【作者】 江一波；

【导师】 张淑君； 孔德信；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 猪繁殖呼吸综合征PRRS (Porcine reproductive and respiratory syndrome,又称蓝耳病)是由PRRSV (Porcine reproductive and respiratory syndrome virus)感染引起的,目前在世界范围内广泛流传,并且造成了感染猪繁殖障碍、免疫力低下、发病率和死亡率较高,给养猪业造成巨大经济损失,并带来肉产品安全及环境安全等隐患。PRRSV感染而导致猪免疫抑制及其免疫机制复杂,至今还没有防治PRRSV的有效药物与疫苗。PRRSV具有极强的宿主偏嗜性,只感染家猪和野猪,也不感染其它动物。目前在猪PRRSV主要感染猪宿主PAM (Porcine Alveolar Macrophage)肺泡巨噬细胞,也从该细胞中鉴定出来了有CD163, CD169(SN, Sialoadhesin)等受体分子。在猪的SN受体具有黏附病毒唾液酸分子,介导病毒感染细胞的能力,受体基因位点直接影响与PRRSV结合力,对PRRSV受体研究是致病机理和抗病机理研究的趋势之一。因此,本研究从PPRSV细胞受体SN基因角度,以受体分子与PRRSV相互作用为重点,分析受体SN基因与PRRSV结合的重要氨基酸位点,研究该位点在与PRRSV结合和在PRRSV物种感染偏嗜性中的作用,探讨受体和PRRSV的相互作用的机理,为抗PRRSV感染的防治药物研制提供分子靶标,为抗病分子育种提供参考。一、利用生物信息学预测与PRRSV结合的SN氨基酸位点:1、将鼠SN中PDB数据库所有SN蛋白模板和唾液酸的配体进行了结构重叠和比对,确定了在6.5A范围内对结合唾液酸最关键的氨基酸；明确了唾液酸分子衍生物都有差异,但是鼠唾液酸分子都有保守的Neu5Ac的母核,而且在不同的PDB(Protein Data Bank)模板中,该母核位置保守和其形成氢键相互作用的氨基酸也是保守的。2、根据猪SN基因cDNA及氨基酸序列,在Signal IP信号肽预测和蛋白结构功能Smart预测的网站上进行预测,参照PDB上鼠SN的模板结构,猪SN信号肽剪接位点可能在第19-20位氨基酸之间。3、利用Smart对SN蛋白的结构和功能进行预测,确定猪SN参与病毒唾液酸结合的功能区域为N末端的150个氨基酸以内的区域。4、利用糖基化预测网站和软件,预测猪pSN的S107, T3,T78等位点,在PRRSV感染的过程中可能发生了糖基化。5、根据猪SN和鼠SN的多序列进行比对和结构重叠的结果,R97,R105氨基酸在PRRSV感染的过程中可能发生侧链方向的改变,而且提供氢键以利于病毒唾液酸的结合。W106,W2氨基酸在结合唾液酸时,因为巨大侧链空间阻碍和方向不变,可能限制PRRSV的唾液酸母核结合SN时的方向。6、利用多序列比对和同源模建及分子动力学优化等,分别构建了猪SN和牛SN的蛋白结构模型；然后与PDB数据库中报道的鼠SN的蛋白结构模型进行了结构比对和重叠；再用Chimera软件构建分子突变体,最后观察猪SN、牛SN蛋白结构的变化。结果表明：猪SN蛋白分子表面形成一个明显的较深的孔洞,而牛的则不明显；将猪SN的S107突变为牛SN的V107时,猪SN的孔洞空间缩小,反之将牛SN的V107突变为猪SN的S107时,牛SN的孔洞空间略有增加,因此预测猪SN的S107在PRRSV结合时可能具有重要作用。7、通过比较分析猪SN的结构,发现猪SN相对于鼠和牛的SN有其特异性,其表面的107位点是特异的亲水氨基酸；在结合病毒唾液酸的过程中,很可能提供了氢键和一个特异的亲水区域以容纳病毒的唾液酸,从而增加了病毒和猪SN的结合能力,这也许是猪个体间存在PRRSV易感性或抗性差异、不同物种间(猪、牛、鼠等)存在PRRSV感染偏嗜性差异的原因之一。此外,猪SN的2,44,45,97,105,106,109等位点的氨基酸,都参与了亲水空间区域的形成。二、利用分子生物学技术分析和验证生物信息预测的PRRSV结合的重要SN氨基酸位点及其作用：1、为了研究生物信息学预测的SN受体氨基酸位点(3、78和107)与PRRSV相互作用,根据猪pSN受体和牛cSN受体(不结合PRRSV唾液酸)序列,利用pEGFP-N1载体和定点突变技术分别构建了猪和牛SN的2个野生型和4个突变体。它们包括2个野生型(pSN-GFP、cSN-GFP)、4个突变体(pSN-S107V-GFP, pSN-T3V-GFP, pSN-T78V-GFP和cSN-V3T-V78T-V107S-GFP)。2、利用pEGFP-N1载体分别构建了GFP和SN融合蛋白的表达载体,并在293T细胞中进行融合表达,利用采用超滤离心技术从细胞培养液中获得了纯度较高的6个SN-GFP融合蛋白(pSN-GFP、cSN-GFP、pSN-S107V-GFP, pSN-T3V-GFP, pSN-T78V-GFP和cSN-V3T-V78T-V107S-GFP),用于在分子水平研究受体SN与PRRSV的结合力的研究。3、在SN-GFP载体的后端加上SN的跨膜片段M,分别构建了6个表达SN-GFP-M载体(pSN-S107V-GFP-M, pSN-T3V-GFP-M, pSN-T78V-GFP-M, cSN-V3T-V78T-V107S-GFP-M, pSN-GFP-M, cSN-GFP-M)和1个对照GFP-M载体,在293T细胞中表达7个SN-GFP-M融合蛋白,用于在细胞水平分析受体SN与PRRSV的相互作用的分析。4、利用WB (Western Blot)技术证明和鉴定了本研究所表达蛋白分别为6个SN-GFP融合蛋白、7个SN-GFP-M融合蛋白。5、分别利用FAR-WB (FAR-Western Blot)技术、ELISA(Enzyme Linked Immunosorbent Assay)技术分析了SN-GFP蛋白和PRRSV的结合活性。分别分析了2个SN野生型融合蛋白(pSN-GFP、cSN-GFP)和4个突变体融合蛋白(pSN-S107V-GFP, pSN-T3V-GFP, pSN-T78V-GFP和cSN-V3T-V78T-V107S-GFP)与PRRSV的结合力,结果表明：猪野生型pSN-GFP蛋白的结合高于其它5种蛋白的结合,发生突变后的猪SN蛋白(pSN-S107V-GFP, pSN-T3V-GFP, pSN-T78V-GFP)的结合力都降低,特别是pSN-S107V-GFP蛋白结合力显著的下降；而牛野生型cSN-GFP蛋白不能与PRRSV结合,但发生突变(cSN-V3T-V78T-V107S-GFP)蛋白能与少量PRRSV结合。6、利用免疫细胞荧光技术,分析了SN-GFP-M蛋白和PRRSV的结合活性。在293T细胞中分别表达了6个带SN的跨膜片段M的SN-GFP-M和1个对照GFP-M蛋白,利用免疫细胞荧光技术,分别探讨了猪和牛SN野生型融合蛋白、3个猪突变体融合蛋白、1个牛突变体融合蛋白、1个对照GFP-M蛋白与PRRSV的结合力,试验结果与利用FAR-WB技术分析的结果相类似。7、以上所有结果表明猪SN基因中第T3、T78和S107氨基酸在与PRRSV结合中具有一定的作用,特别是S107位具有重要的作用。8、猪pSN野生型具有强的PRRSV结合能力,本试验将猪相应氨基酸替换成牛的氨基酸,猪pSN突变型pSN-T78V、pSN-T3V降低了结合PRRSV能力,pSN-S107V显著地降低了结合能力。而将牛SN进行cSN-V107S突变,将牛替换成了猪的氨基酸,生物信息分析显示可以恢复部分的结合PRRSV的能力；并且牛的突变体cSN-V3T-V78T-V107S-GFP在一定程度上也能与PRRSV结合,因此推测：这些位点在不同猪个体对PRRSV敏感性或抗性存在差异、不同物种对PRRSV感染偏嗜性中具有一定的作用。更多还原

【Abstract】 PRRS (Porcine reproductive and respiratory syndrome)disease is caused by PRRSV (Porcine reproductive and respiratory syndrome virus)infection worldwide. Infection of PRRSV for pig leads to reduce reproduction, low immunity, high mortality. It causes meat product safety, environment and serious problems.The mechanism for PRRSV infection immunity and immunity restraint is very complicate and unclear. The traditional vaccine and drug is ineffective for PRRSV. The PRRSV has a strong host tropism which can infect domestic and wild pigs but not infect other animals. PAM (Porcine Alveolar Macrophage) cells are main host cell in pigs, and CD163and CD169(SN, Sialoadhesin) are as main receptor molecules on PAM cells. pSN (Pig SN) receptor adheres to sialic acid molecules on the surface of virus, which mediates the virus-infected cells. Some sites on receptor directly affect receptor binding activity of PRRSV, so it is a direction for mechanism research on infection immunity and immunity restraint.Interaction of receptor and PRRSV is the novel idea of this paper to analysis specificity of pSN molecule, which is an important amino acid sites for PRRSV infection tropism. Therefore, the receptor resistant mechanism is the main focus of this study which is also an important molecular target and reference of drug design, genetics and breeding subject.1. Predicted PRRSV binding site on SN based on bioinformatics analysis(1) According to the mSN (mouse SN) PDB database, SN protein templates and sialic acid ligands structure of the template structure were superposed and compared to critical amino acid within6.5A. The results showed that the sialic acid molecule derivatives had great variation, but the mouse sialic acid molecule has a conservative Neu5Ac mother nucleus. In PDB (Protein Data Bank) template, the position of the mother nucleus and the formation of hydrogen bonding interactions of amino acids is also conservative. (2)According to pig SN gene cDNA and protein amino acids sequence, pSN signal peptide cutting sites were predicted on Signal IP and Smart protein structure domain websites. Referring the PDB on mSN template structure, pSN signal peptide cutting sites might be were between amino acids19-20sites.(3)By Smart protein domain prediction, pSN and virus sialic acid-binding region was within the N-terminal,150amino acid region on pSN.(4)Glycosylation prediction has showed on pSN S107, T3, T78etc. Glycosylation may only happen during PRRSV infection.(5)Based on the results of pig and mouse multiple sequence alignment and structure superimposition result. R97, R105amino acid in PRRSV binding process, they may change of side chain direction and hydrogen. And, R106, R2amino acid, with huge side chain as hindrance. They could limit binding direction during the PRRSV sialic acid nucleus when sialic acid combined with SN.(6)Based on multiple sequence alignment, homology modeling and molecular dynamics optimization steps, pSN and cSN (cow SN) protein structure homology models were built. pSN and cSN were superposed with mSN template. SN some of amino acid mutants were built using chimera software for observation. The results determined a cavity could be formed on the surface of pSN and it seems that cavity was not on the surface of mSN and cSN. When S107V on pSN was replaced for V107, cavity became small. On the contrary, it would improve more space for interaction. So,S107was very key site for PRRSV binding.(7)To observe the structure specificity among pSN, mSN and cSN, their surface of107was a specific hydrophilic amino acid, in the process of binding the virus sialic acid. It was possible to provide a hydrogen bond and a specific hydrophilic region to accommodate the sialic acid of the virus. Thereby, it would increase the binding capacity of the virus and pSN. Perhaps, this was the difference in sensitivity and resistant reason between species. Moreover, other amino acid sites of the pig2,44,45,97,105,106,109, etc., were also involved in the formation of the hydrophilic region. 2. Validated key PRRSV binding sites on SN and their function using experiments(1) In order to analyze3,78and107sites role on SN interaction with PRRSV in bioinformatics, we used and site-directed technology the pEGFP-Nl vector for expression SN and GFP fusion protein in293T cells. According to pSN and cow cSN receptor (cSN not binding with PRRSV sialic acid) sequence, results showed2kinds of wild type pSN-GFP, cSN-GFP and4kinds of mutant type pSN-S107V-GFP, pSN-T3V-GFP, pSN-T78V-GFP, SN-V3T-V78T-V107S-GFP.(2) By pEGFP-N1vector, SN-GFP expression vector were built.6kinds of high dose of chimera protein were expressed in293T cells and secreted into cell medium and abstracted by ultra filtration and centrifugation. They are pSN-GFP, cSN-GFP, pSN-S107V-GFP, pSN-T3V-GFP, pSN-T78V-GFP and cSN-V3T-V78T-V107S-GFP, and can be used in molecular level of examination about binding activity of SN and PRRSV.(3) In order to study the SN and PRRSV interaction on cell level, an M transmembrane domain was constructed into SN-GFP vector, so that expression of the vector SN-GFP-M were performed including M transmembrane fragments. They were pSN-S107V-GFP-M, pSN-T3V-GFP-M, pSN-T78V-GFP-M, CSN-V3T-V78T-V107S-GFP-M, pSN-GFP-M, cSN-GFP-M and GFP-M control.(4) Total6kinds of SN-GFP and7kinds of SN-GFP-M target protein were detected by anti-GFP antibody on WB (Western Blot.)(5) Using for Far-Western Blot and ELISA (En2yme Linked Immunosorbent Assay) method, SN wild type protein and the mutant type were detected in vitro interaction with the PRRSV virus including2kinds of wild type pSN-GFP, cSN-GFP and4kinds of mutant type pSN-S107V-GFP, pSN-T3V-GFP, pSN-T78V-GFP and cSN-V3T-V78T-V107S-GFP. The result shown that the binding activity of pSN wild type was better than that of other5kinds of SN-GFP chimera protein. Mutant type pSN-S107V-GFP, pSN-T3V-GFP, pSN-T78V-GFP binding activity were reduced, especially on pSN-S107V-GFP. cSN wild type could not bind with virus well, but another mutant type cSN-V3T-V78T-V107S-GFP also were bind with little PRRSV. (6)Immunofluorescence experiments for PRRSV and SN-GFP-M interaction also were studied. The6kinds of SN-GFP-M vector and a contrast GFP-M containing the SN transmembrane M segments were transfected and expressed into293T cells to examine the binding ability with PRRSV by Immunofluorescence experiments. The pig and cow SN wild type chimera protein,3kinds of pSN mutant type, a cSN mutant type, and a contrast GFP-M were performed binding activity examination. The result was similar with FAR-WB.(7) The above experimental results showed that pSN sites of T3、T78and S107were of great importance for binding with PRRSV, especially S107.(8) The pSN wild-type and the PRRSV virus had a strong binding ability. In our experiment, some amino acids were replaced from pig to cow in pSN. Pig mutant type pSN-T78V, pSN-T3V especially pSN-S107V reduced binding ability of the virus. But, cow cSN of the107replaced for pig as cSN-V107S, it could restore part of the binding ability of the virus from bioinformatics analysis. The mutant type cSN-V3T-V78T-V107S-GFP also could bind with some PRRSV. So, it could be inferred that these key sites are very important and special for resistance in different breeds of pig and other species infection by PRRSV.更多还原