Ser-125突变型人白细胞介素-2在家兔和山羊乳腺中的瞬时表达

发布时间：2018-04-03 00:08

本文选题：Ser-125突变型人白细胞介素-2　切入点：β-乳球蛋白　出处：《南京师范大学》2005年硕士论文

【摘要】：人白细胞介素-2(HuIL-2)主要是由激活的T淋巴细胞产生的一种淋巴因子,在机体免疫应答中起重要作用,能诱导T淋巴细胞增殖(从G_0期到S期)和分化,并且具有刺激T辅助细胞和自然杀伤细胞分泌细胞因子等功能,因而具有广泛的生物学活性,在临床上具有抗肿瘤、抗感染及增强机体免疫功能等作用。天然IL-2由133个氨基酸残基组成,在58、105和125位各有一个半胱氨酸(Cys),其中58位和105位形成二硫键是IL-2生物活性所必需,125位半胱氨酸的存在易形成错配或二聚体,使IL-2活性丧失,因此将125位半胱氨酸突变为丝氨酸可以消除形成错配或二聚体的分子基础,便于纯化。动物乳腺生物反应器以其生产成本低、产量高且表达产物接近天然产物而日益受到人们重视。但建立乳腺生物反应器是一项周期长、耗资大的工作,而且外源基因的随机表达也给这项工作带来很大麻烦,因此在建立大型转基因动物个体之前,先用小动物鉴定所构建载体的可行性十分必要。本研究用PCR定点突变法将天然人白细胞介素-2的125位半胱氨酸突变为丝氨酸,得到Ser-125突变型人白细胞介素-2(Ser-125 HuIL-2)cDNA,并从山羊新鲜血液中扩增到867bp的β-乳球蛋白(BLG)基因的上游调控序列,然后将Ser-125突变型人白细胞介素-2 cDNA与山羊β-乳球蛋白(BLG)基因的上游调控序列融合,构建了pBLG—Ser-125HuIL-2乳腺定位表达载体。表达载体经纯化后用脂质体包裹,经乳腺导管注入妊娠后期的家兔和山羊乳腺内,进行瞬时表达。分别于家兔和山羊分娩后1～10d采奶,用ELISA方法检测乳汁中Ser-125HuIL-2含量。家兔共做了6只,其中1只作为空白对照,1只注射脂质体包裹的pIL-2质粒,2只注射表达载体,2只注射脂质体包裹的表达载体,结果注射空白对照和pIL-2质粒的2只家兔均未表达;2只直接注射表达载体的家兔1只有表达,含量为0.06～0.53μg/L,1只未表达,表达成功率50%(1/2);
[Abstract]:Human interleukin-2 (hIL-2) is a kind of lymphoid factor produced mainly by activated T lymphocytes. It plays an important role in the immune response of the body and can induce the proliferation and differentiation of T lymphocytes (from G0 to S).It has the function of stimulating T helper cells and natural killer cells to secrete cytokines, so it has a wide range of biological activities, and has anti-tumor, anti-infection and enhance the immune function of the body.Natural IL-2 is composed of 133 amino acid residues, and there is a cysteine cysteine in 58105 and 125.The disulfide bond at 58 and 105 is necessary for the biological activity of IL-2. The existence of cysteine at 125th is easy to form mismatch or dimer, which results in the loss of IL-2 activity.Therefore, mutagenesis of 125 cysteine to serine can eliminate the molecular basis of mismatch or dimer and facilitate purification.Animal mammary gland bioreactor has been paid more and more attention for its low production cost, high yield and close to natural products.But the establishment of mammary gland bioreactors is a long and costly task, and the random expression of foreign genes also brings a lot of trouble to the work, so before the establishment of large transgenic animal individuals,It is necessary to identify the feasibility of the constructed vector with small animals.In this study, PCR site-directed mutagenesis was used to mutate 125 cysteine from natural human interleukin-2 to serine.The upstream regulatory sequence of 尾 -lactoglobulin (BLGG) gene of 867bp was amplified from goat fresh blood by Ser-125 mutant human interleukin-125HuIL-2cDNA.Then the upstream regulatory sequence of goat 尾 -lactoglobulin gene was fused with Ser-125 mutant human interleukin-2 cDNA to construct the pBLG-Ser-125HuIL-2 mammary gland localization expression vector.After purification, the expression vector was encapsulated with liposome and injected into the mammary gland of rabbits and goats in the second trimester of pregnancy for transient expression.The content of Ser-125HuIL-2 in milk of rabbits and goats was determined by ELISA method.Six rabbits were made, one of them was used as a blank control, one was injected with liposome-encapsulated pIL-2 plasmid, two were injected with expression vector and two were injected with liposome-encapsulated expression vector.Results No expression was found in two rabbits injected with blank control and pIL-2 plasmid. The expression rate of 1 rabbit was 0.06 ~ 0.53 渭 g / L ~ (-1), and the success rate of expression was 50 / 1 / 2.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

【相似文献】