【作者】 刘宁宁；

【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病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)归属于套式病毒目动脉炎病毒科属,由该病毒引起的猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)已成为世界养猪业生产中主要疫病之一。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。目前PRRS已经遍及全球主要养猪的国家和地区,给世界养猪业造成了巨大的经济损失。PRRSV侵入宿主细胞首先通过与细胞膜表面上的特异性受体结合,利用细胞内吞作用而感染易感细胞。现已报道的PRRSV感染Marc-145细胞的受体有CD163和Vimentin。CD163属于富含半胱氨酸超家族的清道夫受体蛋白,不但能够结合PRRSV,还能介导病毒在细胞内的复制。Vimentin是一个普遍存在于细胞内的波形蛋白,单克隆抗波形蛋白抗体可以阻断PRRSV对Marc-145细胞的感染,对PRRSV非易感的BHK-21和CRFK细胞在表达了猴的波形蛋白之后变成了PRRSV易感细胞,同时波形蛋白与其它中间细丝蛋白组成的多聚体可能参与PRRSV在细胞内的复制和转移。我的导师周恩民教授利用所制备的针对PRRSV GP5蛋白的单克隆抗独特型抗体(Mab2-5G2),从PRRSV易感细胞上发现了一个可能的PRRSV细胞受体蛋白。该蛋白为非肌肉肌球蛋白II型重链A(non-muscle myosin heavy chain II-A,NMHC II-A)。NMHC II-A广泛存在于自然界。在哺乳动物中,它是非肌肉肌球蛋白II型(NM II)重链的一个亚型,在整个细胞周期,包括细胞迁移、胞质分裂、细胞吸附和细胞形态变化过程中发挥重要的作用。已有研究证明NMHC II-A与T细胞上的趋化因子受体CXCR4结合,从而介导趋化因子的信号传导,可溶性CD163蛋白通过与T细胞上NMHC II-A的协同作用而结合T细胞,这些都说明了NMHC II-A在免疫反应中的作用。本文主要研究了NMHC II-A对PRRSV感染Marc-145细胞的阻断作用。首先根据NMHC II-A羧基端基因序列人工合成七条多肽,通过病毒中和实验证明其中三条多肽对PRRSV感染Marc-145细胞有阻断作用。为探求NMHC II-A羧基端的生物学功能,从Marc-145细胞中提取总RNA,利用特异性引物,经RT-PCR克隆得到NMHC II-A羧基端基因(命名为PRA)。将PRA连接至pET-28a(+)原核表达载体,经诱导表达得到重组的NMHC II-A羧基端蛋白(命名为PRA蛋白)。经分段表达蛋白,Western-blot和I-ELISA实验证明了PRA蛋白能与Mab2-5G2特异性结合,并且找到了比较准确的结合位点。通过间接免疫荧光实验证明了PRA蛋白能够与Marc-145细胞的细胞膜特异性结合。更重要的是,通过病毒中和实验,荧光病毒中和实验,荧光定量PCR实验证明了PRA蛋白能够在一定程度上阻断PRRSV对Marc-145细胞的感染。将重组质粒pEGFP-N1-PRA转染至Marc-145细胞,与未转染的Marc-145细胞相比,PRRSV感染转染pEGFP-N1-PRA的Marc-145细胞的能力降低了50%。另外,还证明了NMHC II-A的阻断剂(R)-(+)-Blebbistatin对Marc-145细胞的胞质分裂没有影响,但对PRRSV感染Marc-145细胞有阻断作用。这些实验结果充分证明了PRA蛋白在PRRSV感染易感细胞过程中的作用,为进一步研究PRRSV感染细胞的分子机制提供了一定的依据,也为进一步探求PRRS的防控提供了新的思路。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome (PRRS) is a disease caused by PRRS virus (PRRSV) which belongs to the family Arteriviridae. PRRS mainly causes reproductive disorder, including premature, abortion, fetal death, mummy and porcine respiratory syndrome at variety of ages with high mortality. At present, PRRS has prevailed in the main world while pig-raising country and districts. Nowadays the study to pathogenic and immunologic mechanism of PRRSV is not clear. There is also no proper way for controling of PRRSV infection.PRRSV has a highly specific cell tropism, both in PAM and in the monkey kidney-derived cell lines; the virus enters through a mechanism of receptor-mediated endocytosis. Until now, two PRRSV receptors (CD163 and Vimentin) have been described on Marc-145 cells. CD163 is a cellular protein in the scavenger receptor cysteine-rich superfamily, functioned as interacting with PRRSV and may determine the replication levels of PRRSV. Vimentin is an intermediate-filament protein, which is widely expressed in various cell types. Anti-vimentin Abs block PRRSV infection of Marc-145 cells. When simian vimentin was delivered to BHK-21cells and CRFK cells, they became susceptible to PRRSV. Vimentin is also thought to be involved in the replication and transportation of PRRSV inside the cell by forming a complex with the other components of the intermediate filaments.An internal image Mab2-5G2 which was generated against idiotypic antibodies specific for GP5 antigen of PRRSV identified a soluable protein prepared from Marc-145 cells. The protein was identified to be non-muscle myosin heavy chain II-A (NMHC II-A). NMHC II-A is distributed ubiquitously throughout nature. It is one isoform of NM II heavy chain and it plays a role in a variety of cellular processes including cell migration, cytokinesis, cell adhesion and cell shape change both during development and in the adult organism. An association between the C-termininal of CXCR4 and CCR5 and the motor protein NMHC II-A has been found and this biochemical association may have a key role in lymphocyte migration. Soluble CD163 binds with T lymphocytes coeffected with NMHC II-A. These have proved the function of NMHC II-A in immuno-reaction. In this article, we mainly study the block function of C-terminus NMHC II-A in Marc-145 cells to PRRSV infection. At first, seven peptides which located on the C-terminal of NMHC II-A were synthesized. Virus neutralization experiment showed that three peptides among the seven ones can block PRRSV infection of Marc-145 cells. In order to study the biological function of C-terminus NMHC II-A, total RNA was extracted from Marc-145 cells and specific primes were synthesized. After RT-PCR, the C-terminus NMHC II-A named PRA was cloned and sequenced, the Gene bank number was HM490008. PRA was then cloned to pET-28a (+) vector, after expression, PRA protein was obtained. Expression of truncated proteins, western-blot and I-ELISA experiments showed that PRA protein can specifically bind with Mab2-5G2; also the specific binding site was identified. IFA experiment proved PRA protein can specifically bind the membrane of Marc-145 cells. What was more important was that VN, FFN and Real Time PCR experiments identified that PRA protein can block PRRSV infection of Marc-145 cells in some degree. PRA was also cloned to pEGFP-N1 vector; the ability of PRRSV infecting the transfected Marc-145 cells was decreased by 50% in comparison with non-transfected cells. Otherwise, (R)-(+)-Blebbistatin which was the inhibitor of NMHC II-A not only can not affect the cytokines of Marc-145 cells but also block PRRSV infection by VN experiment. These results showed that C-terminus NMHC II-A can block PRRSV infection of Marc-145 cells. They provide a noval approach for further understanding of PRRSV infection and control of PRRS.更多还原