【作者】 薛霜；

【导师】 常惠芸； 伏小平；

【作者基本信息】 甘肃农业大学 ， 预防兽医学， 2010， 硕士

【摘要】 口蹄疫(Foot-and-Mouth Disease, FMD)是由口蹄疫病毒(Foot-and-Mouth Disease Virus, FMDV)引起的一种急性、热性和高度接触性传染病。FMDV主要利用VP1 G-H环上一段高度保守的精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp, RGD)序列与细胞表面的受体结合以感染细胞。研究表明至少有5种细胞识别机制,即细胞表面的整联蛋白αvβ3、αvβ6、αvβ1、αvβ8和硫酸乙酰肝素(Heparan Sulfate, HS)可作为FMDV的受体,有研究表明整联蛋白αvβ6可能是决定FMDV嗜性的主要功能受体。FMDV受体在动物体组织中的分布情况可以反映FMDV的感染途径、细胞嗜性及传播方式等,因此研究FMDV受体的生物学功能和组织分布情况对阐明其致病、逃避细胞免疫等机制具有重要作用。构建绵羊FMDV受体整联蛋白β6亚基配体结合域(LBD)的重组原核表达质粒pPRO/β6LBD,将其转化到BL21(DE3)宿主菌中并经IPTG诱导表达,SDS-PAGE鉴定重组蛋白的表达并利用镍离子亲和树脂对其纯化,通过酶联免疫吸附试验(ELISA)和Western-Blot方法分析鉴定表达产物。实验结果显示,成功诱导表达了目的蛋白,并利用pPROEXTM HTb上的6个组氨酸(His)标签纯化出高纯度的融合蛋白。纯化后的蛋白经间接ELISA和Western-Blot检测,与特异性抗体具有良好的特异性反应及抗原活性。构建真核表达载体pcDNA3.1(+)/β6S,将其转染至表达人αv亚基的结肠癌细胞株SW480,间接免疫荧光法(IFA)和Western-Blot方法检测整联蛋白αvβ6在转染细胞中的表达。IFA检测结果显示,转染细胞的细胞膜及细胞质内可见致密的绿色荧光,而未转染的SW480细胞和空载体转染细胞未检测到β6亚基的表达。Western-Blot结果显示,转染β6亚基的SW480细胞表达的重组蛋白可被抗FMDV猪源整联蛋白β6亚基的单克隆抗体所特异性识别。所构建的表达绵羊整联蛋白β6亚基的细胞系为深入研究整联蛋白αvβ6在FMDV感染中的生物学作用奠定了基础。间接免疫组化和间接免疫荧光法检测整联蛋白αvβ6在健康绵羊舌、鼻、唇等组织中的表达分布。结果显示,在羊舌、鼻、唇、蹄冠等多种被检组织的细胞膜及细胞质内均出现了特异性的阳性反应物,且阳性反应物主要见于各种上皮细胞。在阴性对照组中观察不到特异性的阳性反应物。表明FMDV整联蛋白受体αvβ6广泛分布于健康绵羊的多种器官组织的上皮细胞中。利用实时定量RT-PCR方法对健康羊体内不同组织中αv、β1、β6亚基的mRNA表达水平进行相对定量分析。实验数据显示,整联蛋白αv、β1亚基在各种组织中均有不同程度的表达,而整联蛋白β6亚基仅在部分组织中检测到了表达,其中在唾液腺、心脏、肺脏、鼻上皮等组织中的表达量最高,在下颌淋巴结、肝脏和脾脏中未检测到表达。结合免疫组化和免疫荧光试验结果总结αvβ6的表达分布规律,从而为深入研究FMDV的感染途径、细胞嗜性及整联蛋白αvβ6在FMDV感染中的作用奠定基础。更多还原

【Abstract】 Foot-and-mouth disease (FMD) is an acute, febricity and highly contagious disease caused by foot-and-mouth disease virus (FMDV), which is classified by Office International des Epizooties (OIE; World Organisation for Animal Health) as the first one of OIE List A diseases. It has been demonstrated that there are at least five receptors for FMDV, including integrinαvβ3,αvβ6,αvβ1,αvβ8 and heparan sulfate(HS). Integrinαvβ6 serves as the major receptor that determines the tropism of FMDV for the epithelia normally targeted by this virus.The fragment coding sheep integrinβ6 ligand-binding domain was amplified by PCR from the recombinant plasmid pGEM-β6. After doubly digested with BamHⅠand XhoⅠ, theβ6LBD fragment was subcloned into prokaryotic expression vector pPROEXTMHTb which was doubly digested with same enzymes. The recombinant expression plasmid pPRO/β6LBD was constructed and transformed into E.coli BL21(DE3) and induced by IPTG. The expression of fusion protein was detected by SDS-PAGE and purified by Ni-NTA His.Bind resins. The expressed product was identified by ELISA and Western-Blot assay. SDS-PAGE demonstrated that the fusion protein was expressed efficiently as inclusion body in E.coli with the expected molecular weight of 33KDa. ELISA and Western-Blot assays showed that the recombinant protein has the good antigenicity and specificity, which will lay a foundation for further research on distribution and the role of the ovine integrinβ6 subunit in FMDV infection.Eukaryotic expression primers forβ6 ligand-binding domain were designed and theβ6 gene was amplified, using pGEM-β6 as temple. The PCR product was ligated with the vector pcDNA3.1(+) to construct eucaryon expression vector pcDNA3.1(+)/β6S, and then transfected into Human Colon Carcinoma Cell line(SW480). The expression of integrinαvβ6 was detected by indirect immunofluorescence assay(IFA) and Western-Blot assay. IFA indicated that green fluorescence was appeared in cell membrane and cytoplasm of the pcDNA3.1(+)/β6S transfected cells, whereas, it can not be observed in SW480 and pcDNA3.1(+) transfected cells. The result of Western-Blot showed that the recombinant protein was expressed and could be recognized by the monoclonal antibody against porcine integrinβ6 subunit LBD.The integrin receptors of FMDV have been studied extensively in cell culture. However, the roles of the various integrins in determining the tissue tropism and pathogenesis of FMDV have not yet to be established. Here, we present analyses, using indirect immunohistochemistry assay (IHA), immuno?uorescence confocal microscopy and Real-time RT-PCR, ofαvβ6 expression within the different tissues that are normally targeted by FMDV in sheep. The results showed that idio-positive reaction was appeared in cell membrane and cytoplasm of tongue, nose, lip and so on,αvβ6 was mainly expressed on the surfaces of the epithelial cells at the sites where FMDV is known to replicate at a high level during a natural infection. Results of the Real-time RT-PCR study showed that theαv,β1 mRNA were transcripted at high or low level in the different tissues. In constrast, theβ6 mRNA is only restricted in the partial tissues, and at high level in the salivary gland, heart, lung and nasal epithelium, and not detected in the mandibular lymph node, liver and speen. These data suggest that integrinαvβ6 serves as the major receptor that determines virus tropism, andαvβ6 is expressed constitutively at levels sufficient to allow initiation of infection, which will lay a foundation for further understanding of the roles of integrinαvβ6 in FMDV infection.更多还原