹泻(porcine epidemic diarrhea,PED)是由冠状病毒科、冠状病毒属的猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)引起,猪的一种以腹泻、呕吐和脱水为主要临床特征的高度接触性传染病。PED给世界各国养猪业均带来极大的危害,目前主要采用灭活或弱毒疫苗预防本病,但免疫保护效果不理想,因此,开展PED的基因工程疫苗研究有广阔应用前景。PEDV S糖蛋白是诱导机体产生中和抗体和提供免疫保护作用的主要结构蛋白。PEDV的部分保护性抗原基因(即COE基因)编码的部分S糖蛋白具有抗原性,能够诱导机体产生中和抗体。实践证明,血清IgG、IgM对PED的免疫保护效果不理想,肠道黏膜免疫所产生的分泌型抗体(sIgA)是抵抗PEDV感染的有效抗体。因此,选择安全无毒,能够在肠道中存活并能表达和传递抗原物质的载体系统,有效地刺激黏膜免疫系统产生黏膜免疫应答对本病的防治具有重要意义。本试验选择乳酸乳球菌NZ3900作为受体菌表达外源基因COE,以期获得PEDV口服活菌疫苗,依靠乳酸菌天然的抗菌、抗腹泻作用和S蛋白刺激的特异性黏膜免疫作用,达到预防PED的目的。乳酸乳球菌NZ3900缺失乳糖操纵基因(lacF基因),与含有lacF基因的乳酸乳球菌表达载体pNZ8149联合使用,以乳糖为选择标记,是一个食品级的外源基因表达系统。本研究用疑似患PED的病猪肠病料,进行RT-PCR扩增,获得531bp的PEDV部分保护性抗原基因,将其克隆入pMD18-T载体后测序,核苷酸序列同PEDV CV777株相应序列的同源性为99.4%。根据测序结果和表达载体特点,设计一对引物,扩增PEDV部分保护性抗原基因(COE基因)501bp。将COE基因与乳酸乳球菌表面表达载体pNZ8149进行连接,电击转化入食品级乳酸乳球菌NZ3900细胞。重组菌以1ng/mL乳链菌肽(Nisin)诱导,通过SDS-PAGE和Western blot分析,PEDV部分S蛋白获得了表达,并具有反应原性。间接免疫荧光实验表明,重组菌表达蛋白定位于菌体细胞表面。试验结果表明,成功的构建了以乳糖为选择标记的食品级表达PEDV部分S蛋白的乳酸菌,重组菌表达蛋白定位于菌体细胞表面并且具有反应原性。为研制预防PED的基因工程乳酸菌口服疫苗奠定了基础。更多还原

【Abstract】 Porcine epidemic diarrhea virus(PEDV) belong to Coronviridae,Coronavirus.PEDV is the etiological of porcine epidemic diarrhea(PED),which is a highly contagious enteric disease in pigs causing vomit, diarrhea,dehydration.For the tremendous economical loss and weak immunoprotection effect of inactivated vaccine,the research on genetically engineering vaccine of PEDV has a wide implemented perspective.The spike(S) glycoprotein of PEDV is the predominant inducer of neutralization antibody.The partial S glycoprotein of PEDV encoded by COE gene also is the inducer of neutralization antibody and has the antigenicity against PEDV.It has been identified that,the immunoprotection effect of IgG,IgM in blood serum wasn’t good,but the antibody(sIgA) induced by enter-membrane immune was effective.For the above,select a carrier system that safe,could multiply in intestine is important to the protecting of PED.In this study,Lactococcus lactis NZ3900 was selected as receptor strain to express COE gene,which combined antibiosis function with specific immunity together.The aim was to lay foundation for developing the gene engineering Lactococcus lactis oral vaccine of PEDV.Lactococcus lactis NZ3900 with a deletion of lacF gene,combined with expression vector pNZ8149 that carrying lacF gene,use lactose as a selective marker,is a real eatable exogenous gene expression system.In this study,the partial S glycoprotein gene of porcine epidemic diarrhea virus(PEDV) was amplified by RT-PCR and sequenced.The gene consisted of 531bp and shared 99.4%nucleotide homology with PEDV CV777 strain.The COE gene(about 501 bp) in S glycoprotein gene was subcloned into the Lactococcus lactis vectors pNZ8149 and transformed into Lactococcus lactis NZ3900 by electroporation.The recombinant protein was detected by Western blot and IFA experiments after the bacteria induced by 1ng/mL nisin.The result indicated that the molecular weight of the expressed recombinant protein was about 20 kDa;the protein had the reactionogenicity with antibody against PEDV as expected;the protein was secreted and located on the surface of the bacteria.The result showed that we successfully constructed the epression rcombinants for PEDV COE gene in Lactococcus Lactis.Furthermore,the recombinant protein was located on the surface of the bacteria and had the reactionogenicity with antibody against PEDV.The study is expected to lay the foundation studies on the gene engineering Lactococcus lactis oral vaccine in their prevention of PED.更多还原