【作者】 张璐；

【导师】 周恩民；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2011， 硕士

【摘要】 猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)引起的高度接触性传染病。该病主要引起妊娠母猪早产、流产、死胎、木乃伊胎等繁殖障碍以及各年龄段猪的呼吸道症状和高死亡率。PRRSV进入Marc-145细胞的过程是依赖受体进行的,目前已鉴定出在细胞上存在PRRSV的4个受体:硫酸乙酰肝素(heparin sulfate. HS )、唾液酸粘附素(sialoadhesin. Sn)、CD 163分子和波形蛋白Vimtin。HS, Sn和CD 163三者之间的关系大体为:HS主要作用是吸附PRRSV到PAM细胞上,Sn介导PRRSV的内吞。CD 163和波形蛋白可能起协助Sn内吞、PRRSV脱衣壳和将病毒基因组RNA释放到细胞浆的作用。我们的研究团队制备了针对PRRSV GP5抗体的单克隆抗独特型抗体(Mab2-5G2),并利用Mab2-5G2从PAM和MA-104细胞中提取出非肌肉肌球蛋白II型重链A蛋白(Nonmuscle myosin heavy chain II-A,NMHC II-A)。随后的研究证实NMHC II-A和NMHC II-B(NMHC II的不同亚类)均与PRRSV感染细胞有关。为了更加深入的研究PRRSV病毒感染细胞的机制,本为以非肌肉肌球蛋白II型重链B(Nonmuscle myosin heavy chain II B,NMHC II B)羧基端为研究对象,进行了以下三部分的实验:第一,为了得到能够特异性识别NMHC II-B的单克隆抗体,根据Marc-145细胞上NMHC II-B羧基端的蛋白质序列,合成多肽,免疫Balb/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,用ELISA筛选阳性克隆;制备腹水并纯化;用间接ELISA方法和IFA检测纯化抗体的特异性。结果表明获得2株能稳定传代并分泌特异性抗体的杂交瘤细胞系;2株细胞系所产生的抗体能够特异性的与NMHC II-B羧基端蛋白反应而且能够与Marc-145细胞产生特异性的结合。两株单抗的制备及鉴定为以后研究易感细胞上NMHC II-B与PRRSV感染的关系奠定了基础。第二,提取Marc-145细胞的总RNA,用RT-PCR的方法克隆出Marc-145细胞上NMHC II-B羧基端长为804bp的基因PRB,并构建pET-28a-PRB重组质粒。将此质粒转化至Rosetta表达感受态中,用IPTG诱导表达出大小约35KD的蛋白。将该蛋白纯化并免疫Balb/c小鼠,得到特异性的抗体。用IFA方法检测复性的纯化PRB蛋白能够和Marc-145结合;PRRSV与复性的PRB蛋白孵育后再感染Marc-145细胞,其感染能力显著性增强。第三,构建pEGFP-N1-PRB重组质粒,分别转染Marc-145细胞和BHK-21细胞,得到了较高的转染效率,为研究PRB蛋白和PRRSV感染细胞的关系打下了坚实的基础。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome (PRRS) is a contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRS mainly caused reproductive disorder, including of premature, abortion, fetal death, mummy and porcine respiratory syndrome at variety of ages and high mortality. PRRSV has a restricted cell tropism in its host (pig).PRRSV enter into cells were dependent on cell receptors.Several candidate molecules have been identified to be the receptors/co-receptors for PRRSV entry including heparin sulfate, CD163, sialoadhesin and vimtin. The relationship of these receptoers were general as follows: In order to make future exploration of the mechanism of PRRSV infection, this research take C-terminal of NMHCⅡB as target to develop 3 parts experiment: HS adsorpted PRRSV to PAM cells; Sn mediated swallow of PRRSV; CD163 and vimentin maybe assist Sn swallow, PRRSV strip in shell and viral genome RNA released into the role of cell plasma.Our research team preparted the Mab2-5G2, an anti-idiotype monoclonal antibody against GP5 of PRRSV, and pulled down Nonmuscle myosin heavy chainⅡ-A(NMHCⅡ-A) by Mab2-5G2. Whereafter, both NMHCⅡ-A and NMHCⅡ-B(different sub-type of NMHCⅡ) were verified related to PRRSV infection cells. In order to make future exploration of the mechanism of PRRSV infection, this research take C-terminal of NMHCⅡB as target to develop 3 parts experiment:Firstly, in order to obtain monoclonal antibodies (Mabs) against nonmuscle myosin heavy chainⅡ-B (NMHCⅡ-B), a synthetic peptide from NMHCⅡ-B C-terminal was used to immunize Balb/c mice. The hybridomas secreting specific Mabs were selected by the ELISA and IFA. The results showed that two Mabs were produced which could react with C-terminus of NMHCⅡ-B and bind with Marc-145 cells. The two Mabs produced and characterized in this study can be used for future study of the role of NMHCⅡ-B in PRRSV infection of permissive cells.Then extracted total RNA of Marc-145 cell and amplified PRB gene, a 804bp gene at C-terminal of NMHCⅡ-B. The PET-28a-PRB gene was been construsted and the a 35KD protein were expressed by Rosetta compETence cells according to IPTG inducing. The PRB protein were injected into Balb/c mouse, and specific antibody were obtained. IFA results showed that PRB protein can bind with Marc-145 cells not noly the membrane but alao the nuclear and it also can induce PRRSV infect Marc-145 cells.pEGFP-N1-PRB plasmid was construsted, and successfully transfected into Marc-145 and BHK-21 cells which were PRRSV permissive and non-permissive cells respectively. The construction of transfected plasmid established the foundation for researching the relationship between C-terminal of NMHC II-B and PRRSV infection.更多还原