本文關(guān)鍵詞： 豬源PTX3蛋白 豬鏈球菌2型 真核表達(dá) pVAX-ptx3 抗菌作用　出處：《南京農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：豬鏈球菌(Streptococcussuis,SS)是一種重要的人畜共患病病原菌。目前對于該病的治療主要采用抗生素療法,但長期大量使用抗生素必然存在抗生素耐藥性的風(fēng)險;另外針對該病,臨床上仍缺乏有效預(yù)防豬鏈球菌的疫苗,故尋求能替代的治療方案以及抗微生物化合物是備受關(guān)注的。PTX3是第一個被確認(rèn)的長正五聚蛋白,具有結(jié)合到某些病原微生物的能力,PTX3也可以促進(jìn)細(xì)胞吞噬功能以及隨后病原體的清除。另外,PTX3具有充當(dāng)調(diào)理素的治療效果,可識別微生物成分,引發(fā)炎癥反應(yīng)的放大。本試驗首先通過構(gòu)建原核表達(dá)載體PET-32a-ptx3,獲得原核表達(dá)豬源PTX3蛋白,并制備兔抗原核表達(dá)PTX3蛋白血清;隨后又構(gòu)建重組真核表達(dá)載體pIRES-EGFP-ptx3,并轉(zhuǎn)染至真核表達(dá)宿主CHO細(xì)胞中,通過G418(750μg/ml)篩選獲得能夠穩(wěn)定、大量表達(dá)PTX3蛋白的單克隆細(xì)胞株,并采用Ni-NTA親和層析方法純化細(xì)胞培養(yǎng)上清獲得純度較高、性質(zhì)穩(wěn)定的真核表達(dá)豬源PTX3蛋白;隨后成功構(gòu)建了重組真核表達(dá)載體pVAX-ptx3,將純化的重組質(zhì)粒肌肉注射BALB/c小鼠(1OOμg/只),在接種后第1,3,5,7,15,20天取外周血及注射部位肌肉,分別用RT-PCR和Western-blot方法進(jìn)行檢測,在第15天左右均可檢測到表達(dá)產(chǎn)物,表明該重組質(zhì)粒可在小鼠組織內(nèi)進(jìn)行有效表達(dá)。通過研究發(fā)現(xiàn)表達(dá)的豬源PTX3介導(dǎo)一系列抗菌反應(yīng),包括提高豬肺泡巨噬細(xì)胞(3D4/21)對豬鏈球菌2型的吞噬能力以及抑制豬鏈球菌2型對Hep-2細(xì)胞的粘附。在小鼠模型中,用pVAX-ptx3預(yù)先接種的小鼠的死亡率降低,并且血、脾、肺、腦中的豬鏈球菌的載量顯著降低;在仔豬模型中,預(yù)先接種pVAX-ptx3的仔豬感染豬鏈球菌2型后,外周血中的細(xì)菌載量顯著降低,而IL-6和IL-8的血漿水平則顯著升高,與此相反,接種組仔豬外周血中的白細(xì)胞(WBC)和中性粒細(xì)胞(NEU)則相對降低。以上結(jié)果表明豬源PTX3以及pVAX-ptx3表達(dá)的PTX3蛋白對豬鏈球菌2型有一定的抗菌作用,同時構(gòu)建的pVAX-ptx3表達(dá)載體可以在動物體內(nèi)穩(wěn)定無害化表達(dá),這可能為豬鏈球菌病的預(yù)防和治療提供了一個新的視角。
[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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.611

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