人源化抗肝癌单链抗体与牛蛙核糖核酸酶融合基因的构建及表达

利用RT-PCR的方法从牛蛙肝脏中克隆牛蛙核糖核酸酶(RC—RNase)基因并进行序列测定;将人源化抗肝癌单链抗体(scFv)基因与RC—RNase基因相连接,制备scFv—RC—RNase融合基因表达质粒,转化大肠杆菌B121(DE3),用IPTG诱导进行表达。以SDS-PAGE和免疫印迹法对表达产物进行分析鉴定。序列分析表明,扩增出的RC—RNase基因片断大小约为405bpc,经SDS—PAGE和免疫印迹分析显示,scFv—RC—RNase融合基因表达质粒在大肠杆菌中的诱导表达产物出现相对分子质量约为38000的一条新生蛋白带,与预期结果相符。融合蛋白表达量占菌体总蛋白量的18．5％,主要以包涵体形式存在。表明成功地构建了抗肝癌scFv—RC—RNase融合基因,并在大肠杆菌中获得有效表达,为进一步进行肝癌的导向治疗研究奠定了基础。     

IL-1ra-Fce融合基因的克隆、表达及鉴定

白细胞介素-1与免疫球蛋白E在过敏性哮喘发病中发挥着重要作用。本试验克隆了白细胞介素-1受体拮抗剂(IL-1ra)及IgE分子恒定区cDNA片段, 构建了融合基因原核表达载体IL-1ra-Fce/pBV220。将其转化大肠杆菌BL21(DE3), 实现了融合蛋白的高效表达, Western blotting结果表明表达蛋白为目的融合蛋白, 主要以包涵体形式存在; 利用分子筛和阳离子交换层析对表达产物经进行了纯化, 纯化的包涵体复性后经体外功能试验表明, 融合蛋白的活性与IL-1ra没有显著性差异; 初步药代动力学分析显示IL-1ra-Fce半衰期比IL-1ra延长了4.78倍。     

IL-Ira-Fcε融合基因的克隆、表达及鉴定

白细胞介素-1与免疫球蛋白E在过敏性哮喘发病中发挥着重要作用.本试验克隆了白细胞介素-1受体拮抗剂(IL-1ra)及IgE分子恒定区cDNA片段,构建了融合基因原核表达载体IL-1ra-Fcg/pV220.将其转化大肠杆菌BL21(DE3),实现了融合蛋白的高效表达,Western blotting结果表明表达蛋白为目的融合蛋白,主要以包涵体形式存在;利用分子筛和阳离子交换层析对表达产物经进行了纯化,纯化的包涵体复性后经体外功能试验表明,融合蛋白的活性与IL-1ra没有显著性差异;初步药代动力学分析显示IL-1ra-FeE半衰期比IL-1ra延长了4.78倍.     

重组人GM—CSF／MCAF融合蛋白的变性,复性及纯化研究

人粒细胞巨噬细胞集落刺激因子(GM-CSF)和单核细胞趋化激活因子(MCAF)融合蛋白在大肠杆菌中高效表达后,表达产物以包涵体形式存在。包涵体经分离和洗涤后,探索了rhGM-CSF/MCAF变性和复性的合适条件。复性后的样品经Sephadex G-75凝胶过滤和CM-Sepharose FF离子交换两步层析,得到了具有生物学活性的SDS-PAGE纯的rhGM-CSF/MCAF。Western blot检测表明,纯化的rhGM-CSF/MCAF能分别与GM-CSF和MCAF抗体发生特异反应。     

丙型肝炎病毒基因组C区、NS3区、NS4区基因的融合、表达及初步应用

通过PCR技术从HCV全长基因组中扩增并克隆到Core区、NS4区的部分优势表面抗原肽的基因和C33a基因。再将它们以Core\,CC3c\,NS4的顺序和C33c、NS4的顺序分别拼接融合成融合抗原CCN、CN。片段之间以Gly-Gly-Gly-Gly柔性连接肽连接。经核苷酸序列分析表明,拼接点序列正确。将融合抗原CN、CCN基因分别克隆至T7启动子控制下的表达质粒PET24(a)+,PET22(b)+中,转化至大肠杆菌BL21(DE3)后,经IPTG诱导可高表达分子量约为43kD、58kD的融合抗原,表达产物以包涵体形式存在。表达产物经Ni²⁺IDA亲和柱纯化后,并对融合抗原CN、CCN的抗原性做了初步的鉴定。     

重组人白细胞介素15融合蛋白的表达、纯化及免疫原性检测

目的:以白细胞介素15(IL-15)为靶点,研制类风湿性关节炎免疫治疗蛋白疫苗。方法:将N端融合破伤风类毒素(TT)表位的人IL-15基因克隆至带有His标签的原核表达载体pQE-30上,转化大肠杆菌M15,经IPTG诱导表达,获得重组人TT-IL-15融合蛋白(简称rtIL-15);目的蛋白经镍柱亲和层析纯化后,用Western印迹和HPLC进行鉴定;将rtIL-15与氢氧化铝佐剂混合,免疫BALB/c小鼠,检测疫苗的免疫原性。结果:双酶切鉴定和核苷酸序列测定结果表明重组融合表达质粒pQE-30-TT-IL-15构建正确,重组蛋白的表达量达到菌体总蛋白的20%,主要以包涵体形式表达,经过纯化、复性后,目的蛋白纯度达到95%以上;蛋白疫苗免疫小鼠后,能诱导高滴度的特异性的IL-15抗体。结论:在大肠杆菌中表达了重组人IL-15融合蛋白疫苗,并具有良好的免疫原性。     

EGF-SEA融合蛋白在大肠杆菌中的表达和纯化

根据基因库中查到的金黄色葡萄球菌肠毒素A(SEA)基因序列和人体表皮生长因子(EGF)基因序列进行密码子优化,以适于大肠杆菌表达.人工合成SEA基因与EGF基因.将两目的基因克隆至原核表达栽体pFT22b中,经测序验证表明成功构建了重组表达质粒pET22b-EGF-SEA.将构建好的pET22b-EGF-SEA质粒转化大肠杆菌BL21(DE3),经IPTG诱导进行表达;SDS-PAGE分析表明融合基因EGF-SEA在大肠杆菌BL21(DE3)中以包涵体的形式得到了高效表达,产物相对分子质量约为44kDa,与理论值大小一致.包涵体经洗涤,变性、复性后用His Bind Kit进行分离纯化,所得蛋白纯度≥95%.高纯度EGF-SEA融合蛋白的获得为进一步研究其生物学活性及肿瘤治疗奠定了基础.     

天蚕素A-蜂毒素杂合肽用pBV220载体的表达、纯化和活性测定

为提高抗菌肽的表达,在抗菌肽的N端融合了1段酸性小肽以中和表达产物对宿主的毒性;并将融合肽基因同向串连成多拷贝,在大肠杆菌中获得了较高的表达。用化学合成法分别合成了编码天蚕素A(1-8)-蜂毒素(1-10)杂合肽和酸性小肽的DNA片段,首先将其拼接成融合肽的完整基因,然后通过前后接头将融合肽基因连接成两侧具有EcoRI和SalI酶切位点的同向串连的多拷贝基因。将5份拷贝的基因克隆至pBV220表达载体,转化E.coliDH5α,温度诱导得到表达量为35%的融合蛋白。表达产物主要以包涵体形式存在,将包涵体溶解,经Ni²⁺-NTA琼脂糖亲和层析获得纯化的融合蛋白。融合蛋白再经CNBr切割和阳离子交换层析,得到纯化的抗菌肽,经蛋白质N端测序确认序列正确。琼脂糖扩散法和液相测定法证明了纯化的抗菌肽具有抗菌活性。     

牙鲆甲状腺激素受体TRαA的原核表达和多克隆抗体的制备及应用

为研究牙鲆甲状腺激素受体TRαA在牙鲆变态发育过程中的调控作用,将TRαA基因克隆插入融合表达栽体pET30a,并在大肠杆菌Escherichia coli DE3(BL21)中进行诱导表达.表达菌株经1 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导4 h后,重组蛋白TRaA表达并形成包涵体.SDS-PAGE和Western blotting检测鉴定表达产物.包涵体经变性后在His-Bind树脂进行亲和层析纯化,柱上复性法对重组蛋白复性,获得纯度较高的目的蛋白,蛋白复性的效果良好.用纯化后的目的蛋白免疫新西兰家兔制备多克隆抗体.Dot blotting检测抗体效价达1:200 000,检测证明抗体特异性良好.此外,通过染色质免疫沉淀技术鉴定了在活体细胞中多克隆抗体与TRαA的特异性结合.表明了甲状腺激素通过其受体在体内参与碱性磷酸酶(ALP)基因的转录调控.     

牙鲆甲状腺激素受体TRaA的原核表达和多克隆抗体的制备及应用

为研究牙鲆甲状腺激素受体TRαA在牙鲆变态发育过程中的调控作用,将TRαA基因克隆插入融合表达载体pET30a,并在大肠杆菌Escherichia coli DE3(BL21)中进行诱导表达。表达菌株经1mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导4h后,重组蛋白TRαA表达并形成包涵体。SDS-PAGE和Western blotting检测鉴定表达产物。包涵体经变性后在His-Bind树脂进行亲和层析纯化,柱上复性法对重组蛋白复性,获得纯度较高的目的蛋白,蛋白复性的效果良好。用纯化后的目的蛋白免疫新西兰家兔制备多克隆抗体。Dotblotting检测抗体效价达1:200000,检测证明抗体特异性良好。此外,通过染色质免疫沉淀技术鉴定了在活体细胞中多克隆抗体与TRαA的特异性结合,表明了甲状腺激素通过其受体在体内参与碱性磷酸酶(ALP)基因的转录调控。     

人重组GM－CSF／HAS－D3嵌合蛋白（GM－HSA）在大肠杆菌中的表达及其产物稳定性的研究

将人粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）和人血清白蛋白第三功能区（ＨＡＳ－Ｄ３）的基因串联后,在Ｅ．ｃｏｌｉ中获高效表达,表达量占菌体蛋白的３２．６％．利用ＴＦ－１体外细胞活性测定表明,ＧＭ－ＨＳＡ的活性单位为１．０４×１０~６Ｕ／ｍｇ,虽然其比活性低于ＧＭ－ＣＳＦ,但比后者具有更高的体外热稳定性和储藏稳定性．     

cDNA cloning and expression of an apoptosis-related gene, humanTFAR15 gene

By means of cDNA-RDA method, some cDNA fragments were found to have high levels of expression during deprivation of GM-CSF (granulocyte macrophage-colony stimulating factor) in a human myeloid cell line, TF-1 cells. One of these fragments was identified as a novel gene. To get the full length of cDNA, rapid amplification of cDNA ends (RACE) and expressed sequence tags (EST) overlapping fragments assembling strategies were used. The novel gene was named TRAF15 (TF-1 cell apoptosis related gene-15), which consists of 1 218 nucleotides and encodes 212 amino acids. The putative protein product of TFAR15 is partially homologous toC. elegans protein C14A4.11. TFAR15 mRNA is expressed in fetal liver, kidney, spleen and lung, and also in some human myeloid cell lines. Both of the TFAR15 mRNA and protein were highly expressed in TF-1 cells after GM-CSF withdrawal.In vitro analysis showed that the recombinant TFAR15 protein could inhibit the natural cell death of 293 cells, an embryonic kidney cell line.     

Inhibition of GM-CSF/IL-3/IL-5 signaling by antisense oligodeoxynucleotides targeting the common beta chain of their receptors.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 play a key role in allergic inflammation. They mediate their effect via receptors that consist of two distinct subunits, a cytokine-specific alpha subunit and a common beta subunit (betac) that transduces cell signaling. We sought to down-regulate the biologic activities of GM-CSF, IL-3, and IL-5 simultaneously by inhibiting betac mRNA expression with antisense technology. Experiments were performed with TF-1 cells (a human erythroleukemia cell line expressing GM-CSF, IL-3, and IL-5 receptors, which proliferates in response to these cytokines), monocytic U937 cells, which require these cytokines for differentiation, and purified human eosinophils. Cells were treated with antisense phosphorothioate oligodeoxynucleotides (ODN) targeting betac mRNA. In contrast to nontreated cells and cells treated by sense or mismatched ODN, antisense ODN inhibited betac mRNA expression and significantly decreased the level of cell surface betac protein expression on TF-1 and U937 cells. Receptor function was also affected. Antisense ODN were able to inhibit TF-1 cell proliferation in vitro in the presence of GM-CSF, IL-3, or IL-5 in the culture medium and eosinophil survival. We suggest that antisense ODN against betac may provide a new therapeutic alternative for the treatment of neoplastic or allergic diseases associated with eosinophilic inflammation.     

HBsAg/GM-CSF融合蛋白在毕赤酵母中的表达及其免疫原性研究

为探索一种提高乙肝病毒表面抗原免疫原性的新方法,用PCR和基因重组技术构建HBsAg与GM-CSF的融合基因,并在毕赤酵母中分泌表达HBsAg/GM-CSF(S-GM)融合蛋白。表达产物用SDS-PAGE检测,W estern b lot分析,离子交换柱纯化后免疫昆明鼠,ELISA检测免疫小鼠血清中抗HBsAg的抗体水平。结果显示S-GM融合蛋白在毕赤酵母中获得了表达,离子交换柱一步纯化即可得到纯度达90%以上的S-GM。W estern b lot分析S-GM可分别与抗HBsAg及抗GM-CSF的抗体特异结合。ELISA检测发现第一次免疫后4w出现抗HBsAg的抗体,加强免疫后融合蛋白组几乎全部阳转,且抗体水平较HBsAg组(P=0.009<0.05)及HBsAg和GM-CSF的混合物组(P=0.032<0.05)高。HBsAg/GM-CSF融合蛋白能够在毕赤酵母中表达,且可增强HBsAg的免疫原性,为提高乙肝疫苗的免疫效果提供了新的思路与方法。     

High-level expression and purification of recombinant huGM-CSF (9-127)/IL-6 (29-184) fusion protein in Escherichia coil

As HuGM-CSF and huIL-6 seem to have synergistic and complementary actions, researchers have proposed that fusion proteins incorporating these two cytokines could show increased biological activity, especially in terms of hematopoietic function. Here, we sought to obtain a functional GM-CSF/IL-6 fusion protein and to investigate its biological activities in vitro. A novel construct encoding a fusion protein of huGM-CSF (9-127) and IL-6 (29-184) was generated in the pBV220 expression vector by step-by-step cloning. Amino acids 1-8 of huGM-CSF and amino acids 1-28 of huIL-6 were deleted by PCR. The mutant huGM-CSF (9-127) and huIL-6 (29-184) cDNAs were linked via a linker sequence encoding 15 amino acid residues (G-G-S-G-S)3. Direct sequencing was used to confirm the validity of the desired construct, and the fusion protein was expressed in Escherichia coli host strain BL21 (DE3) in the form of inclusion bodies (IBs). The expression level was more than 25% of the total cell lysate, and a novel purification and refolding strategy was used to isolate the fusion protein product. Inclusion bodies were purified by Q Sepharose H.P. ion exchange in 8 mol/L urea, followed by in situ refolding by Sephacryl S-200. The renatured fusion proteins were obtained at a purity of >95%, and the strategy of refolding on the gel filtration column was found to be efficient, with a relative refolding rate of 80%. This entire refolding and purification procedure could be performed within one day and may prove applicable to large-scale purification and refolding of recombinant proteins from IBs in E. coli. This new method was used to obtain huGM-CSF (9-127)/IL-6 (29-184) fusion protein with high purity and biological activity. MTT assays in TF-1 and B9 cell lines showed that the specific biological activity of huGM-CSF was 1.14+/-0.10 x 10(8) U/mg, and that for huIL-6 was 1.89+/-0.11 x 10(7) U/mg. The fusion protein exhibited enhanced huGM-CSF, but similar huIL-6 biological activities compared with those of either GM-CSF or IL-6 alone. This suggests that our novel huGM-CSF (9-127)/IL-6 (29-184) fusion protein may hold future promise as a therapeutic agent.     

Establishment and characterization of a unique human cell line that proliferates dependently on GM-CSF, IL-3, or erythropoietin

We have established a novel cell line, designated as TF-1, from a patient with erythroleukemia, which showed complete growth dependency on granulocyte-macrophage colony-stimulating factor (GM-CSF) or on interleukin-3 (IL-3) and carried a homogeneous chromosomal abnormality (54X). Erythropoietin (EPO) also sustained the short-term growth of TF-1, but did not induce erythroid differentiation. These three hematopoietic growth factors acted on TF-1 synergistically. Transforming growth factor-beta and interferons inhibited the factor-dependent growth of TF-1 cells in a dose-dependent fashion, and monocyte-colony stimulating factor and interkeukin-1 enhanced the GM-CSF-dependent growth of TF-1. Ultrastructural studies revealed some very immature features in this cell line. Although TF-1 cells do not express glycophorin A or carbonyl anhydrase I, the morphological and cytochemical features, and the constitutive expression of globin genes, indicate the commitment of TF-1 to erythroid lineage. When induced to differentiate, TF-1 entered two different pathways. Specifically, hemin and delta-aminolevulinic acid induced hemoglobin synthesis, whereas TPA induced dramatic differentiation of TF-1 into macrophage-like cells. In summary, TF-1 is a cell line of immature erythroid origin that requires GM-CSF, IL-3, or EPO for its growth and that has the ability to undergo differentiation into either more mature erythroid cells or into macrophage-like cells. TF-1 is a useful tool for analyzing the human receptors for IL-3, GM-CSF, and EPO or the signal transduction of these hemopoietic growth factors.     

cDNA cloning and expression of an apoptosis-related gene, human TFAR15 gene

By means of cDNA-RDA method. some cDNA fragments were found to have high levels of expression during deprivation of GM-CSF (granulocyte macrophage-colony stimulating factor) in a human myeloid cell line, TF-1 cells. One of these tragments was identified as a novel gene. To get the full length of cDNA, rapid amplification of cDNA ends (RACE) and expressed sequence tags (EST) overlapping fragments assembling strategies were used. The novel gene was named TRAF15 (TF-1 cell apoptosis related gene-15), which consists of 1218 nueleotides and encodes 212 amino acids. The putative protein protein product of TFAR15 is partially homologous to C. elegans protein C14A4. 11. TFAR15 mRNA is expressed in fetal liver, kidney, spleen and lung. and also in some human myeloid cell lines. Both of the TFAR15 mRNA and protein were highly expressed in TF-(?) cells after GM-CSF withdrawal. In vitro analysis showed that the recombinant TFAR15 protein co(?)ld inhibit the natural cell death of 293 cells, an embryonic kidney cell     

粒细胞—巨噬细胞集落刺激因子／白细胞介素—3融合蛋白的表达研究

利用ＰＣＲ扩增得到粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）、白细胞介素－３（ＩＬ－３）完整基因片段,将其分别克隆ｐＧＥＭ－Ｔ构建成ＧＭ－ＣＳＦ／ＩＬ－３融合蛋白基因,ＤＮＡ序列与设计预期一致。将得到的融合蛋白基因克隆对７２ＲＮＡ聚合酶表达载体ｐＴ７ｚｚ,得到表达质粒ｐＦｕ,经转化至表达宿主Ｅ．ｃｏｌｉ ＢＬ２１（ＤＥ３）,在ＩＰＴＧ诱导下获得融合蛋白目的产物的直接表达。经ＳＤＳ－ＰＡＧＥ电泳鉴     

Interleukin-1β suppresses apoptosis in CD34 positive bone marrow cells through activation of the type I IL-1 receptor

Interleukin-1 is a pleiotropic cytokine that has been shown previously to suppress active cell death in T cells. Two cell surface receptors for interleukin-1 have been identified and their genes cloned, type I (IL-RI) and type II (IL-RII) receptors. In the present study, we provide evidence for a role of interleukin-1β in the short-term suppression of cell death both in purified CD34⁺/Lin⁻ bone marrow precursors and in the GM-CSF dependent cell line TF-1. Several lines of evidence suggest that the biologic effects of IL-1β are mediated by activation of type I IL-1 receptors (IL-1RI) and induction of GM-CSF production. First, neutralizing antibodies to IL-1RI but not IL-1RII drastically abrogated cell survival induced by IL-1β in CD34⁺/Lin⁻ cells and TF-1 cells. Second, neutralizing antibodies against GM-CSF abrogate cell survival supported by IL-1 both in CD34⁺/Lin⁻ bone marrow cells and in the cell line TF-1. Furthermore, exposure of TF-1 cells to IL-1β results in a transient accumulation of GM-CSF mRNA, with a peak at 3 h, which was dramatically decreased by neutralizing anti-IL-1RI antibodies. In contrast, neutralizing anti-IL-1RII did not change the IL-1 induced cell survival of bone marrow cells and was followed by a paradoxical increase in viable cell numbers, in c-myc and c-myb mRNA accumulation in IL-1 treated TF-1 cells. Together our results indicate that the increase in cell survival induced IL-1β occurs through binding to IL-1RI and the subsequent production of endogenous GM-CSF. IL-1RII does not appear to be involved in signal transduction in primary CD34⁺/Lin⁻ cells but could negatively regulate the response to IL-1β in TF-1 cells. © 1996 Wiley-Liss, Inc.