【作者】 李燕华；

【导师】 袁世山；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2010， 硕士

【摘要】 猪繁殖与呼吸综合征(Porcine Reproductive and Respiratory Syndrome, PRRS)首次出现于19世纪80年代末,引起严重的母猪流产等繁殖障碍和仔猪呼吸道病等临床疾病。该病给世界养猪业带来了巨大的经济损失。目前,对该病的控制仍以预防为主,疫苗免疫是最重要的防御手段。但是,现有的商品化疫苗仍然存在很多问题,比如,对异源毒株的保护不佳以及安全性问题。本研究旨在优化现有的高致病性PRRSV细胞致弱毒株反向遗传系统,并在此基础上密码子优化GP5共有序列(consensus sequence)的编码序列,研究病毒核苷酸序列的功能和蛋白表达调控,从而为疫苗研发奠定理论基础。首先,为了克服PRRSV感染性克隆采用RNA转染的方式来拯救病毒的一些缺点,比如,病毒拯救效率低、克隆不稳定、操作繁琐及病毒拯救成本较高。本研究构建并优化了基于质粒DNA转染的PRRSV反向遗传系统,将全长cDNA克隆中的T7启动子换成hCMV启动子,同时分析hCMV的最佳转录起始位点以及polyA末端添加丁型肝炎病毒的核酶序列(HDV ribozyme )在感染性克隆中的作用。通过定点突变( site-directed mutagenesis)和融合PCR(Splicing-overlapping-extension PCR,SOE-PCR)等方法,构建了四个cDNA克隆,将这些克隆中hCMV的TATA框与病毒5’端序列间的距离分别设置为21、23、25、27。转染MARC-145细胞后,以细胞病变出现的时间、N蛋白的表达及各时相的病毒滴度为标准评价病毒拯救效率。当距离设为23或25,转染细胞后84h出现典型细胞病变,48h能用TCID50法滴定到病毒;而设置为21和27时,转染细胞后,72h仍滴定不到病毒。需通过传代才能出现典型细胞病变。以上结果表明,hCMV的最佳转录起始位点为23-25nt。当病毒基因组3’末端添加丁型肝炎病毒的核酶序列(HDV ribozyme),转染细胞后,出现典型细胞病变的时间提前了24h,并且各时相的病毒滴度都比亲本克隆高2个log。以上结果说明了优化hCMV与病毒5’序列之间距离和病毒3’序列末端加上核酶能够显著提高感染性克隆的转染效率。其次,在优化的PRRSV弱毒反向遗传系统基础上,采用猪源偏好的密码子优化GP5共有序列的编码序列,以期获得GP5蛋白的高效表达以及增强交叉保护。另外,本研究通过改变编码序列而保持氨基酸序列不变,旨在探索GP5在基因组核酸水平上也会参与PRRSV病毒复制过程。从Genbank中获得15株北美型PRRSV的全基因组序列,其中国外毒株、国内经典毒株和国内高致病性毒株各占5株,并且获得各毒株的GP5氨基酸序列。基于GP5的遗传进化分析表明这些毒株明显的分属几个亚群,可以代表不同的野生毒株。MegAlign序列比对GP5氨基酸序列获得共有序列(consensus sequence),使获得的GP5共有序列跟各个亚群间的编码序列相似性提高。在线软件DyNAVcaS(http://miracle.igib.res.in/dynavac/)密码子优化GP5共有序列的编码序列,并且人工合成优化后的DNA片段。在优化的PRRSV弱毒反向遗传系统pAJX25HB基础上,构建了pAJXM5、pAJXM5S1、pAJXM5S2、pAJXM5S3和pAJXM5S4五个cDNA克隆,这些克隆ORF5密码子优化的区域分别为:1-555nt、1-180nt、181-555nt、132-555nt和104-555nt。转染细胞的结果显示,当优化的范围处于104-555nt区间时,能够拯救出病毒。利用RT-PCR扩增的方法,从第一代和第三代的vM5S2扩增含有密码子优化区域的基因组片段,测序结果表明,病毒基因组序列没有发生回复突变,说明突变病毒遗传稳定。通过比较vM5S2和亲本病毒的多步生长曲线,两者的生长特性相似,说明突变的核苷酸并没有改变病毒复制规律。pAJXM5和pAJXM5S1转染细胞后,未能拯救出病毒,但RT-PCR和间接免疫荧光能分别检测到亚基因组5,6和7的转录以及GP5和N蛋白的表达,说明病毒的主要结构蛋白的转录和翻译没有破坏。以上结果表明,GP5N端的1-103位核苷酸碱基序列对病毒复制也是必需的。GP5在基因组核酸水平上也可能参与PRRSV病毒复制过程。但是,相关机制需进一步的研究解析。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome (PRRS) first reported in the late 1980s, is recognized as a serious swine disease and is characterized with either reproductive failure in pregnant sows, or respiratory tract distress particularly in sucking pigs. PRRS has brought great economic losses to the swine industry every year. Vaccine is the most effective measure to counteract PRRS, but there are still lots of problems with available commercial vaccines, for example, the low protection efficiency to heterologous strain, and safety. In this study, based on the reverse genetic system of low virulent PRRSV which was attenuated by serial passage in MARC-145, we constructed an improved DNA-launched reverse genetic system. Then, based on sequence alignment analysis, we got the consensus amino acid sequence of GP5 and codon optimized the coding sequence using codon preferred in pigs. Codon optimized ORF5 was swapped into the improved reverse genetic system, through which we dissected the function of virus genome coding sequence in virus replication, viral protein expression, and laid a theoretical foundation for the development of PRRSV vaccine.Firstly, a DNA-launched reverse genetics system (RGS) was constructed and optimized. As far as I know, most PRRSV RGS are based on RNA transfection. There were some disadvantages with RNA based RGS, such as stability, low virus rescue efficiency, complicated in vitro transcription and high cost. In this study, in order to improve the virus rescue efficiency and stability of cDNA clone, T7 promoter was replaced with hCMV promoter, the optimal transcriptional initiation site of hCMV was analyzed, and a ribozyme element of delta hepatitis virus and BGH terminal signal at the 3’end of ployA. Five cDNA clones were constructed by site-direct mutation and splicing overlapping extension polymerase chain reaction. And the distances between the TATA box of hCMV and 5’viral genome were 21nt, 23nt, 25nt and 27nt respectively. The virus rescue efficiency was determined by the time when CPE can be observed, the level of N protein detected by indirect fluorescence assay and virus supernatant titers at different time phases. When the distances were 23nt and 25nt, CPE could be observed at 84 hours post transfection (hpt), and virus supernatant could be titrated by TCID50. However, when the distances were 21 and 27, typical CPE could be observed by passaging virus supernatant in fresh MARC-145 cell, and virus supernatant could not be titrated by TCID50. So, it was indicated that the optimal transcriptional initiation site was between 23 nt and 25 nt. Furthermore, pAJX25HB was constructed by adding a delta hepatitis virus ribozyme element based on pAJX25. In comparison with parental clone, typical CPE could be observed 12h earlier, and the virus titers at different time phase were 2 logs higher. To sum up, it indicated that modifications of 5’and 3’of viral genome could improved virus rescue efficiency remarkably.Subsequently, we tried to re-code the GP5 consensus sequence using more preferred codon in pigs for the purpose to raise the level of GP5 expression and improve cross protection efficiency. Also, we wanted to investigate the function of GP5 coding sequence in the process of virus replication by codon optimization. Fifty northern American PRRSV strains, including 5 typical strains of PRRSV abroad, 5 typical strains of domestic classical PRRSV and 5 typical strains of high pathogenic PRRSV, were selected as samples, and the genomic sequences were obtained from Genbank. Based on the sequence analysis of GP5, we found that the viruses belonged to different subgroup and could represent heterologous strains. GP5 consensus sequence was maintained by alignment of the GP5 amino acid sequences of 15 typical PRRSV strains, and codon optimized by online software DyNAVcaS(http://miracle.igib.res.in/dynavac/). Finally, the coding sequence of codon optimized GP5 consensus sequence was synthesized by Genscript Co.. pAJX25HB was used as backbone, pAJXM5, pAJXM5S1, pAJXM5S2, pAJXM5S3 and pAJXM5S4 were constructed by swapping the ORF5, and the regions of 1-555 nt,1-180 nt,181-555 nt,132-555 nt and 104-555 nt were codon optimized respectively. Then, viruses were rescued by DNA transfecting MARC-145 cell and BHK-21 cell. The results of DNA transfection showed that PRRSV could be rescued when the codon optimized region was between 104 nt and 555 nt of ORF5. Then, virological characteristic vM5S2 were identified by virus multi-step growth curve and RT-PCR. The results of RT-PCR and sequencing indicated that no reversal mutation emerged from P1 to P3 virus. And the growth characteristic of vM5S2 was similar to parental virus, which indicated that codon optimization didn’t change the characteristic of virus replication. PRRSV could not be rescued from pAJXM5 and pAJXM5S1. However, subgenomic mRNA5, 6, and 7, and major structural proteins GP5 and N were detected by RT-PCR and indirect fluorescence assay,Which showed that the transcription and translation were not destroyed by codon optimization. Therefore, we could get a conclusion that, not only GP5 amino acid sequence played significant role in virus life cycle, but also N terminal 103 nt was essential for virus replication. On the other hand, it needs further investigation to dissect the mechanism.更多还原