【作者】 赵静；

【导师】 温贵兰；

【作者基本信息】 贵州大学 ， 兽医学硕士， 2024， 硕士

【摘要】 猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)从发现至今,对各国养猪业产生了巨大影响。猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)是引起PRRS的病原,因其独特的遗传多样性和免疫反应复杂性,使得PRRS的根治成为一个长期且艰巨的任务。因此,对PRRSV与宿主的相互作用关系进行深入的研究,探寻其致病的深层机制对于高效防控PRRS措施的制定至关重要。DEAD-box解旋酶以ATP依赖性方式识别和解开RNA双链,在RNA代谢的各个方面都起着核心作用,例如转录、翻译、前m RNA剪接、RNA衰变、编辑和RNA输出。研究表明,DEAD-box家族蛋白通过充当病毒核酸传感器或调节下游信号转导参与抗病毒先天免疫调控。DDX6(DEAD-box polypeptide 6,DDX6)又称RCK/p54,含有保守的Asp-Glu-Ala-Asp基序(DEAD-box),由一个N端和一个C端Rec A样结构域组成,用于与各种因子相互作用,以实现其特定功能。目前,关于DDX6调控PRRSV复制及其机制的相关研究报道较少。因此,本研究以DDX6、PRRSV为研究对象,探究在PRRSV在复制过程中,DDX6与PRRSV相互作用机制。主要研究内容如下:1.DDX6基因克隆和生物信息学分析分别从非洲绿猴胚胎肾细胞(Marc145细胞)和从江香猪脾脏中扩增出猴源DDX6(m DDX6)和猪源DDX6(p DDX6)基因的CDS序列,进行T克隆和测序,通过生物学软件对其进行生物学信息分析。提取从江香猪不同组织RNA反转录为c DNA,通过q RT-PCR分析DDX6在不同组织中的转录水平。结果显示:m DDX6和p DDX6 CDS长度均为1452 bp,编码483个氨基酸,与预期大小结果相符。mDDX6的CDS核苷酸序列与绿猴DDX6(登录号:XM-008021118)相似性最高,为99.9%;p DDX6的CDS核苷酸序列与野猪DDX6(登录号:XM-003129914)相似性最高,为99.9%。而m DDX6与p DDX6的CDS核苷酸序列同源性为97.2%。m DDX6、p DDX6蛋白由DEAD-like解旋酶超家族(DEADc)结构域和C端(HELICc)两个结构域组成。m DDX6与p DDX6蛋白的二级、三级结构主要由α-螺旋、β-折叠及无规则卷曲构成,不存在跨膜结构。DDX6基因在从江香猪的淋巴结、肝脏和脾脏等10种组织中均有表达。其中,DDX6在淋巴结中的转录水平相对最高,在骨骼肌中的转录水平则相对最低。2.DDX6对PRRSV增殖的影响利用qRT-PCR和Western blot方法检测PRRSV感染Marc145细胞、PAM-CD163细胞后不同时间DDX6基因的转录水平和翻译水平,分析PRRSV增殖对内源DDX6的影响。构建猴源与猪源的真核表达质粒p CMV-Myc-m DDX6、p CMV-Myc-p DDX6,采用Western blot和IFA检测重组质粒在Marc145细胞和PAM-CD163细胞中的表达及定位;分别在Marc145细胞与PAM-CD163细胞中过表达不同剂量的m DDX6、p DDX6,采用q RT-PCR和Western blot两种方法,探究过表达m DDX6和p DDX6对PRRSV增殖的影响。转染不同剂量的针对m DDX6、p DDX6基因的si RNA干扰片段,通过q RT-PCR和Western blot检测干扰内源DDX6对PRRSV增殖的影响。结果表明,PRRSV在感染Marc145细胞24 h和36 h时,DDX6基因m RNA水平显著低于对照组,DDX6蛋白的表达量在24 h后出现下降;PRRSV在感染PAM-CD163细胞24 h时,DDX6的m RNA水平显著低于对照组,DDX6蛋白的表达量在24 h后出现下降。Myc-m DDX6和Myc-p DDX6重组蛋白分别在Marc145细胞和PAM-CD163细胞中成功表达,并且IFA特异性荧光检测发现Myc-m DDX6和Myc-p DDX6主要定位于细胞质中;在Marc145细胞与PAM-CD163细胞中过表达m DDX6或p DDX6时,PRRSV的N基因的转录水平和蛋白翻译水平均呈现出显著下降的趋势。相反,当干扰内源DDX6基因表达时,PRRSV的N基因转录水平和翻译水平显著上调,表明m DDX6和p DDX6在Marc145细胞和PAM-CD163细胞中可显著抑制PRRSV的增殖,且与转染剂量呈正相关。3.DDX6与PRRSV的相互作用构建PRRSV编码的GP2a、GP3、GP4、GP5、GP6、N病毒结构蛋白真核表达质粒;在PAM-CD163细胞中过表达PRRSV编码的病毒结构蛋白与非结构蛋白(PRRSV非结构蛋白真核表达载体由本实验室前期构建),通过RT-q RCR分析过表达PRRSV病毒蛋白对DDX6 m RNA水平的影响。采用免疫共沉淀(Co-IP)联合液相色谱串联质谱(LC-MS/MS)技术筛选与DDX6互作的PRRSV病毒蛋白,并且通过Co-IP和荧光共聚焦技术验证其相互作用关系。结果显示,在Marc145细胞中过表达PRRSV GP4、N、NSP1α、NSP1β、NSP11、NSP12蛋白会引起DDX6 m RNA水平显著下降,在PAM-CD163细胞中过表达PRRSV GP4、N、NSP1α、NSP1β、NSP11蛋白会引起DDX6显著下降;免疫共沉淀(Co-IP)联合液相色谱串联质谱(LC-MS/MS)技术结果显示,有85种蛋白可能与DDX6存在互作,其中包括PRRSV N蛋白。DDX6蛋白和PRRSV N蛋白共转染的情况下,两者主要分布于细胞质周围,且存在共定位情况;在PAM-CD163细胞中共转染p CMV-HA-N和p CMV-Myc-p DDX6,Co-IP结果显示DDX6与PRRSV N蛋白存在直接作用。本研究结果表明:DDX6基因高度保守,在从江香猪组织中分布广泛。PRRSV感染Marc145细胞与PAM-CD163细胞,可引起内源DDX6表达量下降。过表达DDX6于Marc145细胞与PAM-CD163细胞可显著抑制PRRSV的增殖;而干扰DDX6表达,PRRSV的增殖水平上调;进一步研究证实DDX6与PRRSV N蛋白存在直接相互作用关系。研究结果为深入研究DDX6在PRRSV感染的机制中提供了基础资料。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome(PRRS)has had a significant impact on the farming industry in various countries since its discovery.The virus that causes PRRS is the Porcine reproductive and respiratory syndrome virus(PRRSV),and its eradication has been a great challenge due to its genetic diversity and the complexity of its immune response.Therefore,an in-depth study of PRRSV’s immune escape strategies and the exploration of its pathogenic mechanisms are particularly important for the development of novel vaccines and potent drugs,as well as for the formulation of preventive and control measures.The DEAD-box deconjugase recognize and unravel RNA double strands in an ATP-dependent manner,and play central roles in various aspects of RNA metabolism,such as transcription,translation,pre-m RNA splicing,RNA decay,editing,and RNA export.Numerous studies have shown that DEAD-box family proteins participated in antiviral innate immunity regulation by acting as viral nucleic acid sensors or regulating downstream signaling transduction.DDX6(DEAD-box polypeptide 6,DDX6),also known as RCK/p54,contains a conserved Asp-Glu-Ala-Asp motif(DEAD-box),comprising of an N-terminal and a C-terminal Rec A-like structural domain,it is used to interact with various factors to achieve its specific functions.Currently,there are few study reports about PRRSV replication and its mechanism by DDX6 regulation.Therefore,in this study,DDX6 and PRRSV were used as research subjects to investigate the interaction mechanism between DDX6 and PRRSV during the replication process of PRRSV.The main research contents of this study were to:1.DDX6 gene cloning and bioinformatics analysisThe CDS regions of monkey-derived DDX6(m DDX6)and porcine-derived DDX6(p DDX6)genes were amplified from Marc145 cells and the spleen of congjiang fragrant pig,After amplification,both them were T-cloned and sequenced,and the biological information of the DDX6 genes of different host origins was analysed by various biological software.The RNA from different tissues of Jiangxiang pig was extracted and reverse transcribed into c DNA,and the transcript levels of DDX6 in different tissues were analysed by q RT-PCR.The results showed that the length of both m DDX6 and p DDX6 CDS regions was 1452 bp,encoding 483amino acids,which was in line with the expected results.m DDX6 gene nucleotide sequence had the highest similarity with green monkey DDX6 gene(accession no.XM-008021118),which was 99.9%;and p DDX6 gene nucleotide sequence had the highest similarity with boar DDX6 gene(accession no.XM＿003129914),which was 99.9%;and p DDX6 gene nucleotide sequence had the highest similarity with boar DDX6 gene(accession no.XM＿003129914).And the nucleotide sequence homology between m DDX6 gene and p DDX6 gene was 97.2%.The DDX6 genes were highly conserved among different hosts with high nucleotide sequence similarity.m DDX6 and p DDX6 proteins consisted of two structural domains,the structural domain of DEAD-like deconjugating enzyme superfamily(DEADc)and the C-terminal(HELICc).m DDX6 and p DDX6 proteins had significant secondary and tertiary structural features,which mainly consisted ofα-helices,β-folds and irregular The m DDX6 and p DDX6proteins have distinctive secondary and tertiary structures mainly consisting ofα-helix,β-folding and irregularly curled,and do not have transmembrane structures,and both proteins possess multiple phosphorylation modification sites.d DX6 genes were expressed in a variety of tissues of the Congjiang fragrant pig in ten sites,including heart,liver,spleen,lung,lymph node,muscle,blood,kidney,small intestine and brain.Among them,the transcript level of DDX6 gene was higher in lymph node,blood and liver tissues,while it was relatively lower in muscle,heart and kidney tissues.2.Effect of DDX6 on PRRSV proliferationThe transcription level and translation level of DDX6 gene at different time periods after PRRSV infection of Marc145 cells and PAM-CD163 cells were detected by q RT-PCR and Western blot to analyse the effect of PRRSV proliferation on endogenous DDX6.Eukaryotic expression plasmids p CMV-Myc-m DDX6 and p CMV-Myc-p DDX6 were constructed,and the expression and localisation of the recombinant plasmids Myc-m DDX6 and Myc-p DDX6 were detected by Western blot and IFA in Marc145 cells and PAM-CD163 cells;overexpression of different doses of Myc-m DDX6 and Myc-p DDX6 combined with both q RT-PCR and Western blot to investigate the effects of overexpression of m DDX6 and p DDX6 on PRRSV proliferation.Different doses of si RNA interference fragments targeting m DDX6 and p DDX6genes were transfected,and the effects of interfering endogenous DDX6 on PRRSV proliferation were detected by q RT-PCR and Western blot.The results showed that the expression of DDX6 gene of PRRSV was significantly lower than that of the control group when infecting Marc145 cells for 24 h and 36 h.The expression of DDX6 protein decreased after 24 h.PRRSV infected PAM-CD163 cells for 24 h.The expression of DDX6 gene was significantly lower than that of the control group when infecting PAM-CD163 cells.Myc-m DDX6 and Myc-p DDX6 recombinant proteins were successfully expressed in Marc145 cells and PAM-CD163 cells,respectively,and IFA-specific fluorescence assay revealed that Myc-m DDX6 and Myc-p DDX6 were mainly localised in the cytoplasm;further experiments showed that when overexpression of m DDX6 or p DDX6 was carried out in both cells,the PRRSV’s N gene showed a significant decrease in both m RNA transcription level and protein translation level.On the contrary,when the endogenous DDX6 gene was interfered with,the transcription level and translation level of the N gene of PRRSV were significantly up-regulated,suggesting that m DDX6 and p DDX6 significantly inhibited the proliferation of PRRSV in Marc145 cells and PAM-CD163 cells,and were positively correlated with the transfection dose.3.Regulation of PRRSV replication by DDX6Eukaryotic expression plasmids of PRRSV-encoded GP2a,GP3,GP4,GP5,GP6,N viral structural proteins were constructed;PRRSV-encoded viral structural and non-structural proteins were overexpressed in PAM-CD163 cells(PRRSV non-structural protein eukaryotic expression vector was constructed by the laboratory in advance),and the effect of overexpression of PRRSV viral The effect of overexpression of PRRSV viral proteins on DDX6m RNA levels was analysed by RT-q RCR.Immunoprecipitation(Co-IP)combined with liquid chromatography tandem mass spectrometry(LC-MS/MS)was used to screen the PRRSV viral proteins that interacted with DDX6,and the interactions were verified by Co-IP and fluorescence confocal techniques.The results showed that overexpression of PRRSV GP4,N,NSP1α,NSP1β,NSP11,and NSP12 proteins in Marc145 cells caused a significant decrease in the m RNA level of DDX6,and overexpression of PRRSV GP4,N,NSP1α,NSP1β,and NSP11proteins in PAM-CD163 cells caused a significant decrease in DDX6;Co-immunoprecipitation(Co-IP)combined with liquid chromatography tandem mass spectrometry(LC-MS/MS)showed that 85 proteins may interact with DDX6,including PRRSV N protein;in the case of co-transfection of DDX6 proteins and PRRSV N proteins,the two were mainly distributed around the cytoplasm and co-localisation was present;in HEK-293T cells co-transfected with p CMV-HA-N and p CMV-Myc-p DDX6,the Co-IP results showed that there was a direct interaction between DDX6 and PRRSV N protein.Translated with Deep L.com(free version)The results of this study showed that the DDX6 gene is highly conserved and widely distributed in Congjiang fragrant pig;PRRSV infection of cells can cause a decrease in the expression of endogenous DDX6 at 24 h or 36 h;overexpression of DDX6 significantly inhibits the proliferation of PRRSV,and interference with DDX6 promotes the proliferation of PRRSV;further studies found that DDX6 interacts with the PRRSV N protein Further studies revealed that DDX6 interacted with PRRSV N protein,and it was hypothesised that DDX6 inhibited PRRSV replication on Marc145 cells and PAM-CD163 cells by interacting with PRRSV N protein.It provides basic information for in-depth study of DDX6 in the mechanism of PRRSV infection.更多还原