【作者】 丛彦龙；

【导师】 何昭阳； 王春凤；

【作者基本信息】 吉林农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 猪轮状病毒(Porcine Rotavirus，PRV)属于呼肠孤病毒科、轮状病毒属，它作为腹泻的主要病原在世界范围内广泛流行，是引起仔猪病毒性腹泻的主要病原之一。该病幼畜最为易感，1-4周龄仔猪发病率超过80％，死亡率7-20％，且症状严重，主要表现为严重腹泻，部分病例因严重脱水、酸碱平衡失调、继发感染而死亡[91]。鉴于其危害严重且无有效治疗手段，世界卫生组织将RV疫苗列为最优先发展的疫苗项目之一，尤其是它的基因工程疫苗的研制更为重要。 动物机体存在着完整的粘膜免疫系统，该系统包括肠道相关的淋巴组织、呼吸道淋巴组织及乳腺、泌尿生殖道粘膜。肠道壁是机体的最大免疫器官，其粘膜产生的抗体远远大于体液。加之粘膜又是大多数病原的入口。因而，有人预测：未来的疫苗将主要发展成为经肠道免疫的疫苗。研究证实，小肠IgA为抗RV保护作用的相关因素，对于RV疫苗的研究者来说，最重要也是最富有挑战性的任务是构建一种能在肠粘膜表面诱导产生病毒特异性IgA的疫苗。RV组特异性抗原VP6蛋白，位于病毒的内壳，全长基因为1356bp，占病毒颗粒的51％，它在病毒的复制装配过程中起着重要作用，被认为是RV产生免疫的最重要蛋白，其虽不能刺激机体产生中和抗体，但可以刺激粘膜分泌sIgA，从而介导粘膜免疫，抵抗再次感染。该蛋白也具有一个能与细胞毒性T淋巴细胞(CTL)产生交叉反应的抗原表位，同时VP6为RV组和亚组特异性抗原，无种属特异性，在疫苗中纳入该抗原可能有助于解决病毒变异问题。 RV主要保护性抗原基因已经被克隆和测序，但迄今为止报道的表达载体系统，多是以抗生素抗性作为外源基因稳定表达的压力。以抗生素为选择压力，虽然对克隆子容易进行筛选，但是由于抗药性基因不断向环境中漂移扩散，可能造成对环境微生态的破坏，带来严重后果，因而这类表达系统也离“食品级”还有很大的距离，不能直接用于人和动物。非抗性表达载体系统日益受到人们的关注。 由于重组保护性抗原受体菌株主要是活的减毒病原菌，而细菌疫苗的免疫保护力往往和菌株的残余毒力成正比。所以试图寻找能良好地表达外源性抗原且安全的受体菌株日益受到人们的关注。乳酸杆菌为人及动物体内正常菌群的重要成员，以乳酸杆菌作为表达外源保护性抗原基因的受体菌株，就可以将乳酸菌的生物学功能和外源功能抗原基因的特异性免疫相结合，同时乳酸杆菌作为正常生理性细菌，又符合新型疫苗的口服安全、方便、廉价等特点。吉林农业大学硕士学位论文猪A组轮状病毒VP6基因的克隆与表达 不难想象，如果将以非杭性载体表达系统基因为选择压力的“食品级”和“饲料级”系统构建成功，就可以把RV的保护性抗原基因克隆入这种载体中，再导入乳酸菌受体菌中，得到的这种重组工程乳酸杆菌将集益生菌和Rv基因产物的功能于一体，其科学意义和应用前景将十分广阔。这类疫苗的研制成功将为Rv疫苗的发展探索一条新路。 本研究用恒河猴胚肾细胞(MA一104细胞系)增殖并扩增了PRv。根据Genebank已发表的PRv vP6基因cDNA序列，利用Prime:5.0软件设计并合成了一对引物，通过反转录聚合酶链式反应(RT一PCR)技术，从PRV的总RNA中扩增出长1 1 72bp的vP6基因片段。通过T4 DNA连接酶将其直接连于克隆载体pGEM一T一easy中，构建了重组质粒PGEM一T一P Rv6，再转化至受体菌JMI 09中。然后进行质粒提取并通过酶切、PCR鉴定、序列测定，证明重组质粒中pT一P RV6中含有轮状病毒vP6基因。经氨基酸序列分析，表明克隆了轮状病毒的主要保护性杭原vP6全基因的杭原表位区。 进一步将上述VP6基因克隆至PW425t的Sacl和KPnl之间的多克隆位点上，该载体是以ThyA基因[”，96]为选择压力的非杭性的可在大肠杆菌和乳酸菌之间穿梭表达的载体，将重组表达载体命名为pw425t一PRv6。经转化、鉴定，将阳性克隆pw425t一PRv6在ThyA基因缺陷的大肠杆菌中进行表达。通过SDS一聚丙烯酞胺凝胶电泳(SDS一PAGE)分析，表明vP6基因在大肠杆菌中得到了表达。we 5 t ern一blot检测，表明其具有免疫活性。从而为下一步在乳酸菌中表达莫定基础。此技术平台为PRV的免疫防制提供新的手段和可能，也为各种外源和内源的功能基因通过此载体在肠道共生乳酸菌中表达而更好地发挥转基因工程乳酸菌的双重功能开创了一条崭新的道路。更多还原

【Abstract】 Porcine Rotavirus (PRV) belongs to the family reoviridae, species rotavirus. It is one of the major pathogens that cause life-threatening diarrhea in piglets. The young animals are the most easily infected. Its infectious ratio is over 80%, and its death rate is 7-20% in 1-4 weekage pigs. Rotavirus diarrhea’s typically clinical manifestation is serious diarrhea. Partial infected piglets were dead due to severe loss of water, imbalance of acid alkali and secondary infection. Accurate diagnosis and effective vaccination are main means for the prevention of porcine diarrhea. World Health Organization (WHO)gives priority to the development of rotavirus vaccine, especially its engineering vaccine.The mucosal surfaces represent a critical component of the mammalian immunologic repertoire. The immunologic network operating on external mucosal surfaces in animal body consists of gut-associated lymphoid tissue (GALT), the lymphoid structures associated with bronchoepimelium and lower respiratory tract (BALT), occular tissue, upper airway, salivary glands, tonsils and nasopharynx (NALT), larynx (LALT), middle ear cavity, male and female genital tracts, mammary glands, and the products of lactation.The mucosal surfaces of the gastrointestinal tracts represent the principal portals of entry for most pathogens. Therefore, the use of natural or induced infections and mucosal immunization with specific vaccine antigens continue to remain an attractive possibility for immunization against infections, especially those acquired through mucosal surfaces. The major antibody isotype in external secretions is secretory immunoglobulin A (slgA). Induction of IgA response provides specific protection against many respiratory, enteric, and genital infections.It has been illustrated that IgA is an associated factor to resist rotavirus. For those researchers studying rotavirus vaccine, the most important and most challenging task is to construct a kind of vaccine which can induce the intestinal mucosal surface to secrete IgA. Research shows that the inner capsid VP6 takes amount to 51% of virus particle, whose whole length is 1356 bp. It places a most important role in virus duplication and assembling. Though VP6can’t induce neutralization antibody, it can induce IgA and thus induce mucosal immunity. There is also an antigenic epitope in VP6, which can induce crossing reaction with CTL. At the same time, it has no specificity between species and genus, so it is possible to solve the problem of virus’ mutation by using VP6 protein as a major antigen.At present, most protective antigenic genes of rotavirus had been cloned and sequenced. But the vectors that have been reported mostly use antibiotics as selective pressure. It becomes important to construct non-antibiotic expression vector systems instead of antibiotic vector systems owing to the spreading problem of resistant genes in the latter ones. Lactobacilli are important members of normal bacteria community in guts of human beings and animals. The use of live microorganisms as an antigen delivery system is an effective means to elicit local immune responses and thus represents a promising strategy for mucosal vaccination. In this respect, Lactic acid bacteria represent an original and attractive approach, as they are safe organisms that are used as food starters and probiotics. Since Lactobacillus is an enteric organism, it colonizes and penetrates the intestinal mucosal following oral administration. The organism has the advantage of being taken up by the intestinal M cells. It should be possible to develop for human or animal use effective, nonliving, recombinant, replicating, transgenic, and microbial vector- or plant-based mucosal vaccines to prevent infections. Mucosal immunity offers new strategies to induce protective immune responses against a variety of infectious agents. Lactobacillus strains which are able to persist in the intestinal tract for several days after administration may be particularly interesting for the mucosal presentation of antigens. It is not difficul更多还原