Improved large scale purification procedure of natural human fibroblast interferon

Human fibroblast interferon, produced in tissue culture by a superinduction procedure, was purified 10,000-fold by chromatography to ca 50% purity as judged by SDS-PAGE. The specific activity of the interferon preparation is 2 X 10(8) and the yield is 70%. The interferon preparation is suitable for clinical use.     

重组酵母鸡γ干扰素的抗病毒活性测定及临床初步应用

为了获得具有天然抗病毒活性的重组酵母鸡γ干扰素,以Con A(刀豆素A)诱导培养4~10h的鸡全血中提取的淋巴细胞总RNA为模板,通过RT-PCR的方法扩增出鸡γ干扰素成熟蛋白基因。通过EcoRⅠ和XbaⅠ两个酶切位点把鸡γ干扰素成熟蛋白基因插入到酵母表达载体pPICZa-A上,得到了重组酵母鸡γ干扰素表达载体pPICZa-A-CHIFN-γ,经BstxⅠ线性化后的重组载体被转入酵母菌株X33中,通过PCR的方法来筛选重组酵母菌,在甲醇诱导表达后,SDS-PAGE结果显示有两株重组菌在诱导72h后其表达上清中有大小为16kDa的目的条带。干扰素生物活性测定经典实验(微量病变抑制实验)和临床初步应用结果皆说明重组酵母鸡γ干扰素具有较强的抗病毒的生物活性和较好的临床使用前景。     

The IFN-alpha-inducing capacity of Sendai Virus in human leukocytes is independent from the hemagglutinating and hemolytic activities and from the viral proteins of the Sendai Virus preparations

About 30 Sendai Virus (SV) preparations, examined for their capacity to induce natural human interferon alpha from fresh human leukocytes (Le-IFN-alpha) of healthy donors, were characterized for hemagglutinating (HA) and hemolytic (HemA) activities and for SDS-PAGE proteic pattern. The SV preparations were produced by a single passage or by serial propagations through eggs in different conditions of multiplicity of infection (m.o.i.).The produced SV subpopulations showed variable IFN-inducing capacity, the values of which are distributed over a 6-fold range resembling a Gaussian distribution. No detectable difference was observed comparing the SV preparation obtained by serial propagations with those obtained by a single passage. The variability of the measured HA activity and HemA activity and as well as the SDS-PAGE proteic pattern of the SV preparations did not correlate with the induced amount of IFN per cell. In the same experimental conditions to produce Le-IFN-alpha, u.v.-treated SV preparations were unable to induce interferon depending on the u.v.-treatment. So it can be concluded that the distinct nHu-IFN-alpha-inducing capacity of SV subpopulation could be mainly associated with divergent compositions of the viral RNAs rather than with a different contents of viral proteins, among those detectable in SDS-PAGE and those responsible for HA activity and for HemA activity.     

Anomalous behavior of human leukocyte interferon subtypes on polyacrylamide gel electrophoresis in the presence of dodecyl sulfate

35S-labeled human leukocyte interferon (IFN) subtypes produced in a cell-free system derived from Escherichia coli were analyzed by polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE). Some IFN subtypes anomalously showed lower electrophoretic mobilities than those expected from their formula molecular masses. The results with hybrid IFNs and esterification suggest that this anomaly of IFN subtypes on SDS-PAGE is due to the introduction of one or two negative charges in the middle of the molecule.     

疏水相互作用层析分离重组人干扰素α2b

目的采用疏水相互作用层析分离重组人干扰素α2b,去除干扰素样品中的二聚体,得到高纯度的干扰素用于进一步的研究。方法首先采用阳离子交换层析纯化复性重组人干扰素α2b,去除了大部分的杂蛋白,然后采用疏水相互作用层析纯化重组人干扰素α2b,去除复性过程中产生的错误折叠体和二聚体,并考察盐浓度、pH值、流速和洗脱液中尿素对疏水相互作用层析纯化效果的影响。结果硫酸铵初始浓度1.2 mol/L、缓冲液pH值6.0、流速2.5 mL/min、洗脱液中添加尿素浓度为2 mol/L时疏水相互作用层析纯化效果最佳。最终得到的重组人干扰素α2b非还原型SDS-PAGE电泳均呈单一条带。结论确定了疏水层析纯化重组人干扰素α2b的最优条件,成功提取到具有高活性、高纯度的重组人干扰素α2b纯品。     

DNA shuffling技术提高干扰素比活性的研究

DNA shuffling技术可以定向进化干扰素,获得比rhIFN-α2b标准品更高比活的干扰素.rhIFN-α2b基因与Infergen基因用DNaseI酶切成30-50 bp小片段,回收之后进行无引物PCR 重聚和有引物PCR扩增基因.将重组基因连接到载体pUC19中,转化至DH5α并挑选阳性克隆测序.将测序正确的突变基因连接到载体pET30a中并转化至BL21（DE3）中,然后诱导蛋白表达、SDS-PAGE凝胶电泳和Western blot分析.经过表达细胞诱导、细胞裂解、包涵体变性、复性和单克隆抗体亲和层析之后,获得高纯度的重组干扰素.经测定纯化后的重组干扰素比活达到5.8×108IU/mg,比rhIFN-α2b标准品高出5倍左右.实验结果证明此实验方法对提高干扰素的比活是有效的,可以获得比标准品更高比活的干扰素.     

北极狐g-干扰素cDNA的克隆、表达及活性测定

为研究狐g-干扰素的生物学活性, 应用反转录聚合酶链式反应(RT-PCR)从北极狐外周血淋巴细胞中扩增出g-干扰素(VuIFN-g)cDNA。序列分析表明VuIFN-g cDNA全长501 bp, 编码23个氨基酸的信号肽和144个氨基酸的成熟肽蛋白, 与已发表的银黑狐和犬IFN-g 核苷酸序列同源性为99.8%和99.4%; 氨基酸同源性均为100%。应用原核表达系统高效表达北极狐g-干扰素成熟肽蛋白, SDS-PAGE和Western blotting分析表达的融合蛋白分子量约为19 kD, 以不溶性的包涵体形式存在。重组蛋白经纯化和复性, 在Vero和MDCK细胞上可明显抑制VSV病毒的复制, 并测出北极狐重组g-干扰素的活性单位分别为1.0×106 u/mg和1.56×105 u/mg, 为进一步开发基因工程狐干扰素奠定基础。     

聚乙二醇修饰重组人干扰素α-2b的碘染色法

目的：建立检测聚乙二醇位点特异性修饰重组人干扰素α-2b反应的方法。方法：采用分子量20000的甲氧基聚乙二醇马来酰亚胺修饰重组人干扰素α-2b,反应混合物样品经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）后,碘染色法判断反应产物组成。结果：该修饰反应产物除含有单PEG化的干扰素α-2b外,还有不同修饰程度的多PEG化干扰素。结论：本方法方便快捷、分辨率高、特异性强,同时可用于其它聚乙二醇修饰蛋白质的分析研究。     

斑马鱼干扰素γ的克隆表达研究

目的：通过人工诱导斑马鱼干扰素的产生,证实两种斑马鱼干扰素γ基因ifng1-1和ifng1-2的存在,并克隆这两种干扰素基因,构建高效表达载体并做原核表达。方法：通过conA药浴,诱导斑马鱼干扰素γ的表达,即刻提取组织总RNA,并通过RT-PCR方法扩增两种干扰素γ基因。将目的基因连入表达载体pET24a,构建重组载体pET24a-ifng1-1和pET24a-ifng1-2。将载体转化BL21,并用IPTG诱导融合蛋白的表达。产物经SDS-PAGE检测,分析蛋白表达情况。结果：成功克隆了两种斑马鱼干扰素γ基因,构建了pET24a-ifng1-1和pET24a-ifng1-2重组载体,并实现了原核融合表达。结论：两种干扰素γ基因都能够被conA所诱生,且具有相似特性,表明两种干扰素γ都不是假基因,都有可能在免疫系统中发挥作用,本实验为进一步研究两种干扰素γ的功能奠定了基础。     

Activation of human monocyte cytotoxicity by natural and recombinant immune interferon

Human T cell hybridomas were established by fusion of SH9 cells, the 6-thioguanine-resistant mutant line of human T lymphoma Hut 102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. Hybridoma line L38 produced a macrophage activating factor (MAF) with the ability to activate human peripheral blood monocytes to show enhanced cytotoxicity against human colon adenocarcinoma HT-29 cells in a 72-hr 125iododeoxyuridine-release assay. The L38 line was then cloned by the limiting dilution technique and two sublines, L38B and L38D, were found to produce high levels of MAF constitutively. Interferon activity was also detected in L38B and L38D supernatants. When interferon activity was neutralized with specific antiserum to purified human immune interferon (IFN-gamma), MAF activity was abrogated. To confirm that the MAF activity is indeed due to IFN-gamma, IFN-gamma was purified from the culture supernatant of another human T cell hybridoma, L265K2, a cell line known to produce high levels of IFN-gamma. Two highly purified IFN-gamma fractions with m.w. of 20,000 and 25,000, respectively, were obtained by NaDodSO4/polyacrylamide gel electrophoresis (SDS-PAGE). Similar fractions were obtained from IFN-gamma derived from human peripheral blood lymphocyte (PBL) cultures induced with 12-0-tetradecanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA). In comparison, Escherichia coli-derived recombinant human IFN-gamma separated by SDS-PAGE yielded two major active fractions with m.w. of 17,000 and 34,000. With all three types of preparations, a close correlation was found between the presence of IFN-gamma activity demonstrable in an antiviral assay and MAF activity in individual fractions. Substantial quantitative differences were observed in the ability of various human IFN to activate monocytes. Although no MAF activity was detected with IFN-alpha and IFN-beta at concentrations up to 200 U/ml, both natural and recombinant IFN-gamma showed marked MAF activity at concentrations as low as 0.3 to 1 U/ml.     

A 38,000-dalton antigen found in Namalwa cells induced by Newcastle disease virus

An antigen has been isolated from Namalwa cells, a Burkitt lymphoma line, that was induced by Newcastle disease virus (NDV) for interferon production. The antigen was extracted by 3 M NaCl from ribonucleoprotein particles (RNP), obtained from the nuclear 0.01 M Tris extract, and was purified by hydroxylapatite chromatography, phosphocellulose chromatography, and preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its molecular weight was 38 kilodalton (kDa) as determined by SDS-PAGE. The tryptic peptide map of 125I-labeled antigen contained seven major peptides. The antigen was not found in HeLa cells, normal human liver or in Namalwa cells that had not been induced by the virus. This result suggests that this antigen was produced in Namalwa cells as a result of induction by the NDV virus.     

猪β干扰素在毕赤酵母中的分泌表达及其对伪狂犬病毒的抑制作用

为了研制高活性的重组猪β干扰素,对PoIFN-β成熟蛋白第3、7和164位的3个氨基酸密码子进行毕赤酵母偏嗜性改造并构建了酵母表达载体pPICZαA-PIB。pPICZαA-PIB经SacⅠ酶切线性化后电转化导入毕赤酵母菌株X-33。多株PCR鉴定为阳性的酵母转化子经甲醇诱导发酵分泌表达了PoIFN-β,其中B1株酵母的PoIFN-β产量最高,约为2.5×105U/mL,其表达量约为60μg/mL,比活为4.17×106U/mg。将发酵上清液用PEG20000浓缩后进行SDS-PAGE和Western blot检测,结果表明表达产物是分子量约为28kDa和25kDa蛋白的混合物,两者均可与PoIFN-β阳性抗血清发生特异反应。表达产物比PoIFN-β理论推导分子量(约20.8kDa)大,推测可能是表达产物发生了不同程度的糖基化。重组PoIFN-β对伪狂犬病毒在细胞中增殖可呈现抑制作用,并且rPoIFN-β对伪狂犬病毒在MDBK细胞上早期增殖的抑制效果最为明显。     

兔早期胚胎发育相关新基因IFRG的克隆和表达

近来的研究表明干扰素在哺乳动物早期胚胎发育中有重要的作用。我们首次克隆了兔早期胚胎发育相关新基因IFRG（干扰素应答基因）的全长cDNA序列（AJ584672）,根据该cDNA序列以兔卵巢cDNA为模板经PCR扩增后克隆了兔IFRG cDNA的完整开放阅读框(396bp)。将其克隆到原核表达载体pGEX-4T-2上,在大肠杆菌BL21中进行了GST-IFRG融合蛋白的表达。 经IPTG诱导培养后,SDS-PAGE电泳检测结果显示在41kDa处有特异性表达蛋白,回收融合蛋白作为抗原免疫小鼠,制备多克隆抗体,通过Western杂交表明该融合蛋白具有生物免疫活性。     

Differential inactivation and separation of homologous and heterologous antiviral activity of human leukocyte interferon by a proteolytic enzyme.

Human leukocyte interferon (HuLeIF) can express its antiviral activity on both human and bovine cells. The rates of inactivation of HuLeIF by α-chymotrypsin, as expressed on human and bovine cells, are not the same: the ability to induce activity on human cells is lost significantly more rapidly than the activity detected on bovine cells; usually a margin of greater than one hundred-fold exists after α-chymotrypsin treatment. HuLeIF, when subjected to analysis on 10% SDS-PAGE, can be separated into two molecular weight species, one having apparent molecular weight of approximately 21,000 daltons, the other 18,000 daltons. A more rapidly migrating form (molecular weight 16,500 daltons) can also be isolated, which is considerably more active on bovine cells than on human cells. α-chymotrypsin-treated samples analyzed by SDS-PAGE show a clear separation of the activities expressed on human and bovine cells. The residual activity detected on human cells is isolated only in the 21,000 component while the activity found on bovine cells is recovered only as the 16,500 dalton species.     

Radioactive human lymphoblastoid interferon. One-step purification, regulation of heterogeneous species production and its use for radioimmunoassay

Human lymphoblastoid interferon (alpha type), labeled with [3H]leucine added to virus-induced Namalwa cells, was purified quantitatively and in one step from the culture fluid by immune precipitation. The material showed, upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, only four radioactive bands with molecular weights ranging from 17000 to 21000, which coincided well with interferon activity. They coincided also with the four interferon protein bands in the electropherogram of unlabeled interferon purified by a different method. The purity of the labeled interferon was ascertained also by polyacrylamide gel electrophoresis in the absence of dodecyl sulfate. Pulse-labeling of interferon with [3H]leucine for 1 h at various times after induction indicated that the cells always synthesized and secreted the four interferon species in parallel during the interferon production period. Competitive radioimmunoassay for human interferon alpha was achieved by the use of purified radioactive interferon, anti-(interferon alpha) serum, and bacterial adsorbent. The immune precipitation of the labeled interferon was inhibited by unlabeled interferon alpha, and 100 international reference units of interferon alpha could be measured in this way.     

Action of interferon on cell membrane of mouse lymphocytes : Inhibition of ligand-induced redistribution of surface receptors

Mouse interferon at 50–100 U/ml or higher concentrations inhibited cap formation of surface components on mouse lymphocytes induced by anti-lymphocyte serum, concanavalin A (ConA), phytohemagglutinin (PHA) and soybean agglutinin (SBA). Interferon from species other than mouse were not effective, suggesting that interferon itself in the preparations used was responsible for the observed effect. The inhibition was observed immediately after addition of interferon, without preincubation of cells with it. The effect was reversible, disappearing after a short lag when interferon was removed. The effect may thus be a reflection of a rapid change in cell membrane upon binding of interferon molecules. On the other hand, the cap formation by anti-immunoglobulin, anti-thy 1, 2, anti-H-2 sera and wheat germ agglutinin (WGA) were not influenced by interferon at all, indicating that the inhibition by interferon was not a general phenomenon. The cap formations susceptible to interferon are those that are enhanced by pretreatment of cells with colchicine and are accompanied by marked uropod formation, but those insusceptible to interferon occur readily, becoming sharp spots which are then shed. It is suggested that interferon may modify the microtubule-containing structure, it it may selectively affect those membrane components that are anchored to it.     

基因工程犬干扰素α的制备及纯化工艺

目的：运用基因工程技术制备高活性的重组犬仪干扰素。方法：用发酵罐大量培养工程菌,经超声破碎菌体获粗制包涵体,用1％ TritonX-100洗涤去除部分杂蛋白后,以8mol／L尿素溶解包涵体,稀释复性并超滤浓缩,用阴离子柱层析法纯化目的蛋白,测定重组犬干扰素d的活性。结果：得到的重组犬干扰素仅的相对分子质量为19×10^3,蛋白含量为0.55mg／mL,纯度95.65％,目的蛋白回收率为13.75％,活性1.78×10^7U／mL,比活性3.24×10^7U／mg。结论：制备了高纯度且具有高活性的重组犬α干扰素。     

Sarcolectin: complete purification for molecular cloning.

A great variety of vertebrate cells contain detectable amounts of lectins, able to stimulate the initiation of cellular DNA synthesis. One of them, sarcolectin (SCL) can block interferon (IFN) action, by inhibiting the synthesis and the expression of the IFN dependent secondary proteins. As a result, the IFN-induced antiviral state is abolished in the cells, which likely facilitates their replication. We identified a major 65 kDa and a minor 55 kDa protein, which could carry these cellular functions. Their purification, especially that of the 65 kDa, was difficult, because of the proximity of albumin. We devised therefore a two-step primary separation, followed by a four-step final purification, which are reported here. The purification was controlled by high pressure liquid chromatography (HPLC), SDS-PAGE electrophoresis and identified by Western blots. We found that only the minor 55 kDa protein can be considered as being sarcolectin, while the major 65 kDa band results from the binding of some SCL molecules to albumin. The major biological functions, namely, stimulation of DNA synthesis and cell agglutination were preserved to the end of the last purification step. This work is requisite for establishing the molecular structure of SCL by recombinant DNA technology.     

鸡α干扰素基因的克隆、原核表达及抗病毒效果研究

通过PCR从三黄肉鸡的肝脏基因组中扩增了鸡α干扰素(ChIFN-α)全长基因。序列分析表明ChIFN-α基因全长582bp,亚克隆其成熟蛋白编码基因(489bp),利用基因重组技术构建了E.coli/pET-28a( )-IFNα,使IFN-α置于pET-28a的T7启动子下游并同6×His(多聚组氨酸标签)-Tag融合。经酶切鉴定,DNA测序证实重组质粒构建正确;将重组质粒转化大肠杆菌BL21(DE3),IPTG诱导表达,SDS-PAGE,Western-blot分析证实表达出22kD左右的融合蛋白,表达的蛋白以不溶性的包涵体形式存在并且具有良好的免疫学活性。提纯的包涵体纯度可达70%以上,用镍亲和层析方法纯化蛋白则可达到95%。经透析复性后的蛋白在鸡胚成纤维细胞上能够抑制H9N2禽流感病毒的复制。鸡胚试验中重组干扰素抗病毒效果好在H9N2禽流感病毒攻毒组中,能保护鸡胚并使其孵出率达到100%的重组干扰素最小蛋白含量为2μg;重组干扰素对新城疫病毒复制也有一定的抑制能力,延迟该病毒复制时间为12h至48h;雏鸡试验表明重组干扰素也能较好地抵抗H9N2禽流感病毒对雏鸡的感染。两组试验均表明,亲和层析纯化蛋白是包涵体蛋白活性的20倍左右。