本文選題：干擾素γ + 蛋白轉(zhuǎn)導(dǎo)域��； 參考：《第三軍醫(yī)大學(xué)》2006年碩士論文

【摘要】： 干擾素γ(Interferon gamma,IFNγ)具有抗腫瘤和抗病毒等活性,但血腦屏障(blood-brain barrier,BBB)能阻礙其進(jìn)入腦組織,因而限制了IFNγ在腦部腫瘤和腦部病毒感染治療中的應(yīng)用。蛋白轉(zhuǎn)導(dǎo)域(protein transduction domains,PTD)是HIV I反式激活因子(TAT)的一小段多肽,當(dāng)TAT PTD與蛋白融合后能促進(jìn)其高效跨越包括BBB、細(xì)胞膜和皮膚等在內(nèi)的多種膜結(jié)構(gòu)。而且,TAT PTD的分子量小,無細(xì)胞毒性,免疫原性極低,不影響融合的蛋白功能。因此,TAT與IFNγ融合后有望使IFNγ獲得了跨越BBB的能力而用于腦部腫瘤和腦部病毒感染等疾病的治療。為此,本實(shí)驗(yàn)首先克隆了編碼小鼠與人IFNγ(mIFNγ和huIFNγ)及相應(yīng)TAT修飾的IFNγ(IFNγ-TAT)的cDNA,然后將其構(gòu)建于pQE-80L表達(dá)載體,進(jìn)而行蛋白表達(dá)與純化;采用MTT法和BrdU-ELISA體外鑒定重組蛋白抑制腫瘤細(xì)胞增殖的能力;免疫組化法鑒定重組蛋白進(jìn)入細(xì)胞的能力;采用ELISA法測(cè)定SD大鼠腦脊液中小鼠IFNγ-TAT(mIFNγ-TAT)濃度,從而鑒定其跨BBB的能力。取得的主要研究結(jié)果和結(jié)論如下: 1.克隆了編碼mIFNγ和huIFNγ的cDNA,所得序列與GenBank中mIFNγ和huIFNγcDNA的序列完全一致。 2.構(gòu)建了mIFNγ-TAT和huIFNγ-TAT cDNA,克隆于pUC19質(zhì)粒。 3.將上述各DNA分別克隆于pQE-80L表達(dá)載體并轉(zhuǎn)化于E.coli DH5α。經(jīng)IPTG誘導(dǎo)后,mIFNγ、mIFNγ-TAT、huIFNγ和huIFNγ-TAT蛋白均獲得了較高水平的表達(dá)。mIFNγ、mIFNγ-TAT、huIFNγ和huIFNγ-TAT蛋白的相對(duì)分子量分別為1.6×104、1.7×10~4、1.7×10~4和1.8×10~4。針對(duì)mIFNγ和mIFNγ-TAT優(yōu)化表達(dá)參數(shù)為1mmol/L的IPTG在30℃誘導(dǎo)表達(dá)4h。而針對(duì)huIFNγ和huIFNγ-TAT優(yōu)化表達(dá)參數(shù)為1mmol/L的IPTG在35℃誘導(dǎo)表達(dá)4h。mIFNγ、mIFNγ-TAT和huIFNγ蛋白主要以可溶性形式存在,huIFNγ-TAT蛋白主要是以包涵體的形式存在。 4.經(jīng)Ni~(2+)-NTA親和層析系統(tǒng)純化后,各重組蛋白均達(dá)到了較高的純度,純化蛋白在SDS-PAGE上呈現(xiàn)單一條帶。 5.生物學(xué)活性分析表明,mIFNγ和mIFNγ-TAT能在體外抑制小鼠H22細(xì)胞增殖;
[Abstract]:Interferon gamma-IFN- 緯 (IFN- 緯) has antitumor and antiviral activities, but blood-brain barrier B (BBB) can block its entry into brain tissue, thus limiting the application of IFN- 緯 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- 緯 is expected to enable IFN- 緯 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- 緯 (mIFN- 緯 and huIFN- 緯) and the corresponding ta-modified IFN- 緯 (IFN- 緯 -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 緯 -TAT (mIFN- 緯 -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- 緯 and huIFN- 緯 was cloned, and the sequence was identical to that of mIFN- 緯 and huIFN- 緯 cDNA in GenBank. 2. MIFN 緯 -TAT and huIFN 緯 -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 偽. After induced by IPTG, the relative molecular weights of mIFN- 緯 -TATH-huIFN- 緯 and huIFN- 緯 -TATH-TAT protein were 1.6 脳 1041.7x 1041.7 脳 1044 and 1.8 脳 1044.The relative molecular weights of mIFN- 緯 -tIFN- 緯 -TAT- 緯 and huIFN- 緯 -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 緯 and mIFN 緯 -TAT were 1 mmol / L IPTG at 30 鈩，

本文編號(hào)：2084943