Affinity capture of a biotinylated retrovirus on macroporous monolithic adsorbents: towards a rapid single-step purification process

A streptavidin derivitised macroporous monolith was developed to enable single-step capture of chemically biotinylated Moloney Murine Leukaemia Virus (MoMuLV) from crude, unclarified cell culture supernatant. Monoliths were prepared by aqueous cryopolymerisation of acrylamide with N,N'-methylene-bis (acrylamide) and glycidyl methacrylate (Arvidsson et al. [2003] J Chrom A 986:275-290). Streptavidin was immobilised to the epoxy functionalised monoliths. Particulate-containing cell culture supernatant was passed through the monolith without preclarification of the feedstock and adsorption capacities of 2 x 10(5) cfu/ml of adsorbent were demonstrated (cf. Fractogel streptavidin, at 3.9 x 10(5) cfu/ml of adsorbent). The specific titre of the recovered fraction was increased by 425-fold; however, recoveries of less than 8% were achieved. Adsorption of nonbiotinylated MoMuLV on the streptavidin-coated monolith was not observed.     

An RNA Structural Switch Regulates Diploid Genome Packaging by Moloney Murine Leukemia Virus

Retroviruses selectively package two copies of their RNA genomes via mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base-pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5′ untranslated region that contains all residues necessary for efficient RNA packaging (Ψ^WT; residues 147-623) also exhibits a dimerization-dependent affinity for NC, with the native dimer ([Ψ^WT]₂) binding 12 ± 2 NC molecules with high affinity (K_d = 17 ± 7 nM) and with the monomer, stabilized by substitution of dimer-promoting loop residues with hairpin-stabilizing sequences (Ψ^M), binding 1-2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (∼ 100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.     

Affinity recovery of lentivirus by diaminopelargonic acid mediated desthiobiotin labelling

Desthiobiotin-tagged lentiviral vectors have been metabolically produced by DBL producer cells in a 7,8-diaminopelargonic acid (7-DAPA) dependent manner for envelope independent, single-step affinity purification. 7-DAPA, which has little or no affinity for avidin/streptavidin, was synthesised and verified by NMR spectroscopy and mass spectrometry. By expressing the biotin acceptor, biotin ligase and desthiobiotin synthase bioD, DBL cells converted exogenous 7-DAPA into membrane-bound desthiobiotin. Desthiobiotin on the DBL cell surface was visualised by confocal microscopy and the desthiobiotin density was quantified by HABA-avidin assay. Desthiobiotin was then spontaneously incorporated onto the surface of lentiviral vectors produced by the DBL cells. It has been demonstrated by flow cytometry that the desthiobiotinylated lentiviruses were captured from the crude 7-DAPA-containing viral supernatant by Streptavidin Magnespheres^® and eluted by biotin solution efficiently whilst retaining infectivity. The practical, high yielding virus purification using Pierce monomeric avidin coated columns indicates a highly efficient biotin-dependent recovery of infectious lentiviruses at 68%. The recovered lentiviral vectors had a high purity and the majority were eluted within 45 min. This 7-DAPA mediated desthiobiotinylation technology can be applied in scalable production of viral vectors for clinical gene therapy.     

带preS1的乙肝表面抗原融合蛋白的亲和纯化

乙肝表面抗原大分子蛋白ＬＨＢｓ的ｐｒｅＳ１区域２１～４７位肽段为肝细胞受体的结合位点;由ｐｒｅＳ１诱发的抗体有可能阻断病毒对肝细胞的侵蚀。因此,含有ｐｒｅＳ１的新型乙肝疫苗可能会引起机体更广泛、可靠的保护作用。但基因工程表达产物用于生产的一个关键问题是产品的纯化。用抗ＨＢｓＡｇｐｒｅＳ１单抗制成了亲和凝胶载体,并用它纯化了基因工程表达的带ｐｒｅＳ１的乙肝表面抗原融合蛋白．此法有操作简便;纯化效果好,回收率高等优点．一步层析产品纯度可达９０％以上,回收率在５０％左右,具有很好的应用前景。     

肾综合征出血热病毒糖蛋白的提纯和鉴定

以感染肾综合征出血热病毒(HFRSV)的Vero E6细胞为材料,用免疫亲和层析结合制备聚丙烯酰胺凝胶电泳(PAGE)从感染细胞中提纯了HFRSV两种糖蛋白。先用免疫亲和层析从感染细胞的粗制抗原中获得含有四种蛋白的混合液,用[~3H]-氨基葡萄糖在感染细胞中标记病毒糖蛋白,观察到[~3H]-氨基葡萄糖只结合入78K和57K的病毒蛋白。再用制备SDS-PAGE从HFRSV混合液中提纯78K和57K两种蛋白。实验证明这两种糖蛋白均具中和抗原决定簇,57K的糖蛋白尚具血凝活性,初步鉴定表明这两种糖蛋白相当于文献报道的HFRSV G_1和G_2。     

Virus inactivation by protein denaturants used in affinity chromatography

Virus inactivation by a number of protein denaturants commonly used in gel affinity chromatography for protein elution and gel recycling has been investigated. The enveloped viruses Sindbis, herpes simplex-1 and vaccinia, and the non-enveloped virus polio-1 were effectively inactivated by 0.5 M sodium hydroxide, 6 M guanidinium thiocyanate, 8 M urea and 70% ethanol. However, pH 2.6, 3 M sodium thiocyanate, 6 M guanidinium chloride and 20% ethanol, while effectively inactivating the enveloped viruses, did not inactivate polio-1. These studies demonstrate that protein denaturants are generally effective for virus inactivation but with the limitation that only some may inactivate non-enveloped viruses. The use of protein denaturants, together with virus reduction steps in the manufacturing process should ensure that viral cross contamination between manufacturing batches of therapeutic biological products is prevented and the safety of the product ensured.     

Functional Disruption of the Moloney Murine Leukemia Virus Preintegration Complex by Vaccinia-related Kinases

Retroviral integration is executed by the preintegration complex (PIC), which contains viral DNA together with a number of proteins. Barrier-to-autointegration factor (BAF), a cellular component of Moloney murine leukemia virus (MMLV) PICs, has been demonstrated to protect viral DNA from autointegration and stimulate the intermolecular integration activity of the PIC by its DNA binding activity. Recent studies reveal that the functions of BAF are regulated by phosphorylation via a family of cellular serine/threonine kinases called vaccinia-related kinases (VRK), and VRK-mediated phosphorylation causes a loss of the DNA binding activity of BAF. These results raise the possibility that BAF phosphorylation may influence the integration activities of the PIC through removal of BAF from viral DNA. In the present study, we report that VRK1 was able to abolish the intermolecular integration activity of MMLV PICs in vitro. This was accompanied by an enhancement of autointegration activity and dissociation of BAF from the PICs. In addition, in vitro phosphorylation of BAF by VRK1 abrogated the activity of BAF in PIC function. Among the VRK family members, VRK1 as well as VRK2, which catalyze hyperphosphorylation of BAF, could abolish PIC function. We also found that treatment of PICs with certain nucleotides such as ATP resulted in the inhibition of the intermolecular integration activity of PICs through the dissociation of BAF. More importantly, the ATP-induced disruption was not observed with the PICs from VRK1 knockdown cells. Our in vitro results therefore suggest the presence of cellular kinases including VRKs that can inactivate the retroviral integration complex via BAF phosphorylation.     

表达H5N1亚型禽流感病毒HA蛋白的重组鼠白血病病毒的特性

通过反转录 聚合酶链式反应 (RT PCR)扩增了H5N1亚型鹅源禽流感病毒 (AIV)完整的血凝素 (HA)基因并进行了克隆与鉴定。序列测定结果已经登陆GenBank ,登陆号为AY6 394 0 5。序列分析表明所扩增的HA基因开放性阅读框架 (ORF)由170 7个核苷酸组成 ,共编码 5 6 8个氨基酸 ,裂解位点的氨基酸组成为RKKR↓GLF ,含连续的碱性氨基酸 ,具有高致病性AIVHA基因裂解位点的特征。构建了含HA基因的真核表达载体pcDNA HA ,通过与鼠白血病病毒 (MuLV)假病毒构建体系的两种质粒pHIT6 0和pHIT111共转染人胚肾细胞 2 93T ,4 8h后收集假病毒上清 ,超离后通过Western blot证明HA蛋白能够在假病毒颗粒表面表达 ,表明HA能够整合到此病毒粒子表面。通过感染 2 93T、COS 7和NIH3T3三种不同的靶细胞 ,证实所构建的假病毒粒子具有感染性和泛嗜性。本研究成功构建了具有感染性的MuLV HA假病毒体系 ,为研究鹅源禽流感病毒侵入细胞的机理及其组织嗜性的变异提供一种新方法。     

用琼脂糖亲和层析一步纯化蓖麻毒蛋白

本文报道了一种单用琼脂糖(Sepharose4B)来纯化蓖麻毒蛋白的快速简便的方法。我们发现在pH5的条件下,蓖麻毒蛋白和与其密切相关的蓖麻凝集素对琼脂糖的结合能力有很大的差别。在有0.2mol/LD-半乳糖存在下可将蓖麻毒蛋白从Sepharose上洗下,同样条件下蓖麻凝集素仍牢固地结合在柱上。从而经一步柱层析便可得到电泳纯的蓖麻毒蛋白。此法不需另行合成亲和胶,适合于蓖麻毒蛋白的大规模纯化。     

猪繁殖与呼吸综合征病毒M蛋白介导的E蛋白假型鼠白血病病毒的构建

利用RT-PCR法扩增猪繁殖与呼吸综合征病毒(PRRSV)ORF5和ORF6基因,分别构建pcDNA-ORF5、pcD-NA-ORF5/6和pcDNA-ORF5-ORF6真核表达载体,磷酸钙共沉淀法瞬时转染293T细胞,48h后收集细胞,流式细胞仪检测,结果表明:PRRSV ORF5基因编码囊膜蛋白(E蛋白)能在在293T细胞表面表达,而由共表达M蛋白(ORF6基因编码的基质蛋白)介导的E蛋白的表达量比单独E蛋白表达量高,串联表达与单独E蛋白相比较低.将pcDNA-ORF5、pcDNA-ORF5/6和pcDNA-ORF5-ORF6分别与MuLV假病毒构建体系的两种骨架载体pHIT60(包括MuLV的结构蛋白基因,即gag和pol)和pHITlll(为MuLV的基因组,还包括一个报告基因LacZ)瞬时共转染293T细胞,48h后收集假病毒上清,超速离心后通过Western blot证实E蛋白能够在此假病毒颗粒表面表达,证明E蛋白已整合到此假病毒粒子表面.将整合PRRSVE蛋白的假病毒粒子分别感染Marc-145和PAM宿主靶细胞,均能检测到LacZ基因的表达,结果表明:所构建的假病毒粒子具有感染性,且由M蛋白介导的MuLv-E/M感染性比MuLV-E假病毒感染性高.     

腺苷转运体亲和层析分离

本文合成了一种腺苷亲和层析凝胶,并采用亲和层析法从牛脑细胞膜上分离出了几种膜上结合的腺苷结合蛋白质。这些蛋白质在ＳＤＳ－ＰＡＧＥ电泳凝胶上为单一或主要的蛋白带,分子量分别为６４ｋｄ,４５ｋｄ,３５ｋｄ。腺苷转运体抑制剂潘生丁和ＮＢＭＰＲ对６４ｋｄ蛋白与＾３ｈ－腺苷的结合抑制作用远强于腺苷受体的激动剂ＮＥＣＡ和Ｒ－ＰＩＡ;这表明６４ｋｄ蛋白为牛脑细胞膜上结合的腺苷转运体。     

传染性喉气管炎病毒gB基因和新城疫病毒F基因在重组禽痘病毒中共表达

在重组禽痘病毒中表达多个禽类病原的主要免疫原基因是构建多价基因工程疫苗的前提,但相关研究很少。在表达传染性喉气管炎病毒(ILTV)gB基因重组禽痘病毒的转移载体的基础上,构建了含有ILTV gB基因和新城疫病毒(NDV)F基因的重组禽痘病毒转移载体pSY-gB-F,采用脂质体转染禽痘病毒感染的鸡胚成纤维(CEF)细胞后,通过蓝斑试验筛选出重组禽痘病毒(rFPv-gB-F),并进行了6轮蚀斑纯化。Western-blot试验和间接免疫荧光试验证明ILTV gB基因和NBVF基因在rFPV-gB-F感染的CEF细胞中获得表达。为传染性喉气管炎、新城疫与鸡痘活载体多价疫苗的研制奠定基础。     

仙台病毒F基因的克隆及原核表达

目的利用原核表达系统表达仙台病毒(Sendai Virus)F蛋白主要抗原片段FP(S),并对表达产物进行免疫学初步研究。方法根据GenBank公布的仙台病毒F蛋白(gi:9627219)的基因序列设计特异性引物,通过RT-PCR扩增出F基因的主要抗原片段FP(S),插入pMD-18-T载体中,鉴定正确后克隆入pQE31原核表达载体中,将鉴定正确的pQE31-FP(S)转化大肠埃希菌M15,IPTG诱导表达,对大肠埃希菌裂解物进行SDS-PAGE和Western-blot验证。结果大肠埃希菌表达的FP(S)相对分子质量约26×103,与预期相符;能与SeV阳性血清发生特异性反应,出现单一条带。结论原核表达的FP(S)蛋白有良好的抗原性,为检测仙台病毒抗体的ELISA检测方法的研究奠定了基础。     

Binding of ATP to Brain Glutamate Decarboxylase as Studied by Affinity Chromatography

Abstract: The interactions of two forms of porcine brain glutamate decarboxylase (β-GAD and γ-GAD) with the effector ATP were studied by affinity chromatography. A third form, γk-GAD, was only slightly retarded by the affinity matrix and was eluted in the buffer wash. The interaction of GAD with the ATP affinity matrix was qualitatively similar to its interaction with free ATP as reported in previous kinetic studies. The rank order of adenine nucleotides as eluting agents and affinity ligands was ATP > ADP > AMP. GAD was also eluted by its cofactor, pyridoxal 5'-phosphate, and this was enhanced by 1 mM P_i In contrast, a high concentration (140 mM) of P_i by itself was required to elute the enzyme. GAD remained active while bound to the affinity column and was eluted in the holoenzyme form by ATP, indicating that the affinity ligand did not bind in the active site and did not displace catalytically active cofactor from the enzyme.     

用壳聚糖作为配基载体亲和层析提纯淀粉糖化酶的研究

以自制的壳聚糖作配基载体,植物血球凝集素(PHA)作配基,通过戊二醛交联研制成一种用于淀粉糖化酶提纯的新型亲和吸附剂。对淀粉糖化酶的亲和层析研究表明:提纯倍数为1.8,酶活性收率达80％,纯度经聚丙烯酰胺凝胶等电聚焦电泳(IEF-PAGE)鉴定为一条带,没有非特异性吸附作用。具有简单、安全、快速和高收率等优点。     

哺乳动物细胞中基因工程人生长激素的提纯和性质研究

 在哺乳动物细胞中克隆表达的人生长激素,经等电点和乙醇沉淀后,再用亲和层析法,获得产品。经对该产品的各项生化指标分析鉴定和活力测定,所得结果都与天然人生长激素相符合,证明其性质相同。     

Rapid Purification of Annexin V from Human Placenta by Affinity Chromatography

Abstract

Affinity purification of annexin V from human placenta on column with appropriate monospecific antibodies is developed. The procedure permits purification of the protein to a highly purified state by a two stage procedure. The yield of the protein is about 5 mg per 100 g of wet tissue. Because of high homologies between various annexins, it was supposed that this procedure can be also applied for purification of other annexins from other tissues.     

整合禽流感病毒血凝素糖蛋白的假型鼠白血病病毒

本研究通过一个瞬时转染系统将H5N1亚型鹅源禽流感病毒囊膜表面的血凝素(HA)糖蛋白整合到鼠白血病病毒(MuLV)颗粒表面并进行了感染性测定。将包含HA基因的真核表达质粒pcDNA-HA与MuLV假病毒构建体系的两种质粒pHIT60(包括MuLV的结构蛋白基因,即gag和pol)和pHIT111(为MuLV的基因组,还包括一个报告基因LacZ)瞬时共转染转化了SV40大T抗原的人胚肾细胞293T,48小时后收集假病毒上清进行了一系列鉴定。将假病毒上清超速离心后用抗H5亚型禽流感病毒的多抗通过Western-blot证实HA 蛋白能够在此假病毒颗粒表面表达,表明HA能够整合到此病毒粒子表面。通过感染293T、COS 7和NIH3T3 三种不同的靶细胞,均能检测到LacZ基因的表达,证实所构建的假病毒粒子具有感染性。本研究成功构建了具有感染性的MuLV-HA假病毒,为研究鹅源禽流感病毒侵入细胞的机理及其组织嗜性的变异提供一种新方法。     

基于亲和层析方法研究栀子苷的药物靶标

目的:运用亲和层析方法初步探索栀子苷的特异结合蛋白。方法:采用环氧氯丙烷(Epichlorohydrin,ECH)作为活化试剂对琼脂糖凝胶进行环氧基修饰,通过偶联栀子苷与环氧活化的琼脂糖凝胶(epoxy activated Sepharose CL-6B,EAS6B),制备以栀子苷为配基的亲和介质,以高效液相色谱法验证其偶联。利用栀子苷-EAS6B亲和介质,从小鼠脑组织总蛋白中筛选特异结合蛋白。结果:优化偶联条件后,得到最佳的活化反应条件:15%ECH、0.8 mol/L Na OH于37℃反应3 h,环氧基密度达到122μmol/m L,琼脂糖凝胶亲和介质的偶联量为19.53%,偶联率为17.08μmol/m L。从小鼠脑组织总蛋白中筛选得到3个栀子苷特异结合蛋白的条带,经质谱鉴定,发现主要是由热休克蛋白、脑酸溶性蛋白和谷氨酰胺合成酶等组成。结论:确立了琼脂糖凝胶环氧活化的最佳条件,制备了栀子苷-EAS6B亲和介质并应用其筛选出栀子苷的结合蛋白。     

Isolation of Putative Benzodiazepine Receptors from Rat Brain Membranes by Affinity Chromatography

Abstract: The benzodiazepine receptor from rat brain was solubilised and purified 5200-fold by affinity chromatography. The affinity column contained an immobilized benzodiazepine (delorazepam) and biospecific elution with 6 m m -chlorazepate was achieved. The purified receptor is apparently homogeneous in SDS-polyacrylamide gel electrophoresis. The native protein had a molecular weight of 240,000, and the subunit one of 60,000. The dissociation constant ( K _D) is 8 n m for [³H]diazepam. A correlation exists between the value of affinity obtained for benzodiazepine derivatives and their known pharmacological effectiveness.