猪源PTX3蛋白的真核表达及其对猪链球菌2型的抗菌作用研究

发布时间：2018-01-24 00:05

本文关键词： 猪源PTX3蛋白猪链球菌2型真核表达 pVAX-ptx3 抗菌作用　出处：《南京农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：猪链球菌(Streptococcussuis,SS)是一种重要的人畜共患病病原菌。目前对于该病的治疗主要采用抗生素疗法,但长期大量使用抗生素必然存在抗生素耐药性的风险;另外针对该病,临床上仍缺乏有效预防猪链球菌的疫苗,故寻求能替代的治疗方案以及抗微生物化合物是备受关注的。PTX3是第一个被确认的长正五聚蛋白,具有结合到某些病原微生物的能力,PTX3也可以促进细胞吞噬功能以及随后病原体的清除。另外,PTX3具有充当调理素的治疗效果,可识别微生物成分,引发炎症反应的放大。本试验首先通过构建原核表达载体PET-32a-ptx3,获得原核表达猪源PTX3蛋白,并制备兔抗原核表达PTX3蛋白血清;随后又构建重组真核表达载体pIRES-EGFP-ptx3,并转染至真核表达宿主CHO细胞中,通过G418(750μg/ml)筛选获得能够稳定、大量表达PTX3蛋白的单克隆细胞株,并采用Ni-NTA亲和层析方法纯化细胞培养上清获得纯度较高、性质稳定的真核表达猪源PTX3蛋白;随后成功构建了重组真核表达载体pVAX-ptx3,将纯化的重组质粒肌肉注射BALB/c小鼠(1OOμg/只),在接种后第1,3,5,7,15,20天取外周血及注射部位肌肉,分别用RT-PCR和Western-blot方法进行检测,在第15天左右均可检测到表达产物,表明该重组质粒可在小鼠组织内进行有效表达。通过研究发现表达的猪源PTX3介导一系列抗菌反应,包括提高猪肺泡巨噬细胞(3D4/21)对猪链球菌2型的吞噬能力以及抑制猪链球菌2型对Hep-2细胞的粘附。在小鼠模型中,用pVAX-ptx3预先接种的小鼠的死亡率降低,并且血、脾、肺、脑中的猪链球菌的载量显著降低;在仔猪模型中,预先接种pVAX-ptx3的仔猪感染猪链球菌2型后,外周血中的细菌载量显著降低,而IL-6和IL-8的血浆水平则显著升高,与此相反,接种组仔猪外周血中的白细胞(WBC)和中性粒细胞(NEU)则相对降低。以上结果表明猪源PTX3以及pVAX-ptx3表达的PTX3蛋白对猪链球菌2型有一定的抗菌作用,同时构建的pVAX-ptx3表达载体可以在动物体内稳定无害化表达,这可能为猪链球菌病的预防和治疗提供了一个新的视角。
[Abstract]:Streptococcus suis (SSSS) is an important zoonotic pathogen. At present, antibiotic therapy is mainly used in the treatment of Streptococcus suis. However, the risk of antibiotic resistance is bound to exist in the long-term use of antibiotics; In addition, there is still a lack of effective vaccine against streptococcus suis in clinic. Therefore, the search for alternative treatments and antimicrobial compounds are the first identified long pentagglutinated proteins, which have the ability to bind to some pathogenic microorganisms. PTX3 also promotes cell phagocytosis and subsequent pathogen clearance. In addition, PTX3 has therapeutic effects that act as a mediator, recognizing microbial components. In this experiment, the prokaryotic expression vector PET-32a-ptx3 was constructed to express porcine PTX3 protein. Rabbit antigens were prepared to express PTX3 protein serum. Then the recombinant eukaryotic expression vector pIRES-EGFP-ptx3 was constructed and transfected into eukaryotic expression host CHO cells. A stable monoclonal cell line expressing PTX3 protein was obtained by G418G 750 渭 g / ml. The high purity and stable eukaryotic expression of porcine PTX3 protein was obtained by Ni-NTA affinity chromatography. Subsequently, the recombinant eukaryotic expression vector pVAX-ptx3 was successfully constructed. The purified recombinant plasmid was injected intramuscularly into BALB/c mice with 1OO 渭 g / g. The peripheral blood and muscle of injection site were collected for 1520 days and detected by RT-PCR and Western-blot respectively. The expression products were detected about 15 days after injection. The results showed that the recombinant plasmid could be effectively expressed in mouse tissues. The expressed porcine PTX3 mediated a series of antimicrobial reactions. It included enhancing phagocytic ability of porcine alveolar macrophages (3D / 4 / 21) to Streptococcus suis type 2 and inhibiting the adhesion of Streptococcus suis type 2 to Hep-2 cells. The mortality of mice inoculated with pVAX-ptx3 was decreased, and the load of streptococcus suis in blood, spleen, lung and brain decreased significantly. In the piglet model, the bacterial load in peripheral blood of the piglets infected with streptococcus suis type 2 was significantly decreased, while the plasma levels of IL-6 and IL-8 were significantly increased. In contrast. WBC) and neutrophils in peripheral blood of inoculated piglets. The results showed that PTX3 and PTX3 protein expressed by pVAX-ptx3 had a certain antibacterial effect on Streptococcus suis type 2. At the same time, the constructed pVAX-ptx3 expression vector can be stable and innocuous expression in animals, which may provide a new perspective for the prevention and treatment of swine streptococcosis.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.611

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