首页 > 文献资料
-
犬瘟热疫苗弱毒融合蛋白基因5′端的序列特点
犬瘟热是我国养犬业、毛皮动物饲养业及野生动物保护业的头号大敌.鉴于本病目前尚无特效的治疗药物和方法,疫苗接种是唯一有效可行的防治措施.但是疫苗毒株是否适用于所有动物?如何解释实际生产中出现的免疫失败现象?我国现用疫苗毒株本身的背景怎样?
-
小鼠AFP-CTLA4融合蛋白真核表达载体的构建及鉴定
目的:克隆小鼠甲胎蛋白(mAFP)基因并构建小鼠甲胎蛋白-细胞毒性T淋巴细胞抗原4(mAFP-CTLA4)融合蛋白真核表达载体.方法:从Hepal-6细胞中提取总RNA进行RT-PCR,扩增出mAFP基因,亚克隆于pcDNA3.1载体.PCR法从质粒pmCTLA4-Ig中克隆出mCTLA4膜外部分基因并通过重叠PCR法添加接头,重组连接于pmAFP质粒中mAFP基因后,转化大肠杆菌DH5α,筛选阳性克隆酶切、测序鉴定.用质粒瞬时转染CHO细胞,Westernblot检测融合蛋白的表达.结果:利用RT-PCR从Hepal-6细胞总RNA中成功克隆出1.8kb的nAFP基因;重组阳性克隆经酶切鉴定证实连有接头的CTLA4膜外部分基因已正确插入pmAFP质粒中,测序结果证实各片段连接方向及阅读框正确.用质粒瞬时转染CHO细胞,Western blot检测到预计大小分子量蛋白的表达.结论:mAFP-CTLA4融合蛋白真核表达载体的构建成功,为进一步研究其在肝癌免疫治疗中的作用奠定了基础.
-
Single-chain variable domain fragment (scFv) 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pastoris. The afifnity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunolfuorescence staining. The ability of the fusion protein to block myas-thenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for speciifc immunosuppressive therapy of myasthenia gravis.
-
Objective :To develop an agent that is more active against receptor-bearing target cells without increasing the toxic effect on non-target cells. Methods :By the use of molecular biology techniques,we designed and constructed a fusion protein 5'IL6-TNF△ by connecting the human interleukin-6 (hIL-6) gene and a human tumor necrosis factor α derivative (TNF△) gene througha synthetic linker sequence followed by subsequent expression in E. Coli. Results: In cytotoxicity assay with myeloma cell line U266, the normal type of 5' IL6-TNF△ showed an antitumor activity 3 times higher than that of TNF△;and the antitumor activity of 5'IL6-TNF△ blocked by IL-6Rwas only 1/30 of that of normal type of 5' IL6-TNF△. Meanwhile,the 5'IL6-TNF△ blocked by an ti-TNF antibody did not show any cytotoxicity to U266 cells. In activity assay with L929 cells ,the toxic effect of the fusion protein was found 1/22 of that of TNF△. Conclusion: The 5'IL6-TNF△fusion protein might be a useful cytotoxic agent in cancer treatment.
-
人端粒酶催化亚单位诱饵融合蛋白表达载体的构建及初步鉴定
端粒是真核细胞染色体末端的一个特殊结构,由一段具有特定重复序列的DNA和端粒结合蛋白组成,是维持染色体结构稳定的重要因素.正常细胞随着细胞分裂活动的进行,端粒DNA逐渐缩短,当缩短到一定程度时,染色体结构被破坏,细胞进入衰老期并以死亡而告终.但肿瘤细胞似乎丧失了这种能力,表现出永生化而无限制增殖的特性.尽管端粒研究为肿瘤、衰老难题提供了一个重要的研究方向,但有关端粒蛋白组分的结构及其调控机制仍不十分清楚.因此,寻找新的、关键性的端粒、端粒酶调控分子是非常重要的[1].本课题拟以已发现的端粒相关蛋白中较为重要的人端粒酶催化亚单位(Human telomerase reverse transcriptase, hTRT)为诱饵,构建hTRT诱饵融合蛋白表达载体,为从cDNA文库中筛选与hTRT作用的端粒相
-
pGEX-e23 (scFv) PE40融合基因的构建及其在大肠杆菌中的表达
目的 在大肠杆菌中表达抗原癌基因c-erbB-2表达产物p185的单 链抗体e23(scFv)/假单孢菌属外毒素活性片 段PE40免疫毒素的融合蛋白,为乳腺癌、胃癌等多种c-erbB-2呈过量表达的恶性肿瘤的免疫治疗奠定基础. 方法 去除克隆在真核表达载体pLNCX中的e23 (scFv) PE40基因5′端编码信号肽的核苷酸序列,并将改建后的融合基因克隆到原核融合表达载体pGEX-4T中表达. 结果 序列测 定 表明. 改建后的抗p185 e23(scFv)/PE40序列正确. 融合基因经IPTG诱导表达4 h后,经SDS -聚丙烯酰胺凝胶电泳分析,在Mr 90 000处出现一条新生蛋白带,表达量约占菌体总蛋白的15%. 结论 成功改建并在原核中表达了抗p185 e23 (scFv)/ PE40融合蛋白.