猪细小病毒vp2基因的表达和类病毒颗粒的构建

利用PCR方法从病毒基因组中获得了vp2完整的编码区并经测序后克隆到原核表达载体pET-32(a)上.在大肠杆菌中诱导表达了VP2与Trx-His-S Tag的融合蛋白,通过免疫家兔获得了高特异性的兔抗血清.另外,将vp2克隆到pFastBacDUAL载体上,利用昆虫表达系统,即AcMNPV的Bac-to-Bac系统对vp2进行了表达,经SDS-PAGE和Western blot分析,表达产物为64kD,并且该蛋白能在昆虫细胞中形成类病毒颗粒.     

基于猪细小病毒病毒样颗粒的结肠癌靶向纳米载体的构建

【目的】获得具有结肠靶向的纳米载体。【方法】采用SOE-PCR方法将具有结肠靶向的TK肽序列插入到猪细小病毒(PPV)结构蛋白VP2的环2和环4区域得到TK-vp2(?vp2)基因,在Bac-to-Bac?杆状病毒表达系统中构建、表达和自组装。【结果】通过SOE-PCR方法扩增获得?vp2基因,在Bac-to-Bac?杆状病毒表达系统中构建得到Bacmid-?vp2,经脂质体转染至Sf9昆虫细胞得到重组杆状病毒。直接免疫荧光试验、SDS-PAGE和Western blot检测结果表明?VP2蛋白在Bac-to-Bac?杆状病毒表达系统中获得融合表达,目的蛋白约70 k D;透射电子显微镜结果显示?VP2能自组装形成病毒样颗粒(TK-VLPs),直径范围在22 nm-30 nm。【结论】获得纳米载体TK-VLPs,为进一步研究其作为结肠靶向的纳米载体奠定物质基础。     

人微小病毒3个主要蛋白基因真核表达载体的构建

目的:分别克隆人细小病毒B19三个主要蛋白VP1、VP2、NS1全长基因,构建真核表达载体。方法:利用PCR和分子克隆技术,分别将B19病毒vp1、vp2、ns1基因全长片段扩增后,构建带荧光标签的真核表达载体;在人体细胞中表达并通过荧光、RT-PCR和Western Blot、测序等方法鉴定。结果:成功构建了包含B19病毒vp1、vp2、ns1全长基因,并在人体细胞中表达了VP1、VP2、NS1蛋白。结论:人微小病毒B19三个主要蛋白基因得到克隆和表达,为进行相关的研究奠定了基础。     

猪瘟病毒E2基因在昆虫细胞/杆状病毒中的表达

目的利用昆虫细胞/杆状病毒系统表达猪瘟病毒(CSFV)E2蛋白,用于E2蛋白功能、开发CSF新型疫苗以及建立相关血清学诊断方法等研究。方法采用RT-PCR扩增CSFV E2基因,将PCR产物克隆到pGEM-T-Easy载体,将该基因插入到pFast-BacHT A载体中,构建重组转座载体后转化DH10Bac感受态细胞,获得重组Bacmid质粒后转染sf9昆虫细胞,传毒3代,对表达蛋白进行Western-blot及免疫组化鉴定。结果成功克隆CSFVE2基因,其核苷酸序列为1119 bp。SDS-PAGE电泳结果显示表达E2蛋白相对分子质量约为43×103,Western-blot和免疫组化结果证实表达蛋白能够被CSFV标准阳性血清识别。结论在Bac-to-Bac杆状病毒系统中的成功表达了CSFV E2蛋白,与CSFV标准阳性血清具有较好的反应性。     

人细胞周期蛋白D1/CDK4基因的真核表达及生物活性鉴定

通过生物工程获得人重组细胞周期蛋白 (cyclinD1 )及细胞周期蛋白激酶CDK4蛋白 ,作为抗癌药物筛选的分子靶点 .从人HL 6 0细胞中获得细胞周期蛋白D1 CDK4基因的cDNA ,先克隆至pGEMT Easy载体上 ,再经重组构建供体质粒pFastBac D1和pFastBac CDK4 .重组供体质粒转化感受态DH1 0Bac细胞 ,挑取确证为白色克隆的菌落振荡培养 ,分离制备高纯度杆粒DNA .以重组病毒适量感染昆虫细胞Tn 5B1 4 ,利用Bac to Bac杆状病毒表达系统在昆虫细胞Tn 5B1 4 (Hi5 )中表达相应的重组蛋白 .应用昆虫杆状病毒表达系统 (Bac to Bac)在昆虫细胞Tn 5B1 4中分别高效表达了人细胞周期蛋白D1和CDK4蛋白 .SDS PAGE分析表明 ,表达量占细胞可溶性蛋白质的 2 0 %左右 ,表达产物经Ni2 + NTA亲和层析纯化后纯度达 85 %以上 .研究表明 ,昆虫细胞表达的细胞周期蛋白D1和CDK4蛋白能促进Rb蛋白的磷酸化 ,具有生物活性 .成功构建了细胞周期蛋白D1及CDK4真核杆状病毒表达载体 ,并且在昆虫细胞中正确表达了具有生物活性的细胞周期蛋白D1及CDK4融合蛋白 .     

鹅细小病毒vp2基因片段在原核系统中的表达及多克隆抗体的制备

根据鹅细小病毒(Goose parvovirus,GPV)中国分离株HG5/82基因序列,设计引物,利用PCR技术扩增出HG5/82株vp2基因,将其克隆到pMD18-T载体后,转化入感受态细胞TG1中增殖.筛选阳性质粒,将其与原核表达载体pPROEXTMHTb分别用Nco Ⅰ酶切后回收目的片断,进行定向连接,产物转化入感受态DH5α,重组质粒经酶切和测序证实目的基因正确克隆到表达载体的预期位点且插入方向正确,构建了含有HG5/82主要结构基因vp2 5'端969bp片段的原核表达载体.经IPTG诱导后表达出与预期大小相符的约36kDa的融合蛋白,表达形式为包涵体.薄层扫描结果表明表达产物约占菌体总蛋白的21.4%.包涵体通过6mol/L盐酸胍裂解后,利用镍离子亲和树脂进行纯化,用纯化的分子量为36kDa的融合蛋白免疫新西兰白兔,制备兔抗鹅细小病毒部分结构蛋白多克隆抗体.Western blot分析表明该多克隆抗体与HG5/82毒株具有反应性,说明该融合蛋白具有抗原性.     

对虾白斑综合症病毒囊膜蛋白VP19的融合表达及其抗病毒感染作用

根据GenBank上WSSV囊膜蛋白基因vp19的序列,设计并合成引物,PCR扩增得到vp19基因并克隆到pGEM‐T载体中,经过BamHⅠ/HindⅢ酶切、连接并将vp19插入到pET32b表达载体中。用重组质粒pET32b-vp19转化大肠杆菌Origam(iDE3)pLysS,在IPTG诱导下,融合蛋白Trx-VP19以可溶性的形式得到表达,经SDS-PAGE和Western-blot检测显示其分子量与预期的大小相符合。目的蛋白经Ni2 柱纯化并定量后分别直接注射鳌虾和包被饲料投喂鳌虾。实验结果表明注射Trx-VP19可以提高鳌虾个体抗WSSV感染力的作用。     

鹅细小病毒vp2基因片段在原核系统中的表达及多克隆抗体的制备

根据鹅细小病毒(Gooseparvovirus,GPV)中国分离株HG5/82基因序列,设计引物,利用PCR技术扩增出HG5/82株vp2基因,将其克隆到pMD18-T载体后,转化入感受态细胞TG1中增殖。筛选阳性质粒,将其与原核表达载体pPROEXTMHTb分别用NcoI酶切后回收目的片断,进行定向连接,产物转化入感受态DH5α,重组质粒经酶切和测序证实目的基因正确克隆到表达载体的预期位点且插入方向正确,构建了含有HG5/82主要结构基因vp25’端969bp片段的原核表达载体。经IPTG诱导后表达出与预期大小相符的约36kDa的融合蛋白,表达形式为包涵体。薄层扫描结果表明表达产物约占菌体总蛋白的21.4%。包涵体通过6mol/L盐酸胍裂解后,利用镍离子亲和树脂进行纯化,用纯化的分子量为36kDa的融合蛋白免疫新西兰白兔,制备兔抗鹅细小病毒部分结构蛋白多克隆抗体。Westernblot分析表明该多克隆抗体与HG5/82毒株具有反应性,说明该融合蛋白具有抗原性。     

对虾白斑综合症病毒囊膜蛋白VP19的融合表达及其抗病毒感染作用

根据GenBank上WSSV囊膜蛋白基因vp 19的序列,设计并合成引物,PCR扩增得到vp19基因并克隆到pGEM‐T载体中,经过BamHⅠ/Hind Ⅲ酶切、连接并将vp19插入到pET32b表达载体中.用重组质粒pET32b-vp19转化大肠杆菌Origami(DE3)pLysS,在IPTG诱导下,融合蛋白Trx-VP19以可溶性的形式得到表达,经SDS-PAGE和Western-blot检测显示其分子量与预期的大小相符合.目的蛋白经Ni2+柱纯化并定量后分别直接注射鳌虾和包被饲料投喂鳌虾.实验结果表明注射Trx-VP19可以提高鳌虾个体抗WSSV感染力的作用.     

人FGFR1的BacMam系统构建、表达、纯化与鉴定

目前激酶类蛋白的表达大多由昆虫细胞表达系统实现,采用BacMam系统来表达受体型酪氨酸激酶人FGFR1,旨在获得更接近天然结构与功能的重组蛋白.首先,将pDEST20载体的多角体蛋白启动子替换成CMV enhancer启动子,获得BacMam表达载体pDEST20-CMVe.通过Gateway系统,将FGFR1具有酪氨酸激酶活性的区域克隆至改造后的载体中.利用昆虫细胞Sf9产生病毒,P2代病毒转导哺乳动物细胞FreeStyle 293-F进行表达,经亲和纯化后,成功获得了75 kD的融合蛋白.通过磷酸化底物的反应检测此FGFR1激酶的活性.结果表明,BacMam系统是一个良好的表达重组激酶FGFR1的平台,并且所表达的激酶活性高于昆虫细胞表达的激酶蛋白.     

Cloning and preliminary characterization of a 121 kDa protein with multiple predicted C2 domains.

In the course of characterizing proteins present in a preparation of vesicles from rat adipocytes containing glucose transporters, we examined a protein that migrated at 115 kDa upon SDS gel electrophoresis (designated vp115). Sequences of tryptic peptides were obtained, and from this information the cDNA for rat vp115 was cloned. The cDNA encodes an open reading frame for a protein of 121 kDa. Computer-aided sequence analysis predicted that vp115 has a potential membrane-inserted or membrane-spanning domain toward its amino terminus, followed by five C2 domains. Immunoblotting revealed that vp115 was not actually a component of the glucose transporter-containing vesicles, was most abundant in the plasma membranes and high density microsome fractions of rat adipocytes, and was expressed in all the major rat tissues.     

苜宿银纹夜蛾核型多角体病毒vp39基因的克隆及其对S_f9细胞形态的影响

最近的研究发现：ＡｃＮＰＶ的ｖｐ３９基因与侵染密切相关［１］．在侵染过程中,ＶＰ３９蛋白与宿主的肌动蛋白结合,使其重排形成缆索（ｃａｂｌｅ）．导致细胞骨架发生变化有利于病毒编码的蛋白酶的水解．最后,子代病毒颗粒大量形成,宿主昆虫体全部液化成为脓水．可...     

丙型肝炎病毒结构蛋白E1在昆虫细胞中的表达及定位

本文在大肠杆菌中表达了与GST融合无跨膜区的丙型肝炎病毒(Hepatitis C Virus,HCV)E1蛋白,并通过免疫兔制备了兔抗E1的抗血清。然后利用Bac-to-Bac杆状病毒表达系统构建了含有HCV结构蛋白E1基因的重组杆状病毒vAcHCVE1。通过Western blot分析,E1蛋白在Sf9细胞中表达分子量大小为30kDa大于预测的20kDa,表明存在翻译后修饰如糖基化等。通过Confocal显微镜观察当感染48h后E1蛋白定位在细胞质和细胞膜上。     

重组人卵泡刺激素在昆虫细胞中的表达

为了研究在昆虫细胞中表达重组人卵泡刺激素,我们以人胎盘组织提取的染色体DNA为模板,利用重叠PCR方法扩增出hFSHβ亚基的cDNA的编码区。将此cDNA克隆入核型多角体病毒(AcNPV)非融合蛋白基因表达载体pVLl393,我们得到了表达载体pVLl393-hFSHβ,然后与BaculoGold^TM线性杆状病毒DNA共转染昆虫细胞SF9,经多次扩增后获得高滴度的重组病毒AcNPV-hFSHβ。将此重组病毒感染昆虫细胞,我们得到了在胞浆中表达的hFSHβ亚基,Western blot显示分子量大约为21kDa。以重组病毒AcNPV-hFSHβ与AcNPV-hCGoL一同感染昆虫细胞得到了具有分泌性的重组hFSH异二聚体,在非还原的条件下Western blot显示分子量大约为33kDa。     

Cloning and high-level expression of scorpion toxin BmKITa1 in Escherichia coli and insect cells

BmK ITa1 cDNA was cloned and highly expressed in E. coli and insect cell. SDS-PAGE and western blot analysis revealed that subunit molecular weight of expression products is about 40 kDa and 10 kDa respectively. The expression product purified by a Ni(2+)-IDA-sepharose 6B column was toxic for insect, which indicated that it was biologically activity. Furthermore, Quantitative estimation show that the biological activity of recombinant BmK ITa1 from Tn cells was more powerful than from E. coli.     

Engineering of baculovirus vectors for the manufacture of virion-free biopharmaceuticals

A novel baculovirus-based protein expression strategy was developed to produce recombinant proteins in insect cells without contaminating baculovirus virions. This novel strategy greatly simplifies the downstream processing of biopharmaceuticals produced in insect cells. The formation of these virions is prevented by deletion of a baculovirus gene essential for virion formation. The deletion is trans-complemented in a transgenic insect cell line in which the baculovirus seed stock is produced. The Autographa californica multicapsid nucleopolyhedrovirus vp80 gene was selected for this purpose, as absence of VP80 prevented the formation of budded virus as well as occlusion-derived virus, while foreign gene expression was not affected. Sf9 insect cells were engineered to functionally complement the vp80 deletion in the expression vector virus during seed stock production. The trans-complemented vp80-deletion baculovirus seed produced an amount of recombinant protein similar to that produced with conventional baculovirus vectors but without contaminating virions. This novel expression method obviates the need to purify the virions away from the biopharmaceuticals.     

Expression of processed envelope protein of hepatitis C virus in mammalian and insect cells.

The putative envelope protein of hepatitis C virus (HCV) was expressed in insect cells by using a baculovirus expression vector and in monkey COS cells under the control of exogenous promoters. The expressed envelope proteins, identified by immunoblot analysis using sera from patients with chronic HCV infection, were a series of glycoproteins of 35 to 24 kDa (gp35-24) in insect cells and a single species of glycoprotein of 35 kDa (gp35) in monkey cells. The size difference of these proteins was due to the different degrees of glycosylation. The envelope proteins expressed in these cells were produced by common specific cleavage from the precursor protein, and cleavage positions of the envelope protein were mapped at about amino acids 190 and 380. The gp35-24 proteins expressed in insect cells were used for detection of antibody against HCV envelope protein in patient sera. The results showed that (i) the antibody is detected in 2 to 17% of various patients with hepatitis C, (ii) three patients were apparently cured after acquiring the antienvelope antibody, and (iii) in sera of patients with more than a 20-year history of infection, the antibody sometimes coexisted with HCV. These results suggest that the antienvelope antibody is neutralizing only in limited number of patients with hepatitis C.     

Expression of non-structural protein NS3 gene of Bombyx mori densovirus （China isolate）

The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 (China isolate),named BmDNV-3,is a kind of bidensovirus.It is a new type of virus with unique replication mechanisms.To investigate the effects of the NS3 gene during viral DNA replication,a pair of primers was designed for amplifying NS3 gene of Bombyx mori densovirus (China isolate).Gene NS3 amplified was cloned into a prokaryotic expression vector pET-30a and the donor plasmid pFastBacHTe,respectively.The NS3 protein was expressed in Escherichia coli BL21.The pFastBacHTe-NS3 was transformed to E.coli DH10Bac.The recombinant bacmid baculoviruses (rBacmid-EGFP-NS3)isolated from the white colonies were transfected into BraN-4 cells using a transfection reagent.BmN-4 cells were infected with recom-binant virus to express fusion proteins.The expression of fusion protein around 30 kDa in E.coli BL21 was identified by SDS-PAGE,Western blotting,and mass spectrometry.The expressed NS3 protein by B.mor/nucleopolyhedrovirus bacmid system was confirmed byWestern blotting using an anti-NS3 polyclonal antibody.And about 45 kDa protein was found.The expressed fusion protein was smalleithan the expected size of EGFP-NS3,55 kDa.Western blotting analysis indicated that EGFP-NS3 protein was expressed in infected lar-vae with smaller molecular size.     

Baculovirus expression and bioactivity of a soluble 140 kDa extracellular cleavage fragment of L1 neural cell adhesion molecule

L1 neural cell adhesion molecule is the founding member of the L1 subfamily of the immunoglobulin superfamily and plays an important role in the overall development of both the central and peripheral nervous systems, making it an attractive candidate for promoting neural regeneration following injury. Currently, L1 used for experimental studies is primarily mammalian-derived; however, the insect cell expression system described here provides an alternative source of recombinant L1 with equivalent bioactivity. A 140 kDa L1 fragment based on a physiological plasmin cleavage site in the extracellular domain was cloned and expressed with a C-terminal 6x histidine tag. Recombinant insect cell-derived L1 was analyzed by Western blot using an antibody to human L1 to confirm immunogenicity and to optimize infection conditions for recombinant L1 production. The recombinant protein was secreted by insect cells, efficiently purified under non-denaturing conditions using dialysis followed by metal affinity chromatography, and analyzed by SDS-PAGE to produce a single band of the expected approximate 140 kDa size. The bioactivity of insect cell-derived L1 was compared to mammalian-derived L1-Fc and poly-L-lysine (PLL) using chick embryonic forebrain neurons. The results show comparable, robust neurite outgrowth at 24h on insect cell-derived L1 and mammalian-derived L1-Fc, with significantly longer neurites than those observed on PLL. Future studies will examine the immobilization of L1 to biomaterial surfaces in physiologically appropriate orientation via the C-terminal 6x histidine tag and will investigate their application in promoting axonal regeneration in the injured nervous system.