【作者】 任晓峰；

【导师】 刘宝全；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2003， 博士

【摘要】 猪传染性胃肠炎病毒（Transmissible Gastroenteritis Virus，TGEV）属于冠状病毒科冠状病毒属成员，可导致猪的传染性胃肠炎（Porcine Transmissible Gastroenteritis，TGE），该病是猪的一种急性高度接触性肠道传染病，主要临床特征是呕吐、严重腹泻和脱水，不同年龄和品种的猪对其均易感，其中1、2周龄以下的仔猪致死率可高达100％。包括抗毒素和抗生素在内的常规药物对本病无任何治疗作用，预防该病的有效方法是疫苗接种。 在该病发生的很长一段时间里，世界上许多国家的兽医科研人员都在致力于TGEV疫苗的研制。然而，常规疫苗由于本身的内在缺陷而存在着不可避免的弊端，如减毒活疫苗虽然免疫效果好，却有排毒散毒、毒力返祖和疫情扩大的危险；灭活苗虽然安全性好，却存在着免疫效果不理想、需多次注射、常需添加佐剂等费事费力的缺点。因此，研制新型、安全、高效的疫苗一直是当代预防医学科研人员的奋斗目标。随着分子免疫学、分子病毒学和分子生物学等相关技术的发展，利用分子生物学手段研制新型疫苗已成为可能。据此，进行了以下几个实验： 实验一：使用相应的限制性核酸内切酶从猪传染性胃肠炎病毒国内分离株TH-98已克隆的主要免疫保护性抗原基因重组质粒pUC—S和SaPCI中分别回收带有不同粘性末端编码主要抗原位点片段Sa，利用不同的原核表达载体，按照既定阅读框架，构建了多个重组原核表达载体，并通过质粒酶切及套式PCR鉴定，为其深入研究奠定了基础。 实验二：用相应限制性内切酶从pUC—S中回收完整的S基因，将其按正常读序亚克隆于昆虫细胞/杆状病毒表达质粒PF_ASTB_ACHTb的EcoR Ⅰ、Pst Ⅰ多克隆位点上，并进行酶切鉴定，阳性重组子命名为BAC-S。用DH10B_AC对其进行转位，得到的重组子PBAC-S。根据S基因酶切位点分析结果及杆状病毒转位区的结构特点，用相应的限制性内切酶进行酶切鉴定，并用特异性和通用引物进行PCR分析，证明了重组子的可靠性，同时也证明已经成功地构建了能直接在昆虫细胞中进行表达的感染性质粒。 实验三：根据pUC-S序列分析结果，设计PS1、PR1N和PS2N、PR3两对引物（其中上游引物PR1N和下游引物PS2N均有碱基改动，并形成了新的Sca Ⅰ酶切位点，且PR1N中有人工碱基改动，使其序列发生了改变，消除了存在于其中的潜在的早期终止密码子。同时利用了核苷酸的简并性，使其编码的氨基酸并未发生变化），并以pUC-S为模板，扩增出约1.3kb和1.0kb两个片段，分别命名为SNa和SNb。将SNa克隆于T载体后，筛选反向连接重组子TSNa,并用Sca Ⅰ和Kpn Ⅰ进行部分酶切，回收约3.9kb的目的片段。将SNb片段用相同的限制性内切酶处理后，定向连接于TSNa中，酶切鉴定后，命名阳性重组子为TSNab。用EcoR Ⅰ和Kpn Ⅰ回收含SNa和SNb的片段，并亚克隆于EcoR Ⅰ和Kpn Ⅰ回收的pUC-Sb重组质粒中，并经酶切和套式PCR鉴定，阳性质粒为pUC—SN。再一次将改建成功的pUC—SN用EcoR Ⅰ和Pst Ⅰ回收SN，插入到克隆载体PCR3.1的多克隆位点上，鉴定后，将其按正确读框顺序克隆于毕赤酵母（Pichia） 东北农业大学农学博士学位论文一表达载体 pPIC3．5的 Ecoki和 NOn多克隆位点上，经酶切和 PCR筛选后，获得了能够在酵母中直接表达的穿梭质粒SNPIC3．5。 实验四：通过亚克隆重组技术，利用真核表达载体PCI和pCDNA3，构建了含完整S基因的SPCI和含S基因主要抗原位点片段的SaPCI和SaPCDNA3核酸疫苗。并对实验动物进行了免疫应答检测。 将昆明鼠随机分成A、B、C三组，A为SaPCI实验组、B为SPCI实验组、C为PCI载体对照组。每组35只，三次肌肉免疫接种，每次间隔2周。初次免疫第0、14、2、28、35、42天采血分离血清，用ELISA检测抗体动态变化，并检测外周血CD4”。CDS“丁细胞数量动态变化。 本实验首次在国内对猪传染性胃肠炎病毒S基因进行了较为深入和全面的探索，构建了 TGEV S基因多系统表达载体，为其基因工程亚单位疫苗的研制打下了重要的物质基础。在国内外首次构建了 TGEV S基因核酸疫苗，并对其免疫效力进行了系统研究，证明重组核酸疫苗能够诱导小鼠体液和细胞免疫应答c 该研究为今后TGEV的进一步了解奠定了坚实的基础，为其基因工程疫苗的制备提供了必要的物质材料，同时为TGEV核酸疫苗的深入研究提供了翔实、可靠的实验材料和理论依据。总之，对TGEV生物学性状研究、疫苗开发和TGE防治有着重要的现实意义。更多还原

【Abstract】 Porcine transmissible gastroenteritis virus (TGEV) belongs to the family coronaviridae. The virus causes transmissible gastroenteritis (TGE) in all kinds and ages of pigs. Particularly, its mortality in pigs under 1 to 2 week old may reach 100%. TGE is a kind of acute, highly infectious disease. The clinical manifestations of typical TGE consist of omitting, serious diarrhea and loss of water. Vaccination is the effective means for the prevention of TGE.Since the discovery of TGE, many veterinary researchers devoted themselves to the preparation of vaccine against it. However, both attenuated and inactive vaccines exist inherit deficiencies. For example, attenuated vaccines could result in dissemination of virus, recovery of virulence and widespread of disease, even though its excellent vaccination effect; inactive vaccine is safer than the former, but it also exists unfavorable immunization effect, needing of many-times vaccination and adding of adjuvant etc. So the exploitation of novel vaccine, especially genetic engineering vaccine is the urgent task currently. With the development of immunology, virology and biology in molecular level, the task become more and more available, therefore, the following experiments are finished:Assay 1: Sa fragment encoding major antigenic sites of S gene was obtained from cloned recombinant plasmid pUC-S of TGEV Chinese isolate TH-98, and then was inserted into different prokaryotic expression vectors according to correct ORF (open reading frame). These recombinant vectors were identified and analyzed with RE (restriction endonuclease) and by nested PCR, which provided solid basis for further study.Assay 2: Complete S gene obtained with RE from pUC-S was subcloned into EcoRI and PstI sites of vector pFASTBACHTb in insect/baculovirus expression system in appropriate transcription sequence. The transposition of positive recombinant named BAC-S was achieved with competent cell DH10BAC. The recombinant named pBAC-S was verified with RE and by PCR with specific and universal primers according to DNASIS analysis results of S gene and transposition region characteristics of the vector. The result showed that theinfectious recombinant was successfully constructed, which could be expressed directly in insect cell.Assay 3: With Two pairs of primers PS1, PR1N and PS2N, PR3 (There are nucleotide changes led to a new Seal recognized site in sense primer PS2N and antisense primer PR1N. Besides, There are artificial nucleic acid modifications in PR1N, aiming at eliminating potential pre-termination codon of S gene. The deduced amino acid sequence is the same as original one due to utilizing nucleic acid degeneracy) and template PUC-S, two PCR products named SNa and SNb. of 1.3kb and 1.0kb were produced respectively. SNa was cloned into T vector and negative direction recombinant was partially digested with Seal and KpnI, and then about 3.9kb fragment named TSNa was obtained. SNb was inserted into corresponding sites of TSNa after digestion of the same RE and recombinant was named TSNab. The favorite fragment containing SNa and SNb was obtained with EcoRI and KpnI, and then subcloned into plasmid pUC-Sb derived from pUC-S. The recombinant named pUC-SN was verified by nest PCR and with RE and was . cloned into EcoRI and PstI sites of pCR3.1 cloning vector. Eventually, the recombinant SNPCR3.1 was digested with EcoRI and NotI and subcloned into the corresponding sites of Pichia yeast expression vector pPIC3.5 in correct ORF. which could be used to express in yeast cell.Assay 4: Through subclonal recombination technique, complete S gene and major antigenic site fragment of S gene named Sa were cloned into corresponding sites of eukaryotic expression vector PCI and pcDNA3 respectively. The nucleic acid vaccine SPCI and SaPCI as well as SPCDNA3 were constructed and animal experiments were finished.KunMing mice were divided into 3 groups randomly, group A, B, C were injected intramuscularly with SaPCI, SPCI and PCI control respectively for three times with 2 weeks interval.更多还原