本文選題：蛹蟲草 + 豬藍(lán)耳病�。� 參考：《吉林農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：豬繁殖與呼吸綜合征又名豬藍(lán)耳病(Porcine reproductive and respiratory syndrome,PRRS)是由豬繁殖與呼吸綜合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)引起的一種豬的嚴(yán)重傳染病,不僅嚴(yán)重影響著全世界養(yǎng)豬業(yè)和食品的安全,也給全世界養(yǎng)豬業(yè)造成了嚴(yán)重經(jīng)濟(jì)損失。目前,能夠有效預(yù)防和防治PRRS的重要手段是疫苗接種,其中,不僅能夠達(dá)到免疫效果,還能減少人力物力的疫苗是新型的基因工程可飼疫苗。因此,選擇一些含有醫(yī)藥及生理活性成分的食藥用菌,如蛹蟲草等作為受體系統(tǒng),開發(fā)豬藍(lán)耳病可飼疫苗具有應(yīng)用前景。蛹蟲草(Cordyceps militaris)是目前東亞國(guó)家中最具有代表性的昆蟲病原藥用真菌,根據(jù)中國(guó)傳統(tǒng)和現(xiàn)代醫(yī)學(xué)研究表明,蛹蟲草中的蟲草素、麥角甾醇及蟲草酸等具有抗腫瘤、抗炎和免疫調(diào)節(jié)等作用。作為一種具有豐富活性成分的食藥用菌,商業(yè)化生產(chǎn)蛹蟲草子實(shí)體的方法已經(jīng)建立,但是其安全及高效的轉(zhuǎn)化系統(tǒng)研究尚處于起步階段。本文首次利用蛹蟲草菌絲體作為受體系統(tǒng),建立蛹蟲草穩(wěn)定高效的轉(zhuǎn)化體系;并成功克隆了豬藍(lán)耳病疫苗基因ORF3與ORF5,并將其亞克隆于真菌特異表達(dá)載體pCB130NG上。通過(guò)PEG轉(zhuǎn)化法對(duì)蛹蟲草原生質(zhì)體進(jìn)行轉(zhuǎn)化,分別獲得了轉(zhuǎn)ORF3基因與ORF5基因蛹蟲草菌絲體。具體研究結(jié)果如下:1、蛹蟲草原生質(zhì)體提取最佳條件為:菌齡4 d,滲透壓穩(wěn)定劑為0.8 mol/L甘露醇pH4.5,1.5%的溶壁酶與蝸牛酶,酶解溫度34℃,酶解時(shí)間4 h。其使用二乙酸熒光素(Fluorescein Diacetate,FDA)染色法檢測(cè)蛹蟲草原生質(zhì)體活性,其活性可達(dá)90%以上,形狀正常,大小均一。2、通過(guò)對(duì)蛹蟲草再生培養(yǎng)基的篩選,確定最優(yōu)再生培養(yǎng)基為含有0.8 mol/L甘露醇的PDA培養(yǎng)基。3、通過(guò)潮霉素濃度梯度試驗(yàn),確定蛹蟲草原生質(zhì)體潮霉素篩選壓力為500 mg/L。4、利用帶有綠色熒光蛋白(Green fluorescent protein,GFP)基因的pCAMBIA1302載體,建立蛹蟲草原生質(zhì)體轉(zhuǎn)化體系,確定蛹蟲草轉(zhuǎn)化最佳條件為原生質(zhì)體數(shù)量為107個(gè),質(zhì)粒質(zhì)量30μg,25%的PEG,冰浴時(shí)間10 min。5、查閱相關(guān)文獻(xiàn)確定PRRSV的結(jié)構(gòu)蛋白為GP3與GP5,其編碼基因?yàn)镺RF3和ORF5,經(jīng)序列設(shè)計(jì)后,將兩基因串聯(lián)合成到真菌特異表達(dá)載體pCB130NG表達(dá)載體中,構(gòu)建pCB130NG-ORF3-ORF5表達(dá)載體。6、克隆GP3蛋白基因ORF3與GP5蛋白基因ORF5,采用酶切和連接的方法,成功構(gòu)建了表達(dá)載體pCB130NG-ORF3與pCB130NG-ORF5。7、利用PEG轉(zhuǎn)化法分別將表達(dá)載體pCB130NG-ORF3、pCB130NG-ORF5和pCB130NG-ORF3-ORF5轉(zhuǎn)化蛹蟲草原生質(zhì)體,轉(zhuǎn)化子通過(guò)PCR檢測(cè)初步證明豬藍(lán)耳病疫苗基因成功轉(zhuǎn)入蛹蟲草中。
[Abstract]:Porcine reproductive and respiratory syndrome (PRRSs) is a serious infectious disease of pigs caused by porcine reproductive and respiratory syndrome virus (PRRS), which not only seriously affects the pig industry and food safety in the world. Also to the world pig industry caused serious economic losses. At present, vaccination is an important method to prevent and treat PRRS effectively. The vaccine, which can not only achieve the immune effect, but also reduce manpower and material resources, is a new type of genetic engineering feedable vaccine. Therefore, it is promising to select some edible pharmaceutical bacteria containing medicine and physiological active ingredients, such as Cordyceps militaris as the receptor system, to develop the feedable vaccine for swine blue ear disease. Cordyceps militaris (Cordyceps militaris) is one of the most representative medicinal entomopathogenic fungi in East Asia at present. According to Chinese traditional and modern medical studies, cordyceps, ergosterol and cordyceps in Cordyceps militaris have anti-tumor properties. Anti-inflammatory and immune regulation and so on. As a kind of edible medicinal fungi with abundant active ingredients, a commercial method for producing fruiting bodies of Cordyceps militaris has been established, but the study of its safe and efficient transformation system is still in its infancy. In this paper, a stable and efficient transformation system of Cordyceps militaris was established by using mycelia of Cordyceps militaris as the receptor system for the first time, and the genes ORF3 and ORF5 were successfully cloned and subcloned into the fungal specific expression vector pCB130NG. The protoplasts of Cordyceps militaris were transformed by PEG and the mycelium of Cordyceps militaris with ORF3 gene and ORF5 gene were obtained. The results were as follows: 1. The optimum conditions for protoplast extraction of Cordyceps militaris were as follows: the culture age was 4 days, the osmotic pressure stabilizer was 0.8 mol/L mannitol (pH 4.5) 1.5%, the enzymatic hydrolysis temperature was 34 鈩，

本文編號(hào)：1931530