【作者】 张文涛；

【导师】 张蕾；

【作者基本信息】 天津大学 ， 生物学， 2020， 硕士

【摘要】 当机体遭受微生物感染等外界刺激时会产生复杂的生物学反应,如炎症和氧化应激等。猪繁殖与呼吸综合症病毒(PRRSV)感染猪之后,会引起严重的间质性肺炎等炎症性疾病,对全球养猪业造成了重大损失。前列腺素还原酶(PTGR1)作为一种双功能酶,在炎症性疾病和氧化应激中发挥着重要的作用。但是PTGR1在PRRSV感染猪的过程中所发挥的作用,至今仍不清楚。本研究以PRRSV感染猪肺泡巨噬细胞为模型,初步探究了PTGR1在PRRSV致炎和氧化应激过程中发挥的作用,主要成果如下:(1)猪PTGR1基因的序列分析、克隆及抗体制备:通过MEGA7.0等生物信息学软件分析,确定了猪PTGR1的遗传进化关系并预测了蛋白质性质和结构;从猪肺泡巨噬细胞cDNA文库扩增获得猪PTGR1基因,构建原核表达载体,表达并纯化带有His标签的PTGR1重组蛋白,并用于免疫小鼠,制备了鼠源多克隆抗体。(2)猪PTGR1在PRRSV感染后的抗炎作用:以PRRSV感染猪肺泡巨噬细胞为模型,利用q RT-PCR、免疫印迹和流式细胞术证明了猪PTGR1在PRRSV感染过程中的表达呈现先上调后下调趋势;利用q RT-PCR和ELISA实验发现在PRRSV感染猪肺泡巨噬细胞中,PTGR1过表达后会抑制促炎性细胞因子TNF-α、IL-1β和IL-6的表达;PTGR1敲低后会促进TNF-α、IL-1β和IL-6的表达,证明PTGR1在PRRSV感染后,具有一定的抗炎作用。(3)猪PTGR1在PRRSV感染后的抗氧化应激作用:通过免疫荧光实验,确定了猪PTGR1主要表达于细胞质中,且定位于线粒体。以流式细胞术等实验发现在PRRSV感染猪肺泡巨噬细胞中,PTGR1可以一定程度上减少活性氧的表达和丙二醛的含量,证明PTGR1在PRRSV感染后,具有一定的抗氧化应激作用。综上,本研究成功克隆了猪PTGR1基因,利用纯化的重组蛋白制备了鼠源多克隆抗体;明确了猪PTGR1在细胞质中表达,并定位于线粒体上;猪PTGR1在PRRSV感染过程中的表达呈现先上调后下调的趋势;同时初步确定了在PRRSV感染后,猪PTGR1具有一定的抗炎和抗氧化应激作用,为研究其抗炎和抗氧化应激机制奠定了基础,也为治疗PRRSV引起的炎症性疾病提供了理论依据。更多还原

【Abstract】 When the body was exposed to stimulation caused by microbial infections and so on,complex biological responses were aroused such as inflammation and oxidative stress.Severe inflammatory diseases like interstitial pneumonia were induced by porcine reproductive and respiratory syndrome virus(PRRSV),which has caused great losses to the global swine industry.Prostaglandin reductase 1(PTGR1),as a bifunctional enzyme,plays an important role in inflammatory diseases and oxidative stress.However,the role of PTGR1 in pigs infected by PRRSV remains unclear.In this study,pig alveolar macrophages infected by PRRSV were used as a model,and the role of PTGR1 was initially explored in the inflammation and oxidative stress in PRRSV infection.The main results are as follows:(1)Sequence analysis,cloning and antibody preparation of porcine PTGR1 gene:The genetic evolution relationship of porcine PTGR1 was determined and the protein properties and structure were predicted by bioinformatics analysis softwares such as MEGA 7.0.Porcine PTGR1 gene was amplified from the porcine alveolar macrophage cDNA library.A prokaryotic expression vector was constructed,and the His-tagged PTGR1 recombinant protein was expressed and purified,which used to immunize mice and prepared mouse polyclonal antibodies.(2)Anti-inflammatory effect of porcine PTGR1 after PRRSV infection: Pig alveolar macrophages infected by PRRSV were used as a model.At the same time,the expression of porcine PTGR1 during PRRSV infection was first increased and then decreased by q RT-PCR,western blot and flow cytometry.The overexpression of PTGR1 inhibited the expression of proinflammatory cytokines TNF-α,IL-1β and IL-6in porcine alveolar macrophages infected with PRRSV by q RT-PCR and ELISA experiments.And knockdown of PTGR1 would promote the expression of TNF-α,IL-1β and IL-6.These results showed that PTGR1 could suppress inflammation after PRRSV infection to a certain extent.(3)Anti-oxidative stress effect of porcine PTGR1 after PRRSV infection: It was determined that porcine PTGR1 was mainly distributed in the cytoplasm and localized to mitochondria by immunofluorescence assay.The expression of PTGR1 could reduce the expression of reactive oxygen species and the content of malondialdehyde to a certain extent in porcine alveolar macrophages infected with PRRSV by Flow cytometry and other experiments.These results indicated that PTGR1 could suppress oxidative stress to a certain extent.In summary,porcine PTGR1 gene was successfully cloned,and purified recombinant protein was used to prepare mouse polyclonal antibody.The porcine PTGR1 was expressed in the cytoplasm and localized on the mitochondria.The expression of porcine PTGR1 during PRRSV infection was first increased and then decreased.At the same time,it was preliminarily determined that porcine PTGR1 had anti-inflammation and anti-oxidative stress effects on PRRSV infection to a certain extent,which was laid the foundation of later research on its anti-inflammation and anti-oxidative stress mechanisms and provided a theoretical basis for the treatment of inflammatory diseases caused by PRRSV.更多还原