【作者】 周俊芳；

【导师】 华修国；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2007， 博士

【摘要】 猪传染性胃肠炎病毒(Porcine Transmissible Gastroenteritis virus, TGEV)属冠状病毒科冠状病毒属成员,可导致猪传染性胃肠炎(Porcine Transmissible gastroenteritis, TGE)。该病是猪的一种急性、高度传染性胃肠道疾病,常在冬春季节呈暴发性流行,病猪以呕吐、水样腹泻、严重脱水为临床特征。不同年龄和品种的猪对本病均易感,其中两周龄以内的仔猪死亡率可达l00%,死亡原因通常为脱水和电解质紊乱。随着年龄的增大,死亡率会逐渐下降,但是耐过猪生长发育迟缓,饲料报酬率降低。本病发病迅速,危害严重,波及范围广,呈世界范围分布,大多数养猪国家都有本病发生,给世界养猪业造成巨大的经济损失。我国自从50年代首次报道TGE以来,部分地区仍有该病发生和流行,甚至在局部地区暴发。针对TGE,常规药物没有明显治疗效果,目前常用的治疗措施是疫苗接种。但是广泛使用的常规疫苗本身存在着各种各样的缺陷:灭活苗制作成本高而保护能力低;弱毒苗存在散毒和返强的危险,而且干扰田间野毒感染的诊断;而作为替代的基因工程疫苗、核酸疫苗等因生产成本及免疫保护能力等问题大多尚处于实验室阶段,还有较长的一段路要走。所有这些均成为有效防治TGE、发展养猪业的瓶颈。继续研制新型、安全和更有效的TGEV疫苗或其他防治措施为必然之势。RNAi是生物体自身固有的抵御外来感染的机制。它通过严格的基因序列互补特异降解靶mRNA。凭借高效特异、经济便捷的优势,RNAi技术已经远远超过了传统的反义基因调控技术,一跃成为哺乳动物体外和体内科学研究的强大工具,在抗癌症、抗病毒感染方面显示出巨大发展潜力。许多疑难疾病和大量已知序列的动植物病毒已经在基因水平被很好抑制。本病病原TGEV,病原学分类明确,一般生物学特性以及基因组结构、功能等研究较为透彻,并且是单股正链RNA病毒,本身即可行使mRNA功能。所以,运用RNAi技术抗TGEV感染有着很好的技术背景和生物学基础。综上,本研究考虑运用特异高效、技术日趋成熟而成本日益下降的RNAi技术来预防TGEV感染,试图为突破TGE防治瓶颈、拓展TGEV防治手段作一有益探索;并希望通过小型猪体内RNAi抗TGEV的试验研究,为临床研制抗病毒制剂做准备,并为大型动物体内RNAi及相关肠道病毒性疾病的RNAi提供参考资料。本研究共分以下三个部分:第一部分(第二章):首先,通过对TGEs-1基因易突变区(S基因5’端)设计跨突变区引物,成功扩增了国内分离株TGEs-1的S基因5’端序列,包括小部分聚合酶基因序列。把测序结果与GenBank中TGEV SC-Y分离株同源序列比对,结果显示同源性为98%。接着,把TGEs-1 S基因5’端所测序列与国内已发表的TGEV SC-Y基因组其他部分较为保守的序列相结合,利用相关设计软件设计了靶向病毒复制相关基因区的7条siRNA,并成功构建了8个带EGFP基因的shRNA表达载体:pEGFP-U6/P1、pEGFP-U6/P2、pEGFP-U6/S1、pEGFP-U6/S2、pEGFP-U6/M、pEGFP-U6/N1、pEGFP-U6/N2以及非特异性干扰对照载体(pEGFP-U6/T)。以上结果均为在后续细胞水平和小型猪体内有效而稳定地干扰TGEs-1的复制打下了基础。第二部分(第三章)本部分在细胞水平研究了shRNA表达载体抗TGEV感染的作用:ST细胞培养到70-80%融合时,对细胞分别转染不同的shRNA表达质粒(0.4μg/孔)。转染28h后,分别对细胞感染TGEs-1(200CCID50),并于感染后40小时对培养物进行分析。CPE分析发现,pEGFP-U6/P2和pEGFP-U6/P1转染孔未出现细胞病变或只在培养孔边缘呈现微弱的疑似“病变”(pEGFP-U6/P1,TGEV病变一般从孔边缘开始向孔中部漫延),而其他各干扰孔均出现了TGEV特征性病变,只是病变程度不同。相比之下,shRNA表达载体pEGFP-U6/S1和pEGFP-U6/N1抗病变的能力较强,只在孔的边缘出现不同程度的病变,而pEGFP-U6/S2、pEGFP-U6/M以及pEGFP-U6/N2孔细胞的病变程度重,几乎整孔细胞出现病变。其中,pEGFP-U6/S2和pEGFP-U6/N2干扰孔的病变与非特异干扰孔pEGFP-U6/T的情况接近。结果表明,shRNA表达载体pEGFP-U6/P1、pEGFP-U6/P2、pEGFP-U6/S1和pEGFP-U6/N1对细胞的保护能力较强。尤其是, shRNA表达载体pEGFP-U6/P1和pEGFP-U6/P2,CPE和MTS的分析结果都表明,两者可以在细胞水平高效保护ST细胞抵御TGEs-1引起的病变。而且RT-qPCR分析结果也显示,这两个质粒与非特异质粒pEGFP-U6/T相比,可显著减少细胞培养物中的病毒RNA含量,其中,pEGFP-U6/P2几乎达到100%抑制,由此证明上述的抗细胞病变效应是源自序列特异的RNAi作用。综合以上结果,我们初步筛选出pEGFP-U6/P1和pEGFP-U6/P2作为进一步实施小型猪体内试验研究的shRNA表达载体。第三部分(第四章)在这一部分我们初步研究了在小型猪体内shRNA表达载体抗TGEV感染的作用。对12头25日龄的小型猪分组,分别设3个干扰组(pEGFP-U6/P1、pEGFP-U6/P2、两种载体协同干扰组)和3个对照组(非特异干扰pEGFP-U6/T、只攻毒、正常),每组2头仔猪。然后分别从前腔静脉注射shRNA表达载体(3mg/20ml/猪),并于24h后攻毒(10ml/猪)。3天后处死,采集肝、肺、肾、空肠、回肠、盲肠、脾和肠系膜淋巴结。每个样品分三份,分别用于制作石蜡切片、冰冻切片和RT-PCR检测。在攻毒26小时后,只攻毒猪就开始拉黄色稀便,站立不稳,脱水、消瘦。这种情况一直持续到采样,而其他猪表观正常,食欲旺盛。病理切片分析结果显示,部分pEGFP-U6/P1、pEGFP-U6/P2干扰和协同干扰猪出现肝脏轻度颗粒变性和/或肾小管肿胀,但是所有猪小肠绒毛完好;而只攻毒猪除了具有以上症状外,还存在肾小球严重肿胀,呈肾小球性肾炎;小肠绒毛严重萎缩脱落。运用IFA和RT-PCR检测技术,所有shRNA表达质粒转染猪的样品中均未检测到TGEs-1及其RNA。结果表明,shRNA表达质粒pEGFP-U6/P1、pEGFP-U6/P2可以在小型猪体内有效减少TGEs-1数量,减轻TGEs-1对组织细胞的致病变作用。总之,通过在细胞水平和小型猪体内分别对shRNA表达载体抗TGEs-1感染的能力进行仔细分析比较,我们发现抗TGEs-1的shRNA表达载体可以在细胞和小型猪体内有效保护组织(细胞)抵御TGEs-1引起的特异性病变。因此,我们相信经过进一步优化细胞水平和小型猪体内RNA干扰的过程,可以在不久的将来把shRNA表达载体pEGFP-U6/P1和pEGFP-U6/P2制成抗TGEs-1制剂而应用于临床。更多还原

【Abstract】 Porcine Transmissible Gastroenteritis virus (TGEV) belongs to Coronavirus genus, Coronaviridae, it is the main cause of transmissible gastroenteritis (TGE) of swine. TGE is acute and hyperinfectious disease, which usually outbreaks in winter and spring. Infected animals are characterized by vomiting, diarrhea and severe dehydration. There are no differences among ages and strains of swine in susceptibility to the disease, the mortality of piglets aging less than 2 weeks can be high up to 100%. Generally, the causes of death are dehydration and disorder of metabolism of electrolyte. The mortality decrease as the animals grow, but the survived animals show growth retardation and low rate of reward to feeding. TGE covers a wide range all over the world. Most countries with porcine industry suffer from this disease, resulting in enormous economic loss. Since the first report in 1950s in our country, the disease has been found in many places. TGE is one of the list B infectious diseases, and should be inspected in market according to the law in our country.No conventional medicine shows efficacy for TGE and vaccination is the commonly used method so far. But the widely used vaccines themselves contain various disadvantages. The inactive vaccine is of both high cost and low protection. The attenuated one has potential risk of virus spread, reverting to a virulent form, and perhaps interfering with the diagnosis of wild strain of TGEV. As the replacements, genetically engineering vaccine and nucleic acid vaccine still need to be improved due to their costs and efficacy in immunoprotection, etc. All these obstacles in the course of treated and prevented pigs from TGE result in less profit in swine industries. Therefore, researching on new, safe and effective vaccines or other measurements for treatment and prophylaxis of TGEV is necessary and urgent.As an ancient defense mechanism of cells against hostile genes, RNA interference (RNAi) cleaves mRNA in a sequence-specific manner. As an efficient, economic and convenient research tool, RNAi has exceeded traditional antisense technology on the level of gene knockdown, becoming a powerful tool in scientific reseach both in vitro and in vivo. RNAi have been shown especially tremendous development potential in anti-cancer and anti-virus diseases, and a lot of cancer-associated genes and viral replications have been effectively silenced.TGEV, the pathogen of TGE, has a clear etiological classification, known well of biological characteristics, general genomic structure and function. In addition, TGEV has a single, positive-strand RNA, which can function as an mRNA itself. Taken together, there are good technical backgrounds and biological bases for employing RNAi technique as an anti-TGEV strategy.In sum, the goal of this study is to protect ST cells and minipigs from TGEV infection by using specific and potent RNAi technology, which advances fast and costs decreasingly, also to break through the bottleneck of therapy of TGE and to find some new therapeutics. In addition, we hope to provide scientific references for researching and manufacturing agents against other viruses and for large animal-associated RNAi experiments.This study contains the following three parts:Part IThe 5’sequence of S gene from domestic isolates TGEs-1, including a part of polymerase gene, was successful amplified with designed primers covering a mutable district of TGEs-1 gene. Sequencing results were blasted with the homologous isolates in GenBank, and the homology is high up to 98%. Then, seven siRNA targeting TGEs-1 genome were generated by combining our identified sequence of TGEs-1 S gene with the other conserved genes among different strains existing in domestic SC-Y strain. After that, 8 shRNA-expressing vectors, pEGFP-U6/P1, pEGFP-U6/P2, pEGFP-U6/S1, pEGFP-U6/S2, pEGFP-U6/M, pEGFP-U6/N1, pEGFP-U6/N2 and a non-specific interference control, pEGFP-U6/T were successfully constructed, all of which contained an EGFP-expressing gene. The above works established the foundation of the following work aiming to efficiently interfer the replication of TGEs-1 in ST cells and minipigs.Part IIThis part focused on shRNAs-expressing plasmids against TGEV on cellular level. ST cells were respectively transfected with different shRNA-expressing plasmids with 0.4μg per well when they reached a confluence of 70-80%. 28 hours later, the cells were infected with TGEs-1(200CCID50), and 40 h after infection they were analyzed by CPE and MTS methods. CPE analysis shows that pEGFP-U6/P2 and pEGFP-U6/P1 transfected cells didn’t emerge disease or only showing weak suspected "lesions" in the margin of well, meanwhile other wells displayed different degree of characteristic lesions. The shRNA-expressing vectors pEGFP-U6/S1 and pEGFP-U6/N1 have more strongly anti-infection, only showing weak lesions in the well margin, than pEGFP-U6/S2, pEGFP-U6/M and pEGFP-U6/N2, which showing lesions almost in the whole well. Among them, the inhibitory effects of pEGFP-U6/S2 and pEGFP-U6/N2 are similar with that of non-specific plasmid pEGFP-U6/T. In sum, the data above show that the ability of shRNA-expressing vectors pEGFP-U6/P1、pEGFP-U6/P2、pEGFP-U6/S1 and pEGFP-U6/N1 to protect cells from TGEV destruction is fairly strong, especially pEGFP-U6/P1 and pEGFP-U6/P2. In addition, the analysis of MTS shows that the number of viable cells in wells transfected with pEGFP-U6/P1、pEGFP-U6/P2、pEGFP-U6/S1 or pEGFP-U6/N1 is obviously bigger than that transfected with the other plasmids, and the data further confirmed the conclusion drawn from CPE analysis. Taken together, we chose pEGFP-U6/P1 and pEGFP-U6/P2 as the interference vectors for the next experiment on minipigs. Besides, before doing experiments in vivo, we also analyzed the ability of decreasing the quantity of TGEs-1 RNA by RT-qPCR method, and the data indicated that comparing with non-specific plasmid pEGFP-U6/T, the plasmids (pEGFP-U6/P1 and pEGFP-U6/P2) could reduce the number of virus RNA in cell culture in a large scale, especially the pEGFP-U6/P2 nearly reach a 100% inhibition, which suggested that the virus inhibition triggered by the plasmids was caused by sequence-specific RNAi.Part IIIIn this part, we studied the effect of shRNA-expressing plasmids on prophylaxis of TGEV in vivo. Piglets aging 25 days were randomly divided into 3 interference groups (pEGFP-U6/P1, pEGFP-U6/P2 and a combination of the two) and 3 control groups (just transfected with pEGFP-U6/T, just infected with TGEs-1 and normal), and each group contained 2 piglets. Then piglets were injected with shRNA-expressing vectors (3mg/20ml/pig) through the precaval vein, and inoculated TGEs-1 24h later. They were sacrificed 3 days after infection, and the liver, lung, kidney, jejunum, ileum, caecum, spleen and mesenteric lymph node of each piglet were collected. Each sample was divided into 3 parts for analysis of histopathological changes, immunohistochemistry and RT-PCR, respectively.Twenty six hours after infection, one of the piglets just infected with TGEs-1 began to diarrhea, flabbiness, dehydration and thinness, and this situation lasted till it was sampled. Meanwhile, piglets in other groups were normal with a good appetite. Histopathological changes analysis showed that there were granular degeneration of hepatocytes and renal tubular swelling in some minipigs transfected with pEGFP-U6/P1、pEGFP-U6/P2 or the combination of them, but all intestinal villi in them were in good shape. Besides the histopathological changes in livers and kidneys above, minipigs just infected with TGEs-1 showed by severe glomerular swelling as well as shorten and dropped intestinal villi. The results of IFA and RT-PCR indicated that virions and virus RNA couldnot be found in all the samples from minipigs transfected with shRNA-expressing plasmids, which suggested that pEGFP-U6/P1 and pEGFP-U6/P2 could reduce TGEs-1 virion efficiently in vivo and thus lessen the histopathological changes triggered by TGEs-1.In short, through complete analyses of the anti-TGEs-1 ability of shRNA-expressing plasmids in vitro and in vivo, we have found that shRNA-expressing plasmids could effectively protect ST cells as well as minipigs from pathological changes triggered by TGEs-1. So, we believe that undergoing further optimization, the pEGFP-U6/P1 and pEGFP-U6/P2 could be utilized in clinic as anti-TGEV agents in the future.更多还原