表达绿色荧光蛋白报告基因重组禽腺联病毒载体系统的构建与鉴定

为了开展重组禽腺联病毒(Recombinant avian adeno-associated virus,rAAAV)介导的基因转移研究,用限制性内切酶消化含AAAV全基因组的重组质粒pCR-AAAV,去除AAAV Rep和Cap蛋白编码序列,将PCR扩增的绿色荧光蛋白(Green fluorescent protein,GFP)报告基因插入AAAV末端反向重复序列(Inverted terminal repeats,ITR)之间,获得表达GFP基因的AAAV转移载体pAITR-GFP;以含AAAV全基因组的质粒为模板,用PCR分别扩增AAAV Rep、Cap和Rep-Cap蛋白基因,将Rep和Cap基因分别插入真核细胞双表达载体pVITRO2-mcs的两个多克隆位点,将Rep-Cap蛋白基因插入真核细胞表达载体pcDNA3,获得AAAV辅助质粒pVITRO2-ARC和pcDNA-ARC;将pAITR-GFP、pVITRO2-ARC或pcDNA-ARC与腺病毒辅助质粒pHelper组成三质粒转染系统,用磷酸钙沉淀法共转染AAV-293细胞,获得了表达GFP的rAAAV.经SDS-聚丙烯酰胺凝胶电泳分离后,纯化病毒出现分子量正确的VP1、VP2、VP3结构蛋白;经PCR检测证明,重组病毒中含有GFP报告基因;用重组病毒分别感染鸡胚成纤维细胞(CEF)和鸡胚肝CEL细胞,可以观察到GFP报告基因的表达,表达时间持续两周以上.这些试验结果表明,成功建立了辅助病毒非依赖性rAAAV体外包装体系,为禽源细胞的基因转移研究和禽重组活载体疫苗的研制打下了基础.     

应用纤维素结合域标签构建谷氨酸棒状杆菌表达系统

为优化谷氨酸棒状杆菌表达系统的纯化工艺,合成里氏木霉的CBD基因,将其与谷氨酸棒状杆菌分泌表达载体pXMJ19-sp连接,构建以CBD为纯化标签的重组载体pXMJ 19-sp-CBD.在该载体中插入GFP基因并转化至谷氨酸棒状杆菌,可获得分泌表达融合蛋白GFP-CBD的重组菌.该菌经IPTG诱导后的发酵液在紫外灯下显示强烈的绿色荧光,重组蛋白的分泌表达量达200 mg/L.利用CBD标签对纤维素柱的可逆性吸附,可直接对谷氨酸棒状杆菌分泌到培养基中的重组蛋白进行纯化,从而简化工艺和降低成本,为工业化大生产奠定基础.     

一种新型IIa类细菌素的克隆表达和活性鉴定

NB-C1为一种潜在的IIa类细菌素基因,为实现其在大肠杆菌中的高效可溶表达,首先构建了NB-C1蛋白与绿色荧光蛋白 (GFP) 的融合表达载体pIVEX 2.4d-GFP-NB-C1,然后将构建的表达载体转化大肠杆菌BL21(DE3) pLysS,经诱导表达后,重组蛋白GFP-NB-C1以可溶的形式存在于细胞内。经Ni-NTA亲和层析柱分离纯化后,重组融合蛋白的纯度大于95％,产量达36.1 mg/L。抑菌试验表明,纯化后的重组蛋白对单核细胞增生李斯特氏菌具有明显的抑制作用。     

δ-睡眠肽与GFP融合蛋白的表达和纯化

构建δ-睡眠肽(DSIP)蛋白与GFP的融合基因表达载体,高效表达和纯化GFP-DSIP融合蛋白。通过SOE-PCR拼接DSIP全长编码基因,并使得DSIP上游具有肠激酶识别位点,经双酶切定向克隆至表达载体pET-28a,构建重组载体pET-28a-DSIP,通过PCR扩增GFP全长编码基因,经双酶切定向克隆至pET-28a-DSIP,构建原核重组表达载体pET-28a-GFP-DSIP,通过双酶切和测序鉴定后,导入E.coli BL21宿主菌中,IPTG诱导表达融合蛋白,采用镍亲和层析和分子筛凝胶层析获得高纯度蛋白,SDS-PAGE分析鉴定。经测序鉴定成功构建了原核重组表达载体pET-28a-GFP-DSIP,在IPTG诱导下获得可溶性的绿色荧光蛋白与睡眠肽的融合蛋白,经Ni-NTA亲和层析纯化成功获得高纯度的融合蛋白。成功构建了DSIP与GFP融合基因的重组表达载体,确定了GFP-DSIP融合蛋白诱导表达的最佳条件,获得了较高纯度的融合蛋白,为进一步研究DSIP蛋白的生物学功能奠定了基础。     

SOCS3基因重组腺病毒的构建及其在猪脂肪细胞中的表达

本研究旨在构建细胞因子信号转导抑制因子3(Suppressor of cytokine signaling 3,SOCS3)的重组腺病毒表达载体,获得有感染性的病毒颗粒。以pcDNA3-SOCS3质粒为模板扩增SOCS3基因,将其亚克隆至腺病毒穿梭载体pAdTrack-CMV,经测序验证后,重组的穿梭质粒用PmeI酶切线性化后转化到BJ5183感受态细菌中与其内的骨架载体pAdEasy-1进行同源重组,获得的重组质粒pAd-SOCS3,经PacI线性化后转染至HEK293细胞中进行包装和扩增,纯化后用TCID50法测定病毒滴度。以重组的病毒感染原代培养的猪脂肪细胞后,荧光显微镜下观察报告基因GFP的表达,RT-PCR和Western blotting检测细胞内SOCS3 mRNA和蛋白的表达。重组腺病毒载体pAd-SOCS3经酶切及PCR鉴定正确,病毒滴度为1.2×109PFU/mL;感染原代培养的猪脂肪细胞后,荧光显微镜观察可见报告基因GFP的表达;RT-PCR和Western blotting检测到细胞中SOCS3 mRNA和蛋白的表达显著提高。本研究成功构建了SOCS3基因的重组腺病毒,感染原代培养的猪脂肪细胞可稳定表达SOCS3蛋白,为深入研究SOCS3的功能奠定了基础。     

表达绿色荧光蛋白的重组CVI988病毒的构建及特性

提取马立克氏病毒Ⅰ型疫苗毒株CVI988的总DNA为模板,利用PCR技术扩增出病毒生长非必需的US2基因并克隆入T—easy载体。将CMV启动子和增强子控制的含GFP基因表达盒克隆入US2基因中,成功构建了含GFP基因的转移质粒载体pGUS2GFP。用脂质体将其与CVI988株共转染CEF细胞,用96孔板稀释法得到纯化的表达绿色荧光蛋白的重组CVI988病毒株rCVIGFP,并分别测定其在体内和体外的生长情况。表达EGFP基因的重组病毒在细胞上生长曲线与亲本毒CVI988类似,体外实验表明,1日龄腹腔接种该重组毒后,可以从鸡体内分离到表达绿色荧光的病毒。     

表达绿色荧光蛋白的重组CVI988病毒的构建及特性

提取马立克氏病毒I型疫苗毒株CVI988的总DNA为模板,利用PCR技术扩增出病毒生长非必需的US2基因并克隆入T-easy载体.将CMV启动子和增强子控制的含GFP基因表达盒克隆入US2基因中,成功构建了含GFP基因的转移质粒载体pGUS2GFP.用脂质体将其与CVI988 株共转染CEF细胞,用96孔板稀释法得到纯化的表达绿色荧光蛋白的重组CVI988病毒株rCVIGFP,并分别测定其在体内和体外的生长情况.表达EGFP基因的重组病毒在细胞上生长曲线与亲本毒CVI988类似,体外实验表明,1日龄腹腔接种该重组毒后,可以从鸡体内分离到表达绿色荧光的病毒.     

小鼠 LRRC 10腺病毒载体构建及其在心肌细胞中的表达

目的：构建含有小鼠Lrrc10（Leucine-rich Repeat Containing protein 10）基因的重组腺病毒表达载体。方法：设计小鼠Lrrc10特异性引物,以小鼠cDNA为模板,通过PCR扩增出mLrrc10的编码区,并引入HA标签蛋白和Sal Ι酶切位点。该片段经凝胶电泳纯化后插入pMD-18 T载体。测序后,用Sal Ι和Hind III酶切,将目的片段亚克隆至pAd-track-cmv穿梭载体中。用PmeΙ线性化后,用100 ng转化细菌BJ5183,在细菌内同源重组后得到 pAd-Lrrc10质粒。pAd-Lrrc10经PacⅠ线性化后用LipofectamineTM2000转染293A细胞,包装得到含Lrrc10基因的病毒重组子。将病毒重组子在293A细胞中扩增后,反复冻融得到滴度较高的含Lrrc10的病毒液。将收集的病毒液感染心肌细胞,绿色荧光观察 GFP、免疫印迹检测Lrrc10-HA蛋白的表达。结果：用病毒液感染原代心肌细胞,24小时后在荧光显微镜下可观察被感染的细胞发出绿色荧光,提取心肌细胞总蛋白,Western可检测到Lrrc10-HA融合蛋白的表达。结论：小鼠Lrrc10腺病毒载体构建成功,并可将编码Lrrc10-HA的目的片段导入心肌细胞中表达。     

紫花苜蓿 DREB 1 基因的原核表达与蛋白纯化

构建 MsDREB1 基因的原核表达载体, 对表达产物进行鉴定和纯化, 为研究 DREB1 基因在植株中功能奠定基础。用冷酚法从紫花苜蓿提取 RNA, 并转化成 cDNA, 然后将其克隆至 pET32a 原核表达载体, 构建重组载体 pET32aDREB1 。用重组质粒转化大肠杆菌 BL21, IPTG 诱导表达后进行 SDS-PAGE 分析, 用 Ni-NTA 亲和层析柱对重组蛋白进行纯化。结果表明 , 原核表达载体构建正确 ; SDS-PAGE 分析, 出现了与 DNAMAN 预测的 43.2 kd 大小一致的蛋白条带;经分析重组蛋白纯化率达到 90%以上。     

小鼠CSRP2基因的克隆、融合表达及蛋白的分离纯化

目的：获得小鼠CSRP2（cysteine rich protein2）基因片段,高效表达、纯化CSRP2。方法：用RT-PCR技术从小鼠心脏组织中提取总DNA并扩增出相应大小的CSRP2DNA片段,将其与pGEM—T—easy载体连接后测序,将测序正确的CSRP2从BamHⅠ和HindⅢ酶切位点克隆入原核表达载体pRSET—A,将连接产物转化大肠杆菌BL21,挑出阳性克隆,IPTG诱导表达重组的6xHis融合蛋白,对重组融合蛋白通过镍柱进行纯化。结果与结论：PCR获得的C职咫序列与GenBank报道的一致（为675bp）;重组载体在大肠杆菌BIJ21中以包涵体形式高效表达;融合蛋白经SDS—PAGE和Western blot分析,在相对分子质量约24×10^3处有特异的蛋白条带,目的蛋白表达量占菌体总蛋白量的25％;重组蛋白经镍柱纯化后,得到了高纯度的CSRP2融合蛋白。     

Use of protein-acrylamide copolymer hydrogels for measuring protein concentration and activity

We report the development and characterization of a polyacrylamide-based protein immobilization strategy for surface-bound protein assays, including concentration detection, binding affinity, and enzyme kinetics. Glutathione S-transferase (GST) fusion proteins have been labeled with an acrylic moiety and attached to acrylic-functionalized glass surfaces through copolymerization with acrylic monomer. The specific attachment of GST-green fluorescent protein (GFP) fusion protein was more than sevenfold greater than the nonspecific attachment of nonacrylic-labeled GST-GFP; 0.32 ng/mm(2) of surface-attached GST-GFP was detectable by direct measurement of GFP fluorescence and this lower detection limit was reduced to 0.080 ng/mm(2) using indirect antibody-based detection. The polyacrylamide-based surface attachment strategy was also used to measure the kinetics of substrate phosphorylation by the kinase c-Src. Michaelis-Menten kinetic constants for the reaction occurring in solution were K(m) = 2.7 +/- 1.0 microM and V(max) = 8.1 +/- 3.1 (arbitrary units). Kinetic values for the reaction utilizing surface-immobilized substrate were K(m) = 0.36 +/- 0.033 microM and V(max) = 9.7 +/- 0.63 and were found to be independent of the acrylamide concentration within the copolymer. Such a surface attachment strategy should be applicable to the proteomics field and addresses denaturation and dehydration problems associated with protein microarray development.     

Electroporation of human embryonic stem cells: Small and macromolecule loading and DNA transfection

Cryopreservation, directed differentiation, and genetic manipulation of human embryonic stem cells (hESCs) all require the transport of exogenous small molecules, proteins, or DNA into the cell. The absence of standard small and macromolecule loading techniques in hESCs as well as the inadequacies of current DNA transfection techniques have led us to develop electroporation as an efficient loading and transfection methodology. The electroporation parameters of pulse voltage, duration, and number have been explored and evaluated in terms of cell viability, molecular loading, and transfection efficiency on a per cell basis. Small molecule loading was assessed using propidium iodide (PI) and the disaccharide trehalose. Additionally, protein loading was investigated using a glutathione-S-transferase green fluorescent protein (GST-GFP) conjugate, and DNA transfection optimization was performed by constitutive expression of GFP from a plasmid. The optimum pulse voltage must balance cell viability, which decreases as voltage increases, and loading efficiency, which increases at higher voltages. Short pulse times of 0.05 ms facilitated PI and trehalose loading, whereas 0.5 ms or more was required for GST-GFP loading and DNA transfection. Multiple pulses increased per cell loading of all molecules, though there was a dramatic loss of viability with GST-GFP loading and DNA transfection, likely resulting from the longer pulse duration required to load these molecules.     

Generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling

We recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of Escherichia coli. Recombinant proteins were covalently coupled to the E. coli ribosome by fusing them to ribosomal protein 23 (rpL23) followed by expression in an rpL23 deficient strain of E. coli. This allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpL23 and the target protein. rpL23-GFP-His is among the fusion proteins used in our previous study for ribosomal coupling of C-terminally His-tagged green fluorescent protein. To assess the efficiency of separation of target protein from ribosomes, by site-specific proteolysis, we required monoclonal antibodies directed against rpL23 and GFP. We therefore purified rpL23-GFP-His, rpL23-His and GFP from E. coli recombinants using affinity, ion exchange and hydrophobic interaction chromatography. These proteins could be purified with yields of 150, 150 and 1500 microg per gram cellular wet weight, respectively. However, rpL23-GFP-His could only be expressed in a soluble form and subsequently purified, when cells were cultivated at reduced temperatures. The purified rpL23-GFP-His fusion protein was used to immunize balb/c mice and the hybridoma cell lines resulting from in vitro cell fusion were screened by ELISA using rpL23-His and GFP to select for monoclonal antibodies specific for each protein. This resulted in 20 antibodies directed against rpL23 and 3 antibodies directed against GFP. Antibodies were screened for isotypes and their efficiency in western immunoblots. The most efficient antibody against rpL23 and GFP were purified by Protein G Sepharose affinity chromatography. The purified antibodies were used to evaluate the separation of ribosomes from GFP, streptavidin, murine interleukin-6, a phagedisplay antibody and yeast elongation factor 1A by centrifugation, when ribosomes with covalently coupled target protein were cleaved at specific proteolytic cleavage sites. We conclude that the generated antibodies can be used to evaluate ribosomal coupling of recombinant target proteins as well as the efficiency of their separation from the ribosome.     

Expression of recombinant actin 5C from Drosophila in the methylotrophic yeast Pichia pastoris

A system for actin expression in cells of yeast Pichia pastoris was constructed. Drosophila actin 5C, by 90% homologous to beta-actin of higher eukaryotes, was used as a target protein. To improve the procedures of target protein biosynthesis in yeast cells and of extraction and purification of recombinant actin the fusion protein GFP-actin 5C, having fluorescence protein GFP as a reporter part, was expressed and purified. The dimensions and resistance of yeast cells producing recombinant actin were characterized. It was shown that the size and form of cells depended on the accumulation of recombinant protein. The purified fusion protein was used for obtaining polyclonal antibody for testing recombinant actin.     

Consequences of membrane protein overexpression in Escherichia coli

Overexpression of membrane proteins is often essential for structural and functional studies, but yields are frequently too low. An understanding of the physiological response to overexpression is needed to improve such yields. Therefore, we analyzed the consequences of overexpression of three different membrane proteins (YidC, YedZ, and LepI) fused to green fluorescent protein (GFP) in the bacterium Escherichia coli and compared this with overexpression of a soluble protein, GST-GFP. Proteomes of total lysates, purified aggregates, and cytoplasmic membranes were analyzed by one- and two-dimensional gel electrophoresis and mass spectrometry complemented with flow cytometry, microscopy, Western blotting, and pulse labeling experiments. Composition and accumulation levels of protein complexes in the cytoplasmic membrane were analyzed with improved two-dimensional blue native PAGE. Overexpression of the three membrane proteins, but not soluble GST-GFP, resulted in accumulation of cytoplasmic aggregates containing the overexpressed proteins, chaperones (DnaK/J and GroEL/S), and soluble proteases (HslUV and ClpXP) as well as many precursors of periplasmic and outer membrane proteins. This was consistent with lowered accumulation levels of secreted proteins in the three membrane protein overexpressors and is likely to be a direct consequence of saturation of the cytoplasmic membrane protein translocation machinery. Importantly accumulation levels of respiratory chain complexes in the cytoplasmic membrane were strongly reduced. Induction of the acetate-phosphotransacetylase pathway for ATP production and a down-regulated tricarboxylic acid cycle indicated the activation of the Arc two-component system, which mediates adaptive responses to changing respiratory states. This study provides a basis for designing rational strategies to improve yields of membrane protein overexpression in E. coli.     

Production of fluorescent single-chain antibody fragments in Escherichia coli

We describe a novel vector-host system suitable for the efficient preparation of fluorescent single-chain antibody Fv fragments (scFv) in Escherichia coli. The previously described pscFv1F4 vector used for the bacterial expression of functional scFv to the E6 protein of human papillomavirus type 16 was modified by appending to its C-terminus the green fluorescent protein (GFP). The expression of the scFv1F4-GFP fusion proteins was monitored by analyzing of the typical GFP fluorescence of the transformed cells under UV illumination. The brightest signal was obtained when scFv1F4 was linked to the cycle 3 GFP variant (GFPuv) and expressed in the cytoplasm of AD494(DE3) bacteria under control of the arabinose promoter. Although the scFv1F4 expressed under these conditions did not contain disulfide bridges, about 1% of the molecules were able to bind antigen. Fluorescence analysis of antigen-coated agarose beads incubated with the cytoplasmic scFv-GFP complexes showed that a similar proportion of fusions retained both E6-binding and green-light-emitting activities. The scFv1F4-GFPuv molecules were purified by affinity chromatography and successfully used to detect viral E6 protein in transfected COS cells by fluorescence microscopy. When an anti-beta-galactosidase scFv, which had previously been adapted to cytoplasmic expression at high levels, was used in this system, it was possible to produce large amounts of functional fluorescent antibody fragments. This indicates that these labeled scFvs may have many applications in fluorescence-based single-step immunoassays.     

Expression of recombinant actin 5C from Drosophila in the methylotrophyc yeast Pichia pastoris

A system of expression for the foreign actin gene in yeast cells Pichia pastoris has been developed. As a target protein, the Drosophila cytoplasmic actin 5C, which has 90% homology to the β-actin of higher eukaryotes, was used. In the present work, in order to develop conditions for biosynthesis of the target protein in yeast cells and a purification procedure for the recombinant protein, a GFP-actin fusion protein containing green fluorescent protein (GFP) as a fusion tag was expressed and purified. The size and survival of P. pastoris cells producing recombinant protein were characterized and shown to depend on the accumulation of recombinant actin. The purified fusion protein was used to obtain a polyclonal antibody necessary for testing for recombinant actin.     

A human kringle domain-based fluorescence-linked immunosorbent assay system

As a non-immunoglobulin protein scaffold, human kringle domain (KD) has attractive properties such as high specificity, stability, and production in bacterial hosts. Here, we developed a rapid and sensitive fluorescence-linked immunosorbent assay (FLISA) system using a fluorescent kringle domain (fluoKD), a fusion protein of a green fluorescent protein (GFP), and a kringle domain variant (KD548). Two kinds of fluoKDs in which KD was fused to the N terminus of GFP (N-fluoKD) or the C terminus of GFP (C-fluoKD) were constructed and characterized. In Escherichia coli host, both fluoKDs were produced in high yield and solubility and were successfully purified by a simple procedure. The purified fluoKDs exhibited strong fluorescent activities and high affinities to the target antigen. Furthermore, it was successfully demonstrated that the FLISA with purified fluoKDs allowed for more rapid detection of target antigens with higher sensitivity compared with conventional enzyme-linked immunosorbent assay (ELISA), indicating that a simple, rapid, and sensitive immunoassay system could be developed by using KD instead of antibody or antibody fragments.     

Soluble expression and characterization of a GFP-fused pea actin isoform（PEAc1）

A pea actin isoform PEAcl with green fluorescent protein (GFP) fusion to its C-terminus and His-tag to its Nterminus, was expressed in prokaryotic cells in soluble form, and highly purified with Ni-Chelating Sepharose^TM Fast Flow column. The purified fusion protein (PEAcl-GFP) efficiently inhibited DNase I activities before polymerization,and activated the myosin Mg-ATPase activities after polymerization. The PEAcl-GFP also polymerized into green fluorescent filamentous structures with a critical concentration of 0.75μM. These filamentous structures were labeled by TRITC-phalloidin, a specific agent for staining actin microfilaments, and identified as having 9 nm diameters by negative staining. These results indicated that PEAc 1 preserved the essential characteristics of actin even with His-tag and GFP fusion, suggesting a promising potential to use GFP fusion protein in obtainning soluble plant actin isoform to analyze its physical and biochemical properties in vitro. The PEAcl-GFP was also expressed in tobacco BY2 cells,which offers a new pathway for further studying its distribution and function in vivo.     

Expression and localization of VCX／Y proteins and their possible in-volvement in regulation of ribosome assembly during spermatogenesis

Variable Charge X/Y (VCX/Y) is a human testis-specific gene family that localized on X and Y chromo-somes. In this study, VCY protein was expressed in E. coli in the form of glutathione-S-transferase (GST)fusion protein. With the purified fusion protein as antigen, the anti-GST-VCY antibody was generated andthe localization of VCY protein in human testis was determined by immunohistochemistry. In the testisseminiferous epithelium, VCY proteins were highly expressed in nuclei of germ cells. Using propidium io-dide staining and green fluorescent protein (GFP) tag technologies, VCY and VCX-8r proteins were mainlylocalized in the nucleoli of COS7 cells. In addition, the colocalization for VCY and VCX-8r in COS7 cellswas also observed. With VCY cDNA as bait, a cDNA fragment of acidic ribosomal protein PO was obtainedusing yeast two-hybrid system. All the information above indicates that VCX/Y protein family might beinvolved in the regulation of ribosome assembly during spermatogenesis.