Identification of domains of the v-crk oncogene product sufficient for association with phosphotyrosine-containing proteins.

The oncogene product of the avian sarcoma virus CT10, P47gag-crk, contains the SH2, SH2', and SH3 domains and binds proteins in a phosphotyrosine (ptyr)-dependent manner. In this study, we have determined the region of P47gag-crk essential for binding to ptyr-containing proteins. Mutant P47gag-crk proteins expressed in Escherichia coli that have the intact SH2 and SH2' regions retained the capacity to bind ptyr-containing proteins obtained from cells transformed by crk and src. The deletion of SH2 resulted in the loss of binding activity. Other mutants that have altered SH2 or SH2' bound few, if any, of the ptyr-containing proteins. Those mutants that bound ptyr-containing proteins associated with tyrosine kinase activity. We also found that polypeptides containing SH2, SH2', and SH3 of p60v-src and p60c-src associated with ptyr-containing proteins from crk-transformed cells. Thus, the SH2 and SH2' domains of P47gag-crk are responsible for their binding to ptyr-containing proteins.     

High-level expression using baculovirus, purification, and characterization of a monomeric form of type II calmodulin-dependent protein kinase.

The type II calmodulin-dependent protein kinase is an oligomer existing in multiple isozymic forms. To facilitate investigations of the regulatory mechanisms of this complex enzyme, we have constructed a truncated, calmodulin-dependent monomer of the alpha subunit. The N-terminal enzyme fragment (alpha 315) was expressed at high levels in a baculovirus/insect cell expression system. The recombinant protein was purified chromatographically using DEAE-cellulose, calmodulin-Sepharose, and AffiGel blue, yielding 4 mg of kinase from 1.5 x 10(8) cells in 4 h. Characterization of the truncated kinase indicated that it is a monomer and that interactions of alpha 315 with calmodulin and substrates are indistinguishable from those observed for purified holoenzyme from rat brain. These results indicate that the baculovirus/insect cell expression system is well suited for producing alpha 315, a structurally simplified model of the type II calmodulin-dependent protein kinase.     

Expression of recombinant anti‐breast cancer immunotherapeutic monoclonal antibody in baculovirus–insect cell system

The anti‐breast cancer monoclonal antibody (mAb) BR55 was expressed in the baculovirus–insect cell expression system, which is advantageous because of its high production capacity, cell culture flexibility and glycosylation capability. The baculovirus–insect cell expression system was successfully established for production of mAb BR55 and mAb BR55 fused with the KDEL (Lys–Asp–Glu–Leu) endoplasmic reticulum (ER) retention signal (mAb BR55K). The heavy chain (HC) and light chain (LC) genes of mAb BR55 were cloned under the control of the polyhedrin (P_PH) and P₁₀ promoters, respectively, in the pFastBacDual vector. The antibody gene‐expression cassettes carrying both the HC and LC genes were transferred into a bacmid in Escherichia coli (DH10Bac). The bacmid carrying the expression cassettes was transfected into Sf9 insect cells to generate baculovirus expressing mAb BR55 and BR55K. Western blot analysis confirmed the expression of mAb BR55 and BR55K in baculovirus‐infected insect cells. Cell direct enzyme linked immunosorbent assay (ELISA) showed that both mAbs from insect cell lysates or cell culture medium bound to MCF‐7 human breast cancer cells. Both mAb BR55 and BR55K were successfully purified using a Protein A affinity column. Collectively, these results suggest that the anti‐breast cancer mAb BR55 can be expressed, properly assembled and purified from the baculovirus expression system, which can serve as an alternative system for antibody production.     

Hydrophobin (HFBI): A potential fusion partner for one-step purification of recombinant proteins from insect cells

Hydrophobins play an important role in binding and assembly of fungal surface structures as well as in medium-air interactions. These, hydrophobic properties provide interesting possibilities when purification of macromolecules is concerned. In aqueous micellar two-phase systems, based on surfactants, the water soluble hydrophobins are concentrated inside micellar structures and, thus, distributed to defined aqueous phases. This, one-step purification is attractive particularly when large-scale production of recombinant proteins is concerned. In the present study the hydrophobin HFBI of Trichoderma reesei was expressed as an N-terminal fusion with chicken avidin in baculovirus infected insect cells. The intracellular distribution of the recombinant fusion construct was analyzed by confocal microscopy and the protein subsequently purified from cytoplasmic extracts in an aqueous micellar two-phase system by using a non-ionic surfactant. The results show that hydrophobin and an avidin fusion thereof were efficiently expressed in insect cells and that these hydrophobic proteins could be efficiently purified from these cells in one-step by adopting an aqueous micellar two-phase system.     

以杆状病毒为载体在鸡原代骨骼肌细胞中表达IBDV VP2基因

摘要：【目的】本研究旨在构建在鸡原代骨骼肌细胞中表达IBDV病毒VP2基因的重组杆状病毒。【方法】从IBDV适应细胞毒中提取RNA,用RT-PCR技术扩增VP2基因,将其克隆到自主构建的杆状病毒转移载体的CMV启动子之下,通过Bac-to-Bac系统获得VP2重组Bacmid,并将其转染Sf9昆虫 细胞,获得了VP2重组杆状病毒。重组病毒经扩增后以50个MOI感染鸡原代骨骼肌细胞,接种72h后裂解细胞收获蛋白。【结果】蛋白样品经SDS-PAGE和Western blot证实VP2蛋白获得表达,分子量约48kDa,与预测蛋白大小一致,且能被IBDV阳性血清所识别。【结论】重组杆状病毒可以有效地将VP2基因导入鸡原代细胞,并在CMV的启动下表达具有抗原性的VP2蛋白,本研究为研制IBDV及其他重要禽类传染病的杆状病毒载体疫苗奠定了基础。     

携带苏云金芽胞杆菌cry1Ac10基因重组杆状病毒的构建及其杀虫活性

用Bac-to-Bac系统,构建了包含极晚期基因ph启动子驱动的带有全长苏云金芽胞杆菌cry1Ac10基因和完整多角体基因的重组质粒pFCP,用该重组质粒感染昆虫Sf9细胞,得到了带有多角体和能够表达cry1Ac10基因的重组杆状病毒vFcph,并在昆虫细胞中表达了Cry1Ac10蛋白。同时构建了含cry1Ac10的穿梭载体．pHTC,并分别转化大肠杆菌、枯草杆菌和苏云金杆菌晶体缺陷型菌株,结果表明此三种工程菌均表达了分子量为133．3kDa的原毒素蛋白,其中在苏云金芽胞杆菌中的表达量最高。生物测定表明重组杆状病毒vFcph的表达产物具有杀虫活性,能增加杆状病毒力,加快杆状病毒杀虫速度,说明利用杆状病毒极晚期基因启动子驱动苏云金芽胞杆菌杀虫晶体蛋白表达,从而改善杆状病毒的杀虫特性是可行的。     

Expression of Human Recombinant CD23 in Insect Cells

Abstract

Human CD23 (low affinity receptor for IgE) has been expressed in insect cells (Sf9) using the baculovirus expression system and the baculovirus transfer vector pAc373. Insect cells infected with a recombinant baculovirus coding for CD23 synthesized a polypeptide not found in wild-type infected insect cells that had antigenic properties similar to natural CD23 produced in RPMI 8866 cells. Surface expression of recombinant CD23 was demonstrated by its ability to bind IgE. Recombinant CD23 expressed in insect cells had a slightly lower molecular weight (4 3 kDa) than that of natural CD23 (4 5 kDa) from RPMI 8866 cells as detected by SDS-PAGE followed by Western-blotting. Affinity-purified recombinant CD23 from in-fected insect cells showed B-cell growth promoting activity. These observations demonstrate for the first time that biologically active recombinant CD23 can be produced by the baculovirus expression system, thus providing a useful source of recombinant material to elucidate the biological functions of CD23.     

人修饰型降钙素和鼠酰胺化酶基因在昆虫细胞中的偶联表达

人降钙素 (hCT)是 32氨基酸的多肽激素 ,C-端为α脯氨酰胺结构 ,具有调节体内钙、磷代谢等许多重要生理功能。用重组昆虫杆状病毒表达系统 ,偶联表达合成的人修饰型降钙素 (hmCT)基因与GST融合基因和大鼠酰胺化酶 (PAM)基因 ,再用抗hmCT或抗PAM抗体 ,既检测到由昆虫细胞表达的GSThmCT产物也检测到PAM产物。经GSH 琼脂糖凝胶亲和层析 ,分离纯化GSThmCT融合蛋白。这种蛋白修饰酶与底物在真核细胞偶联表达也将适用于其他生物活性肽的体外表达     

CFP10-ESAT-6-MPB64在杆状病毒系统中的表达纯化及免疫原性鉴定

目的：在杆状病毒昆虫细胞表达系统（baculovirus expression vector system,BEVS）中表达结核分枝杆菌蛋白CFP10-ESAT-6-MPB64,并鉴定其免疫原性。方法：目的基因CFP10-ESAT-6-MPB64连接到pFastBac转移载体并转化DH10Bac感受态,通过Tn7转座片段将目的基因转座到Bacmid中,得到Bacmid-CFP10-ESAT-6-MPB64穿梭载体,脂质体包被后转染Spodoptera frugiperda（Sf9）细胞收获病毒,病毒转染细胞后收集上清通过Co亲和层析纯化得到目的蛋白。纯化的蛋白免疫Balb/c小鼠并检测血清中特异性抗体滴度及PPD抗体,ELISA检测CFP10-ESAT-6-MPB64蛋白刺激脾脏细胞产生IFN-γ的浓度,MTT法检测目的蛋白对免疫后小鼠脾脏细胞的增殖作用。结果：CFP10-ESAT-6-MPB64在昆虫细胞中成功表达,纯化后蛋白纯度达90%以上,蛋白产量为42mg/L,纯化蛋白能有效刺激Balb/c小鼠产生抗体,提高小鼠脾脏细胞培养基中IFN-γ的含量,目的蛋白在1~50μg/ml之间对脾脏细胞有明显的促增殖作用。     

Production and purification of human indoleamine 2,3-dioxygenase (HuIDO) protein in a baculovirus expression system and production and characterization of egg yolk antibody against the purified HuIDO

The human indoleamine 2,3-dioxygenase (HuIDO) baculoviral construct, for expression of HuIDO protein with a hexa-histidine and FLAG (DYKDDDDK) tag, was produced using the BacPAK Baculovirus Expression System. HuIDO baculovirus was used to infect Sf21 insect cells to produce functionally active protein in large amounts. Conditions for protein purification by metal affinity chromatography were determined and optimized. Addition of haemin ensured optimal activity of the purified heme-containing oxygenase. The soluble purified protein was used to immunize a chicken to produce large quantities of polyclonal IgY against HuIDO. The anti-HuIDO IgY antibody specifically detected HuIDO produced by a range of cell types including transfectants and native HuIDO expression induced in IFN-gamma-stimulated cells. The antibody detected HuIDO in cell lysates by western blotting and in the cytoplasm of cells by microscopy. The antibody was unable to block the function of the enzyme, indicating that this antibody binds outside the active site of HuIDO.     

Characterization of the human transferrin receptor produced in a baculovirus expression system

Recombinant human transferrin receptor has been produced in a baculovirus expression system. Magnetic particles coated with an anti-transferrin receptor monoclonal antibody were used to immunoselect virus-infected Sf9 insect cells expressing the human transferrin receptor on their cell surface. Recombinant virus containing the human transferrin receptor cDNA was then plaque-purified from these cells. Biosynthetic labeling studies of infected cells showed that the human transferrin receptor is one of the major proteins made 2-3 days postinfection. The recombinant receptor made in insect cells is glycosylated and is also posttranslationally modified by the addition of a fatty acid moiety. However, studies with tunicamycin and endoglycosidases H and F showed that the oligosaccharides displayed on the recombinant receptor differ from those found on the naturally occurring receptor in human cells. As a consequence, the human receptor produced in the baculovirus system has an Mr of 82,000 and is smaller in size than the authentic receptor. About 30% of human transferrin receptors made in insect cells do not form intermolecular disulfide bonds, but are recognized by the anti-transferrin receptor antibody, B3/25, and bind specifically to a human transferrin-Sepharose column. Binding studies using 125I-labeled human transferrin showed that insect cells infected with the recombinant virus expressed an average of 5.8 +/- 0.9 X 10(5) transferrin receptors (Kd = 63 +/- 9 nM) on their cell surface. Thus, the human transferrin receptor produced in insect cells is biologically active and appears suitable for structural and functional studies.     

SARS冠状病毒S蛋白在昆虫细胞中的表达和纯化

导致严重急性呼吸综合征(sevcre acute rcspiratory syndrome,SARS)的元凶是一种新型的冠状病毒(SARS coronavirus,SARS-CoV)。SARS-CoV感染入侵宿主细胞关键的一环是病毒自身的棘突蛋白(spike protein,S-protein)与细胞受体的相互作用,故而S蛋白己成为SARS研究的主要热点。     

增殖细胞核抗原蛋白在Spodoptera frugiperda昆虫细胞中的表达及纯化

目的:利用昆虫细胞表达系统生产重组的人增殖细胞核抗原(proliferating cell nuclear antigen,PCNA),并进行纯化和抗体结合特性鉴定。方法:以HeLa细胞逆转录的cDNA为模板,扩增人PCNA基因,并插入杆状病毒载体AcMNPV。利用昆虫细胞得到PCNA基因的重组杆状病毒。病毒感染细胞表达蛋白,联合镍柱亲和层析和离子交换层析获得高纯度的重组人PCNA蛋白。ELISA法测定抗体结合特异性。结果:以HeLa细胞cDNA为模板得到的基因序列同GenBank的人PCNA基因序列一致。草地贪夜蛾细胞(Spodoptera frugiperda,Sf9)表达重组人PCNA(recombinant human PCNA,rPCNA)的最佳感染值(MOI)和感染时间分别为0.05h和144h。rPCNA的产量高达110mg/L细胞,纯度95%。间接ELISA法检测抗体结合特性,rPCNA的敏感性和特异性分别为93.3%和85.0%。结论:建立了rPCNA的表达和纯化方法,获得了高效表达、高度抗体结合特异性的PCNA蛋白,该蛋白质能进一步开发为PCNA相关疾病的体外诊断试剂盒,具较大的应用价值。     

重组含有Epstein-Barr病毒潜伏膜蛋白1(LMP1)基因的杆状病毒在昆虫细胞中的表达

用杆状病毒表达系统重组病毒,在昆虫细胞中表达了完整的含有ＥＢＶ－ＬＭＰ１基因３个外显子开放读码框架的长２．３ｋｂ的ｃＤＮＡ片段。用重组病毒感染Ｓｆ９细胞,用免疫荧光染色,结果表明：４８小时表达重组蛋白,７２小时细胞较完整,免疫荧光染色强阳性,９６小时后细胞出现破碎。我们采集７２小时的组织培养上清和细胞破碎裂解液,分别采用ＳＤＳ－ＰＡＧＥ、ＨＰＬＣ分子筛法,用免疫蛋白印迹法实验证明,表达的蛋白能被抗ＬＭＰ１的单克隆抗体所识别,测定表达蛋白的分子量为６０ｋＤ。经蛋白含量扫描图分析,采用Ｓｅｐｈａｄｅｘ－７５柱初步纯化表达的ＬＭＰ１蛋白,将后者进行裸鼠体内致瘤实验,未见肿瘤生长。     

Biological and biochemical activity of v-Crk chimeras containing the SH2/SH3 regions of phosphatidylinositol-specific phospholipase C-gamma and Src.

The chicken CT10 virus oncogene product, P47gag-crk, contains SH2/SH3 domains that have been identified as conserved domains among proteins involved in signal transduction. We studied the functional similarity of the SH2/SH3 domains by replacing those of v-Crk with those of phosphatidylinositol-specific phospholipase C-gamma, v-Src, or c-Src. The transforming activity of v-Crk was partially retained in a mutant with a v-Src SH3 domain but not in the other mutants with heterologous SH2/SH3 domains. Mutant viruses with Crk-SH2/SH2' domains induced tyrosine phosphorylation of cellular proteins, but mutants with phosphatidylinositol-specific phospholipase C-gamma or Src SH2/SH2' domains did not. However, the mutant proteins with heterologous SH2/SH2' regions were able to weakly associate with some phosphotyrosine-containing proteins in vitro. These results indicate that in the context of the P47gag-crk structure, the requirement of Crk-SH2/SH3 is more stringent for its activity to induce cell transformation than to cause phosphorylation of cellular proteins. The substitution with heterologous sequences least perturbs the capacity to bind phosphotyrosine-containing proteins. In each case, the SH3 domain is more flexible to substitution than is the SH2 domain.     

Expression, purification and sublocalization of SARS-CoV nucleocapsid protein in insect cells

The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is a major viral protein recognized by acute and early convalescent sera from SARS patients. To facilitate the studies on the function and structure of the N protein, this report describe the expression and purification of recombinant SARS-CoV N protein using the baculovirus     

大口黑鲈β-防御素在杆状病毒系统中的表达及抑菌活性

β-防御素是鱼类天然免疫的重要组成部分, 通过杆状表达系统表达大口黑鲈β-防御素, 研究其具有高效、广谱的不同于抗生素的独特抗菌的能力。研究通过序列分析, 发现大口黑鲈β-防御素(MSBdefe)与其他物种β-防御素具有相似的特征, 都包含6个保守的半胱氨酸。将MSBdefe基因进行昆虫密码子优化合成后, 克隆至穿梭载体pYBDM-IM质粒中, 构建成为重组质粒MSBdefe-pYBDM-IM, 重组质粒转化感受态细胞MultiBac/rSW106/asd-/inv+, 阳性重组菌株MSBdefe-pYBDM-IM-Am直接用于Sf9细胞的感染, 获得重组杆状病毒AV-MSBdefe。通过SDS-PAGE和Western Blot检测AV-MSBdefe感染Sf9细胞后蛋白表达, 结果表明成功获得MSBdefe重组蛋白。通过对淡水鱼类最常见的病原菌嗜水气单胞菌的抑菌活性分析, 结果显示当MSBdefe重组蛋白的终浓度为30 μg/mL时, 抑菌效率为83.00%, 并随着蛋白稀释2倍、4倍后, 即终浓度为15和7.5 μg/mL时, 抑菌效果逐渐减弱, 抑菌效率分别为54.33%和33.67%, 进而验证了大口黑鲈β-防御素能够抑制嗜水气单胞菌的生长, 且随着蛋白浓度的降低抑制能力下降。这些结果为利用昆虫生物反应器规模化生产鱼类β-防御素奠定了基础, 以期为开发能够替代或部分替代抗生素的天然抗菌剂提供良好的候选者和技术途径。     

Expression of human papillomavirus type 11 L1 protein in insect cells: in vivo and in vitro assembly of viruslike particles.

The L1 coat protein of human papillomavirus type 11 (HPV-11) was expressed in Sf-9 insect cells with the recombinant baculovirus vector Ac11L1. Viruslike particles (VLPs) were identified by electron microscopy in the nucleus and cytoplasm of Sf-9 cells infected with Ac11L1. The L1 protein was purified from Ac11L1-infected insect cells. The purified protein spontaneously assembled in vitro into various aggregates, including particles appearing similar to empty virions. Reaction of VLP-containing insect cell extracts with antisera directed against either denatured or nondenatured capsid epitopes in Western blot (immunoblot) and immuno-dot blot assays suggested that conformational epitopes present in native HPV-11 infectious virions were also present on the baculovirus-produced HPV-11 VLPs. Immuno-dot blot assays using human sera obtained from individuals with biopsy-proven condyloma acuminatum correlated closely with results previously obtained in HPV-11 whole virus particle-based enzyme-linked immunosorbent assays. These morphologic and immunologic similarities to native HPV-11 virions suggest that recombinant VLPs produced in the baculovirus system may be useful in seroepidemiology and pathogenesis studies of genital HPV infection and that they may also be potential candidates for vaccine development.