【作者】 欧阳永长；

【导师】 庄东红； 胡忠；

【作者基本信息】 汕头大学 ， 生化与分子生物学， 2005， 硕士

【摘要】 苦瓜是药食两用的蔬菜。MAP30(MAP30是Momordica anti-HIV protein of30 Kda的缩写)和α苦瓜素是苦瓜中的两种核糖体失活蛋白。MAP30在苦瓜中纯化出来之后,被证明有抗艾滋病毒,抑制HIV-1整合酶,抗肿瘤的作用。MAP30对许多类型病毒感染的细胞、肿瘤细胞非常有效,但它对未感染的人正常细胞无毒性,并且利用基因工程原核表达的MAP30同样具有天然产物的功能和特性。α-苦瓜素能够选择性杀伤绒毛膜癌细胞和黑色素瘤细胞,对胃癌NKM细胞株的DNA,RNA和蛋白质的合成亦均具有明显的抑制作用。核糖体失活蛋白(ribosome inactivating proteins,RIPs)是广泛存在于植物界的一类蛋白毒素,有抗肿瘤,提高植物抗病的能力,在临床医学和农业方面有着光明的应用前途,因此本文克隆这两个蛋白基因,并对它们在原核中的表达进行了研究,为进一步研究这两个蛋白及应用提供基础。主要研究内容和结果如下:1.本文在构建MAP30基因原核表达载体的过程中,对PCR快速检测阳性克隆进行了研究。结果表明,直接用菌落、菌液、酚氯仿处理过的菌液,以及提取的质粒进行PCR都可以成功地筛选阳性菌落。其中,酚氯仿处理过的菌液PCR与质粒PCR的结果最接近,而且比质粒PCR简单,因此可作为方便可靠的阳性克隆筛选的新方法。2.克隆了MAP30和α苦瓜素基因,并构建了MAP30的原核表达载体pET28a-M和pET32a-M,α苦瓜素的表达载体pET28-MCA。在BL21(DE3)菌株中,这两个基因都未表达,至少肉眼未见明显表达。经分析MAP30和α苦瓜素基因都存在多个大肠杆菌稀有密码子AGG,AGA,AUA,GGA,其含量都超过其基因的5%,严重影响了它们在细菌中的表达。改用能超表达稀有密码子基因的菌株Rosstle~Tm(DE3)pLysS后,MAP30和α苦瓜素基因在原核中表达量激增,并且RMAP30基因在30℃和α苦瓜素基因22℃时都出现可溶性表达。利用离子鳖和层析纯化出了此两个基因的的重组蛋白,虽然重组蛋白在体外无切割超螺旋DNA活性,但重组蛋白MAP30在0.7mg/mL时,能中度抑制金黄色葡萄球菌,溶血葡萄球菌和阴沟肠杆菌。3.构建了MAP30基因的植物表达载体用于改良作物抗病性的转基因研究,同时克隆了菜豆几丁质酶基因,并构建了菜豆几丁质酶的原核表达载体和植物表达载体。构建的这两个载体可以直接用于转基因的研究,也为构建MAP30和菜豆几更多还原

【Abstract】 The bitter gourd is the vegetables, and medicines too. MAP30 and alpha momorcharin both are the ribosome-inactivating proteins (RIPs) in the bitter gourd , which is a cytotoxin ,antitumor and resistant-disease of plant protein existing in the plant kingdom extensively . After MAP30 protein had isolated from bitter gourd, it is proved anti-HIV, anti-virus, anti-tumor activities and little cytotoxin to normal cells. Recombinant MAP30 has full biological actives as well as his counterpart from his original plant source. Alpha momorcharin has inhibitory effect on DNA, RNA and protein synthesis of NKM cell and also selectively injured choriocarcinoma and melanoma. Thereby, they have bright application future in agriculture and clinical medicine. So we cloned these two protein genes and then studied on their expressing in Ecoli. This research includes the following several respects mainly:First, A method by using PCR for rapid identification of positive clone was eveloped. Result showed this screening method could be used to detect positive colonie s from samples of bacterial, purified plasmid, liquid culture, and liquid culture treated with mixture of phenol/ Chloroform. The result from liquid-culture-treated-PCR (LCT-PCR) is very close to that of by plasmid-PCR. LCT-PCR is reliable and much easier to used than plasmid-PCR; therefore the LCT-PCR can be used for clone screening during the molecular cloning.Second, MAP30 and Alpha momorcharin both had successfully expressed in Ecoli in the way of solubility, though their genes have many rare usage coden of bacteria. Recombinant MAP30 has antimicrobial activity against Staphylococcus aureus, Enterobacter cloacae and Staphylococcus hominis.Last, constructed plant- expressing vectors of MAP30 and chitinase genes, which will be used for improving ability of peanut to resistant virus. These are the basis of construction of bivalent genes of plant -expressing vector.更多还原