【作者】 李红；

【导师】 周恩民；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2011， 博士

【摘要】 猪繁殖与呼吸综合征(Porcine Reproductive and Respiratory Syndrome,PRRS)又名“蓝耳病”。是由PRRS病毒(PRRSV)感染所致的一种接触性传染病。该病以繁殖障碍、呼吸道疾病、哺乳仔猪的高死亡率、免疫抑制和持续性感染等为主要特征。其病原具有典型的嗜单核细胞/巨噬细胞特性,在体内,PRRSV主要感染猪肺泡巨噬细胞(Porcine alveolar macrophages, PAM),在体外,主要感染非洲绿猴肾细胞系MA-104及其衍生物Marc-145。PRRSV感染宿主细胞首先通过与细胞膜表面上的特异受体结合,利用细胞的内吞作用而感染易感细胞。至今,已报道了四个独立但功能相关的PRRSV细胞受体。唾液酸粘附素(Sialoadhesin, Sn)、似肝磷脂蛋白(Heparan sulphate, HS)、波形蛋白(Vimentin)和清道夫受体(CD163)。研究发现HS主要作用是吸附PRRSV到PAM细胞上,Sn则介导病毒的内吞,CD163可能协助Sn内吞,病毒脱衣壳和将基因组RNA释放到细胞质中的作用。我们导师周恩民教授通过免疫共沉淀用单克隆抗独特型抗体Mab2-5G2(Mab2-5G2:针对抗GP5抗体)在猪肺泡巨噬细胞和Marc-145中提取到一个分子量约为230kD的蛋白质,可能是PRRSV的一个新的受体,经质谱分析和测序确定为非肌肉肌球蛋白ⅡA(Non-myscle myosinⅡA,NMHCⅡA)。在动物体内,非肌肉肌球蛋白Ⅱ有三种异构体:ⅡA,ⅡB,ⅡC。此蛋白广泛分布在整个生物体内,在细胞的吸附,迁移和胞质分裂中发挥重要的作用。可溶性CD163与非肌肉肌球蛋白重链A型存在着相互作用,但是,迄今为止CD163受体参与病毒结合的部位,病毒上参与受体结合的蛋白及其结合部位,受体与受体之间的关系以及CD163受体与我们发现的新蛋白非肌肉肌球蛋白的结合部位还不得而知。为确定CD163受体在PRRS病毒侵染过程中的作用以及与非肌肉肌球蛋白之间的关系,本实验分别从猪肺泡巨噬细胞(PAM)和非洲绿猴肾细胞(Marc-145)中经RT-PCR扩增出编码CD163受体的全长基因,并克隆到pcDNA3.1/V5-His A载体中,得到表达CD163受体的真核表达载体。猪肾PK-15细胞和幼仓鼠肾细胞BHK-21为PRRSV非易感细胞系,将表达载体转染PK-15和BHK-21细胞后接种PRRSV,然后利用PRRSV的核衣壳蛋白(Nucleocapsid,N)的单克隆抗体进行间接免疫荧光检测病毒的情况。结果发现表达CD163蛋白的PK-15细胞/BHK-21细胞能够感染PRRSV。并且发现,CD163在PK-15和BHK-21细胞中的转染效率是有很大差别的,CD163在BHK-21中的转染效率约为10%,而在PK-15中的转染效率约为30%。在此基础上,共转染全长的CD163和真核表达的非肌肉肌球蛋白ⅡA羧基端330AA的蛋白PRA于非易感细胞系BHK-21,按100TCID50接PRRSV-12#,通过间接免疫荧光实验发现病毒感染量增加。以上实验结果显示CD163和PRA在PRRSV感染过程中可能起协同作用;为了证明NMHCⅡA与PRRSV的关系,Marc-145细胞被PRRSV感染,并在不同时间内固定,通过共聚焦显微镜发现,PRRSV在4℃时,主要吸附在细胞的表面,转入37℃后,大约5min,开始进入细胞内部,随着时间的延长,病毒进入细胞,则发现NMHCⅡA的表达量增多,在60min时PRRSV完全进入细胞,NMHCⅡA的表达量也开始减少。上述实验揭示,NMHCⅡA可能与PRRSV感染细胞有关。使用Scanprosite软件分别对猪肺泡巨噬细胞(PAM)和Marc-145细胞的CD163蛋白结构和功能域进行预测。CD163有1个信号肽和9个SRCR结构域(Scavenger receptor cystein-rich domain),在SRCR 6和SRCR 7之间有一个富含脯氨酸-丝氨酸-苏氨酸(proline-serine-threonine,PST)区域。第二个PST区域则与SRCR 9相连。在CD163的羧基端含有一个跨膜区和一个短的胞质尾部。对于PAM,全长的CD163有1115个氨基酸组成,SRCR 1-9在CD163中的位置分别是54AA-151AA,161AA-258AA,268AA-365AA,375AA-472AA,480AA-577AA,585AA-682AA,721AA-818AA,828AA-925AA,931AA-1028AA;第一个PST区域所在的位置是683AA-720AA;第二个PST以及胞质尾部在1029AA-1115AA之间。对于Marc-145细胞的CD163,还没有人报道其全长序列,根据Vero细胞和PAM的CD163核苷酸序列的高度同源性,设计引物,经RT-PCR扩增出其全长序列是1117个氨基酸,首次在GenBank上注册,其基因序列号是JF753553。其SRCR 1-9在CD163中的位置分别是54AA-152AA,162AA-259AA,269AA-366AA,376AA-473AA,481AA-578AA,586AA-683AA,722AA-819AA,831AA-926AA,932AA-1029AA。第一个PST位置是684AA-721AA;第二个PST以及胞质尾部在1030AA-1116AA之间。通过SignalP信号预测软件预测知CD163氨基端1-16个核苷酸是信号肽,它指导着CD163的跨膜转移。根据CD163基因的结构特征以及相关的文献,本实验进一步将PAM和Marc-145细胞中的CD163受体基因各分割成3段。根据其来源不同将PAM中的CD163片段命名为P1,P2,P3; Marc-145细胞中的CD163片段命名为M1,M2,M3。经RT-PCR将各个片段克隆到PMD18-T载体,并将各个片段分别构建于真核表达载体pcDNA3.1/v5-His A和pEGFP-N1中。命名为Z-P1,Z-P2,Z-P3;Z-M1,Z-M2,Z-M3。Z-P1(798bp),在全长中的位置1-798bp;Z-P2在790bp-2046bp之间,共1257bp;Z-P3是2023bp-3345bp(1323bp);Z-M1含有777bp从1bp-777bp;M-2则从763bp-2049bp,共为1287bp;M3是1317bp,从CD163全长的2035bp到3351bp。分别将它们转染非易感细胞系BHK-21和PK-15,并对转染条件进行优化。结果发现P3和M3转染效率最高,P2,M2次之,P1,M1最低。同时,发现短片段的CD163在BHK-21细胞中的转染效率比在PK-15中的高。转染的细胞接入PRRSV后,通过PRRSV的N蛋白单克隆抗体,发现转染P1,M1的非易感细胞并不被PRRSV感染,而P2,M2和P3,M3则能够被病毒感染,并且发现转染P2,M2的非易感细胞病毒感染量要强于P3,M3。说明CD163的SRCR1-2并不参与病毒的感染,SRCR4-6对PRRSV复制起主要作用。为了进一步研究CD163在病毒感染中的作用以及PRRSV受体与受体、受体与PRRSV的蛋白、受体与非肌肉肌球蛋白(ⅡA,ⅡB)之间的关系,将分段的CD163片段进行原核表达。通过稀有密码子Graphical codon analysis分析软件,发现CD163序列中含有很多的稀有密码子,不利于蛋白的表达,为了蛋白表达,将P1,M1,P3,M3基因进一步的截短,截短的基因分别命名为Y-P1,Y-M1,Y-P3,Y-M3。其大小分别变为639bp,618bp,942bp和978bp,在CD163中的原位置依次变为160bp-798bp,160bp-777bp,2143bp-3084bp和2143bp-3120bp。将构建的重组质粒pET-28a-Y-(P1,P2,P3,M1,M2,M3)测序检测没有移码和突变,转入Rosetta感受态细胞并进行诱导表达。经SDS-PAGE分析,表达的重组蛋白大小分别约为24KD,46KD,35KD,22KD,46KD和36kD,与预期的结果相吻合。经Western-blot检测,所获得的各段重组蛋白均能与抗His-单克隆抗体发生特异性反应。将纯化的重组蛋白分别免疫Balb/c小鼠,三免后,获得高免的小鼠血清。根据血清病毒阻断实验,发现Y-P3和Y-M3小鼠血清在1/20和1/40的时候,能够阻断病毒感染,而Y-P2和Y-M2不仅没有阻断反而稍微增强,而Y-P1和Y-P2则与对照没有明显的区别;蛋白结合实验,发现纯化复性的CD163片段能够与Marc-145结合,通过荧光强度和细胞数量,证明在200μg/ml的条件下P1和M3结合最弱。蛋白结合实验证明CD163各片段可能结合Marc-145细胞上的某蛋白;为了证明CD163片段与NMHCⅡA的关系,通过免疫共沉淀技术以及Western Blot实验,发现M1和M3能够结合NMHCⅡA;在原核表达水平上,通过ELISA实验,证明GP5蛋白不与CD163各个片段相互作用,而当将GP5的几个抗原表位(GP5-1,GP5-2,GP5-3,Gp5-4)分段表达时,发现GP5-1与P1,P3和M1存在着一定的相互作用;GP5-2与P1,P3,M1有相互作用;GP5-4则与P1,M1;而GP5-3则与CD163各片段不存在相互作用。唾液酸粘附素Sn的分段蛋白Sn-1,Sn-3分别与CD163各个片段进行孵育,发现Sn-1与P1,M1,M3存在着相互作用,而Sn-3则与M1和M3有一定的相互作用。同时,还证明PRA与P3,M1相互作用,而PRB(非肌肉肌球蛋白ⅡB羧基端267AA处)则与P1,P2,P3和M1。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS) is a contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). This disease is also called“Blue Ear”. PRRSV mainly caused reproductive disorder in sows and respiratory problems in pigs of all ages and decreased in average weight gain. The virus also infected pregnant sows causing immnosuppresion and persistent infection. PRRSV shares a very narrow host tropism and a marked preference for cells of the monocyte/macrophage lineage. More specifically, in vivo, PRRSV infects subpopulations of differentiated macrophages, with alveolar marcophages being the major target cells during acute infection. In vitro, PRRSV replicates in primary cultures of alveolar marcophages and to some extent in peripheral blood monocytes. Furthermaore, the African green monkey kidney cells MA104 and cells derived Marc-145 are showed to sustain PRRSV infection. Both in PAM and in the monkey kidney-derived cell lines, the virus enters through a mechanism of receptor-mediated endocytosis. The first step in this entry process is the attachment to one or more cellular receptors. Until now, four receptors have been reported: sialoadhesin (Sn), Heparan sulphate (HS), Vimentin and CD163. HS can mediate virus attachment but no internalization; Sn is sufficient for both PRRSV attachment and internalization; CD163 maybe play a role in viral uncoating and genomic RNA releasing. Our supervisor immunoprecipitated a protein from MA-104 and PAM cell lysates by Mab2-5G2. Mab2-5G2 represents internal image anti-idiotope which mimicked the GP5 antigen inhibited the interaction between idiotypic anti-GP5 antibodies and GP5 antigen. This protein is about 230KD and it maybe a new receptor for PRRSV. Using mass spectrometric analysis and sequencing, we kown it is non muscle myosinⅡA. In mammals, three different isoforms of nonmuscle myosinⅡ,Ⅱ-A,Ⅱ-B,Ⅱ-C, are widely distributed throughout the entire organism. These proteins play a role in many fundamental cellular and development processes such as cell-cell adhesion, cell migration and cytokinesis. Soluble CD163 with activated T lymphocytes associates with cellular non-muscle myosin heavy chain type A. But so far, the viral proteins involved in the interaction with CD163 are unkown. And the counterpart on the CD163, which binds to the viral protein, is also unclear. The interaction of recetpor to receptor or CD163 to non-muscle myosin is also known litter. The aim of the present study is to investigate the interaction between CD163 and viral protein or non-muscle myosinⅡA/other PRRSV receptor.In order to perform the function of CD163 for PRRSV receptor or the relationship between CD163 and non-muscel myosin, the full length CD163 gene was generated by RT-PCR from porcine alveolar marcrophages (PAM) and Marc-145 cells. The gene was inserted into a eukaryotic expressional vector pcDNA3.1/V5-His A to yield recombinant plasmid pCD163 and mCD163. PK-15 and BHK-21 cells are PRRSV non permissive cell line. They were transfected by purified plasmid (pCD163, mCD163). Then the cells were exposured to PRRSV-12# strain for the infection of virus after 24h post transfection. In IFA test, the PRRSV infected cells could be detectable by anti-N monoclonal antibody. The result was that the PK-15/BHK-21 cells transfected CD163 could be infected by PRRSV. And the same time, It was found that transfection efficiency differed between cell lines, with 10% and 30% transfected cells for BHK-21 and PK-15 cells, espectively. On this base, non-permissive BHK-21 cells transiently expressing recombinant CD163 and PRA (It is sited on the carboxyl-terminal of non-muscle myosinⅡA ), either separartely or combined, were inoculated with the American prototype PRRSV-12# strain and analysed for expression of viral nucleocapsid protein and production of infectious virus. Immunofluorescence experiments showed that the cells were infected. In contrast, when CD163 and PRA were combined, clearly more infected cells were observed. This result showed that PRA can cooperate with CD163 for PRRSV infection. In order to prove the relationship of non-muscle myosinⅡA and PRRSV, The Marc-145 cells were infected by PRRSV at different time to be fixed. Confocal microscopical analysis of PRRSV during infection showed that PRRSV mainly attached the surface of cells at 4℃. When the cells were shifted to 37℃, the virus entered into the cells after about 5min. As time goes on, A majority of PRRSV entered into cells and non-muscle myosinⅡA (NMHCⅡA) became more and more. After 60 minutes, PRRSV entered cells completely, NMHCⅡA also became decreasing. This experiment illustrated that NMHCⅡA is associated with PRRSV infection.Predict the structure of protein and the the functional domain using the Scanprosite softwore. CD163 is a typeⅠmembrance protein composed of a signal peptide by followed by nine SRCR domain, with a thirty-five amino acid proline-serine-threonine (PST) rich region separating SRCR 6 and 7. A second PST rich region connects SRCR 9 with the transmembrane domain and a short cytoplasmic tail, which contains a functional internalization motif. For PAM, full length sequence is 1115 amino acid (AA). The site of SRCR from 1 to 9: 54AA-151AA, 161AA-258AA, 268AA-365AA, 375AA-472AA, 480AA-577AA, 585AA-682AA, 721AA-818AA, 828AA-925AA and 931AA-1028AA, seperatively. The first PST is located on 683AA-720AA, the second PST and the cytoplasmic tail is between 1029AA and 1115AA. For Marc-145 cells, there is no report about its full length sequence. According to the high homology of CD163 from PAM and Vero cells, the primer was designed and amplified the full length sequence by RT-PCR. The gene is 1117 amino acid and was registered in GenBank. The GenBank accession number was JF53553. Its SRCR 1 to 9 is located on 54AA-152AA, 162AA-259AA, 269AA-366AA, 376AA-473AA, 481AA-578AA, 586AA-683AA, 722AA-819AA, 831AA-926AA and 932AA-1029AA, seperatively on the full length sequence. The first PST was located on 684AA-721AA, the second PST and the cytoplasmic tail was located on 1030AA-1116AA. The signal peptide is from initiation codon to 16AA, it guides the transmembrane of CD163.According to the structure of CD163 and the related references, subsequently, the full length of CD163 from PAM and Marc-145 cells was dicided into a series of fragments. The fragments from PAM were named P1, P2 and P3, seperatively; the fragments from Marc-145 cells were called M1, M2 and M3, seperatively. These fragments were cloned into pMD18-T vector, and then inserted into eukaryotic expressional vector pcDNA3.1/V5-His or pEGFP-N1. These fragments were named Z-P1, Z-P2, Z-P3, Z-M1, Z-M2 and Z-M3, seperatively. Z-P1 was 798 base pair (bp) and lacated on 1-798bp, Z-P2 (1257bp) was between 790bp and 2046bp, Z-P3 (1323bp) was 2023bp-3345bp; For Z-M1, Z-M2 and Z-M3, It was lactaed on 1bp-777bp, 763bp-2049bp and 2035bp-3351bp. The length is 777bp, 1287bp and 1317bp, seperatively. These positive plasmids were transfected into non-permissive cells BHK-21 and PK-15 for the viral infecting test. The results were that P3 and M3 were high transfection efficiency compare to other fragments. P1 and M1 were in the last place, and it was found that the transfection efficiency was higher in BHK-21 than PK-15. The transfected cells were exposured to PRRSV-12# strain. In IFA test, the PRRSV could be detectable by anti-N monoclonal antibody. The experiment showed that P1 and M1 could not been infected by PRRSV and other plasmids could do. The infected degree of P2 and M2 was stronger than P3 and M3. These results illustrated that SRCR 1-2 were not required for PRRSV infection. SRCR 3-6 were involved in PRRSV infection.Make further investigation of CD163 for PRRSV infection /PRRSV receptors or receptor to viral proeins and receptor to NMHCⅡA. CD163 fragments were expressed into prokaryotic vector pET-28a (+). CD163 gene included many rare codons by the Graphical codon analysis. The P1, M1, P3 and M3 fragments were truncated again. The new fragments were named Y-P1, Y-M1, Y-P3 and Y-M3, seperatively. They were 639bp, 618bp, 942bp and 978bp and the sequence coded the amino acid from 160-798bp, 160bp-777bp, 2143bp-3084bp and 2143bp-3120bp. The new CD163 fragemts were correct by sequencing. The plasmids were transformed into Rosetta competent cell and induced by IPTG at different conditions. The recombinant proteins were about 24KD, 46KD, 35KD, 22KD, 46KD and 36KD by SDS-PAGE. Western blot test confirmed that the expressed recombinant protein could specifically react with the anti His-Tag mouse monoclonal antibody. The furified recombinant protein was immunized Balb/c mouse and obtained the hyper-immune serum. Y-P3 and Y-M3 mouse serum could block PRRSV infection Marc-145 cells at 1/20 or 1/40 dilution by the serum block assay, while Y-P2 and Y-M2 could improve the viral infection. For Y-P1 and Y-M1, there is no obvious difference compare to the positive control. For the protein binding cells experiment, CD163 fragment protein chould bind Marc-145 cells in IFA. P3 and M3 proteins bound weaker than other proteins at 200μg/ml concentration. This result clarified that CD163 fragments maybe bind some proteins on the surface of Marc-145 cells. M1 and M3 proteins could bind NMHCⅡA by co-immunoprecipitation and Western Blot. For ELISA test, GP5 protein could not bind CD163 fragments. For GP5 fragments protein (GP5-1, GP5-2, GP5-3, GP5-4), GP5-1 could bind P1, P2, M1 and M3; GP5-2 bound P1, P3 and M1; GP5-4 was interaction with P1 and M1, while GP5-3 could not do with CD163 fragments. For Sn fragmenrs Sn-1 and Sn-3, Sn-1 could bind P1, M1 and M3 and Sn-3 was interaction with M1 and M3.更多还原