【作者】 陈艳玲；

【导师】 高丰；

【作者基本信息】 吉林大学 ， 基础兽医学， 2009， 硕士

【摘要】 猪血凝性脑脊髓炎病毒(Hemagglutinating encephalomyelitis virus,HEV)是引起猪血凝性脑脊髓炎的重要病原,主要引起仔猪呕吐、衰竭或中枢神经系统障碍。1～3周龄的仔猪感染HEV后,死亡率通常高达20～100%。然而,目前还不清楚HEV的细胞受体及由受体介导的感染机制。因此,研究HEV敏感细胞受体的结构组成及其与病毒间的相互作用机制等,对了解病毒感染过程、致病机理以及该病的预防与治疗等都非常重要。为初步鉴定猪血凝性脑脊髓炎病毒(HEV)在敏感细胞上的受体基因,本研究根据本课题组已从构建的PK-15细胞cDNA文库中筛选到一段430bp的基因片段PK-CP1为基础,根据该基因序列设计特异性引物,以含PK-CP1基因的噬菌体DNA为模板,利用PCR反应扩增长411bp的基因片段,将其亚克隆到原核表达载体Pet-28a(+)中,构建重组质粒pET-28a-PKCP1,并转化到宿主菌E.coli BL-21中进行诱导表达,利用SDS-PAGE和Western-Blot对表达产物进行分析和鉴定。重组蛋白经Ni-NTA亲和层析柱纯化,并进行稀释透析复性,用纯化的重组蛋白免疫BALB/c小鼠制备出抗pkCP1蛋白的多抗,之后应用间接免疫荧光对目的蛋白在PK细胞和SK细胞的分布进行定位。结果表明,该基因在原核系统中获得高效表达,表达产物以包涵体形式存在;western-blot试验表明表达的蛋白具有良好的反应原性;免疫荧光检测表明PK-CP1蛋白位于PK-15细胞和sk细胞的细胞上,这些研究结果初步鉴定出PK-CP1基因片段为HEV在PK-15细胞和sk细胞上的结合基因,可能的受体基因,从而为进一步研究HEV感染细胞的过程或其致病机制奠定了基础。更多还原

【Abstract】 Porcine hemagglutinating encephalomyelitis is caused by hemagglutinating encephalomyelitis virus (HEV). It is a acute and strong contagious disease between swines. The mortality rate for piglets under 3 week old high reach 20%-100%. However, the receptor of HEV and the machnism of HEV infect cell are still not clear .we are researching on the frame compose of HEV recepotor and the mechanism of action from HEV to sensitivity cell .because it is very significant to understand the process of effect , mechanism of pathogenesis and to cure virus disease.The virus first to infect cell recptor ,then a series of dymanics will start to help virus enter in to cell.the receptor is proteinic molecule located on the cell membrane or inner cell can specific bind and interact with some definite chemical signal that usually called ligandin .They have syntrophic functions of identification combining transfering signal and producing correspond biological effect .the research and study are not only limite to interaction between recptor and ligandin but also comprised the activate mechanism of recptor .After that we have to research how to start the mechanism of transfering and passway signal and how to cause the correspond biological effect .The recptors of animals and human cells are not prepared to virus invading .they have normal physiologic function .the recptor located on the host cell surface is adsporptioned by virus and recongnized by proteins .At present , Almost all the recptor relatived to host cell membrane we have known are lipoprotein and glycoprotein which participate to execute respective normal physiologic function.some research indicate. Some research proved that coronavirus particles bind to cell recptor by spike protein and hemagglutinin(hemagglutinin-esterase,HE)protein which were located on the surface of coronavirus ,then invad host cells by membrane fusion and cell internalization.while distribution of receptor host cells in different kinds of host cells are correlated with extent of virus infect host animals .To preliminay identify the HEV receptor gene in sensitive cells ,this research was based on the collaborators successed constructed the cDNA library of PK-15cell and screened a segment new possible receptor gene,430bp,according to this gene containing PKCP1,the 411bp gene containing PKCP1 was amplified by PCR using phage DNA as template.The recovered target fragment was subcloned into Pet-28a(+)prokaryoutic high-expressing vector,was transformed to E.coli BL-21 competent cell, then picked white bacterial colony to extract plasmid. The recombinant plasmid was sequenced after the PCR and enzyme digension identification.,the result showed that The recombinant plasmids pET-28a- PKCP1 was constructed and transformed into E.coli BL-21. And the cells were cultured and induced by IPTG. Then the pET-28a-PKCP1 protein was checked by SDS-PAGE and Western blot analysis of the cell lysates.Target protein and multimetric histidine vector were confluently expressed, and a target band is approximative of 14.9kDa. Then we studied the expressing condition of recombinant protein. There are more recombinant proteins expressing with more time and 5h after being induced, recombinant protein reach its maximum. The expressing quantity of recombinant protein is influenced by the concentration of IPTG In the condition of 1mmol/L IPTG being induced 5 hours, at 24℃the extrinsic protein of bacteria is more than in any other conditions. In order to obtain active protein of nature conformation, we optimized the condition for extraction and purification of recombinant protein from cytorrhyctes of bacteria. In the present study, we obtained high purity recombinant protein through Ni2+ affinity chromatography. The purity of protein reached 88.7% by renature; the purified pET-28a-PKCP1 protein was checked by Western Blotting with anti-his goat monoclonal antibodies, A target band is approximative of 14.9kDa. It demonstrated that pET-28a-PKCP1 gene can be expressed in prokaryotic expression system and this protein have biologic activityTo primary idenitify whether the screened PKCP1 gene is a receptor gene of HEV and the distribution in cells, Immunoprotection response induced by PKCP1 protein against cell in BALB/c mice were immunized with PKCP1 protein by intraperitoneal injection.The immunoprotection of PKCP1 protein against PK-15 and SK cell were studied .the localization of PKCP1 in PK-15 and SK cell were determined by immunofluorescence using antibodies for PKCP1 .Immunofluorescence staining of PK-15 and SK cell showed that PKCP1 protein was present in the cytoplasm of PK-15 and SK cell. It primary idenitified the screened PKCP1 gene is a receptor gene of HEV in PK-15. Moreover, these results establish a foundation of studying the infectious mechanism and disoperation of cell or organism mediated by receptors .更多还原