蛹虫草菌丝体转化体系的建立及猪蓝耳病疫苗基因的转化

发布时间：2018-05-25 01:41

  本文选题：蛹虫草 + 猪蓝耳病　； 参考：《吉林农业大学》2016年硕士论文

【摘要】：猪繁殖与呼吸综合征又名猪蓝耳病(Porcine reproductive and respiratory syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)引起的一种猪的严重传染病,不仅严重影响着全世界养猪业和食品的安全,也给全世界养猪业造成了严重经济损失。目前,能够有效预防和防治PRRS的重要手段是疫苗接种,其中,不仅能够达到免疫效果,还能减少人力物力的疫苗是新型的基因工程可饲疫苗。因此,选择一些含有医药及生理活性成分的食药用菌,如蛹虫草等作为受体系统,开发猪蓝耳病可饲疫苗具有应用前景。蛹虫草(Cordyceps militaris)是目前东亚国家中最具有代表性的昆虫病原药用真菌,根据中国传统和现代医学研究表明,蛹虫草中的虫草素、麦角甾醇及虫草酸等具有抗肿瘤、抗炎和免疫调节等作用。作为一种具有丰富活性成分的食药用菌,商业化生产蛹虫草子实体的方法已经建立,但是其安全及高效的转化系统研究尚处于起步阶段。本文首次利用蛹虫草菌丝体作为受体系统,建立蛹虫草稳定高效的转化体系;并成功克隆了猪蓝耳病疫苗基因ORF3与ORF5,并将其亚克隆于真菌特异表达载体pCB130NG上。通过PEG转化法对蛹虫草原生质体进行转化,分别获得了转ORF3基因与ORF5基因蛹虫草菌丝体。具体研究结果如下:1、蛹虫草原生质体提取最佳条件为:菌龄4 d,渗透压稳定剂为0.8 mol/L甘露醇pH4.5,1.5%的溶壁酶与蜗牛酶,酶解温度34℃,酶解时间4 h。其使用二乙酸荧光素(Fluorescein Diacetate,FDA)染色法检测蛹虫草原生质体活性,其活性可达90%以上,形状正常,大小均一。2、通过对蛹虫草再生培养基的筛选,确定最优再生培养基为含有0.8 mol/L甘露醇的PDA培养基。3、通过潮霉素浓度梯度试验,确定蛹虫草原生质体潮霉素筛选压力为500 mg/L。4、利用带有绿色荧光蛋白(Green fluorescent protein,GFP)基因的pCAMBIA1302载体,建立蛹虫草原生质体转化体系,确定蛹虫草转化最佳条件为原生质体数量为107个,质粒质量30μg,25%的PEG,冰浴时间10 min。5、查阅相关文献确定PRRSV的结构蛋白为GP3与GP5,其编码基因为ORF3和ORF5,经序列设计后,将两基因串联合成到真菌特异表达载体pCB130NG表达载体中,构建pCB130NG-ORF3-ORF5表达载体。6、克隆GP3蛋白基因ORF3与GP5蛋白基因ORF5,采用酶切和连接的方法,成功构建了表达载体pCB130NG-ORF3与pCB130NG-ORF5。7、利用PEG转化法分别将表达载体pCB130NG-ORF3、pCB130NG-ORF5和pCB130NG-ORF3-ORF5转化蛹虫草原生质体,转化子通过PCR检测初步证明猪蓝耳病疫苗基因成功转入蛹虫草中。
[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，

本文编号：1931530