TAT修饰的小鼠和人IFNγ的制备及功能鉴定

发布时间：2018-06-30 04:43

  本文选题：干扰素γ + 蛋白转导域　； 参考：《第三军医大学》2006年硕士论文

【摘要】： 干扰素γ(Interferon gamma,IFNγ)具有抗肿瘤和抗病毒等活性,但血脑屏障(blood-brain barrier,BBB)能阻碍其进入脑组织,因而限制了IFNγ在脑部肿瘤和脑部病毒感染治疗中的应用。蛋白转导域(protein transduction domains,PTD)是HIV I反式激活因子(TAT)的一小段多肽,当TAT PTD与蛋白融合后能促进其高效跨越包括BBB、细胞膜和皮肤等在内的多种膜结构。而且,TAT PTD的分子量小,无细胞毒性,免疫原性极低,不影响融合的蛋白功能。因此,TAT与IFNγ融合后有望使IFNγ获得了跨越BBB的能力而用于脑部肿瘤和脑部病毒感染等疾病的治疗。为此,本实验首先克隆了编码小鼠与人IFNγ(mIFNγ和huIFNγ)及相应TAT修饰的IFNγ(IFNγ-TAT)的cDNA,然后将其构建于pQE-80L表达载体,进而行蛋白表达与纯化;采用MTT法和BrdU-ELISA体外鉴定重组蛋白抑制肿瘤细胞增殖的能力;免疫组化法鉴定重组蛋白进入细胞的能力;采用ELISA法测定SD大鼠脑脊液中小鼠IFNγ-TAT(mIFNγ-TAT)浓度,从而鉴定其跨BBB的能力。取得的主要研究结果和结论如下: 1.克隆了编码mIFNγ和huIFNγ的cDNA,所得序列与GenBank中mIFNγ和huIFNγcDNA的序列完全一致。 2.构建了mIFNγ-TAT和huIFNγ-TAT cDNA,克隆于pUC19质粒。 3.将上述各DNA分别克隆于pQE-80L表达载体并转化于E.coli DH5α。经IPTG诱导后,mIFNγ、mIFNγ-TAT、huIFNγ和huIFNγ-TAT蛋白均获得了较高水平的表达。mIFNγ、mIFNγ-TAT、huIFNγ和huIFNγ-TAT蛋白的相对分子量分别为1.6×104、1.7×10~4、1.7×10~4和1.8×10~4。针对mIFNγ和mIFNγ-TAT优化表达参数为1mmol/L的IPTG在30℃诱导表达4h。而针对huIFNγ和huIFNγ-TAT优化表达参数为1mmol/L的IPTG在35℃诱导表达4h。mIFNγ、mIFNγ-TAT和huIFNγ蛋白主要以可溶性形式存在,huIFNγ-TAT蛋白主要是以包涵体的形式存在。 4.经Ni~(2+)-NTA亲和层析系统纯化后,各重组蛋白均达到了较高的纯度,纯化蛋白在SDS-PAGE上呈现单一条带。 5.生物学活性分析表明,mIFNγ和mIFNγ-TAT能在体外抑制小鼠H22细胞增殖;
[Abstract]:Interferon gamma-IFN- 纬 (IFN- 纬) has antitumor and antiviral activities, but blood-brain barrier B (BBB) can block its entry into brain tissue, thus limiting the application of IFN- 纬 in the treatment of brain tumors and brain virus infection. Protein transduction domain (protein transduction domain) is a small peptide of HIV I transactivator (tat). When the protein is fused with the protein, it can efficiently cross many membrane structures, including BBB, cell membrane and skin, etc. Moreover, the molecular weight of tat PTD is small, no cytotoxicity, and the immunogenicity is very low, which does not affect the function of fusion protein. Therefore, the fusion of tat and IFN- 纬 is expected to enable IFN- 纬 to gain the ability to cross BBB and to be used in the treatment of brain tumors and viral infections. Therefore, the cDNA encoding mouse and human IFN- 纬 (mIFN- 纬 and huIFN- 纬) and the corresponding ta-modified IFN- 纬 (IFN- 纬 -TAT) were first cloned, then constructed into pQE-80L expression vector, and then the protein was expressed and purified. MTT assay and BrdU-ELISA were used to identify the ability of recombinant protein to inhibit tumor cell proliferation in vitro, immunohistochemical method to identify the ability of recombinant protein to enter the cell, and Elisa method to determine the concentration of mouse IFN 纬 -TAT (mIFN- 纬 -TAT) in cerebrospinal fluid of SD rats, so as to identify the ability of recombinant protein to cross BBB. The main results and conclusions are as follows: 1. The cDNA encoding mIFN- 纬 and huIFN- 纬 was cloned, and the sequence was identical to that of mIFN- 纬 and huIFN- 纬 cDNA in GenBank. 2. MIFN 纬 -TAT and huIFN 纬 -TAT cDNAs were constructed and cloned into plasmid pUC19. The above DNA was cloned into pQE-80L expression vector and transformed into E. coli DH5 伪. After induced by IPTG, the relative molecular weights of mIFN- 纬 -TATH-huIFN- 纬 and huIFN- 纬 -TATH-TAT protein were 1.6 脳 1041.7x 1041.7 脳 1044 and 1.8 脳 1044.The relative molecular weights of mIFN- 纬 -tIFN- 纬 -TAT- 纬 and huIFN- 纬 -TAT- TAT protein were 1. 6 脳 10 ~ (417) 脳 10 ~ (41.7) 脳 10 ~ (4) and 1. 8 脳 10 ~ (4), respectively. The optimal expression parameters of mIFN 纬 and mIFN 纬 -TAT were 1 mmol / L IPTG at 30 鈩，

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