高效表达可溶性重组蛋白表达载体——pHisSUMO

目的:构建含有IL-1基因的原核表达质粒,并对其原核表达情况进行检测,验 证pHisSUMO表达载体的高效可溶性表达.方法:以质粒pMD18-T-IL-1为模板,利用PCR获得I L-1基因克隆并将其与表达载体pET、pTYB、pHisSUMO连接,重组质粒经鉴定后转化到大肠杆 菌DH5α中,并检测其蛋白表达情况.结果:仅在pHisSUMO表达系统获得了IL-1融合蛋白的 高效可溶性表达.利用Ni-NTA纯化后的融合蛋白经SUMO蛋白酶Ⅰ切割,获得了纯度较高的成熟 蛋白且不残留任何氨基酸残基.结论:实验证明pHisSUMO表达系统有助于增加外源蛋白可溶性和表达量.     

sgna基因小麦高效表达载体的构建

利用在禾谷类作物中表达效率较高的启动子Ubi对含目的基因sgna的现有载体进行了改造,并引入筛选标记基因bar;为提高目的基因的表达水平,在目的基因5′端引入了Ω和kozak序列,3′端引入了poly(A)序列,成功构建了适用于小麦的抗虫基因植物表达载体pGU4AGBar和pGBIU4AGBar,基因pGU4AGBar含有顺向连接的Ubi-sgna及Ubi-bar基因表达盒,pGBIU4AGBar含有T-DNA边界序列,并在其左右边界中间插入了含有顺向连接的CaNV35S-nptⅡ,Ubi-sgna和Ubi-bar基因表达盒,人工合成的雪花莲外源凝集素基因sgna可以编码对同翅目昆虫具有毒杀作用的蛋白。     

金属硫蛋白突变体的植物高效表达载体的构建及其在烟草中的表达

金属硫蛋白有α、β两个结构域（ｄｏｍ ａｉｎ）,其中α结构域优先结合Ｃｄ２＋ 和Ｈｇ２＋ ．小鼠αα突变体在大肠杆菌中已经构建并得到表达,其转基因植株已得到,可在Ｃｄ３００（３００ μｍ ｏｌ／Ｌ）中生长．为了进一步提高外源基因在烟草中的表达量,首先用ＰＣＲ 的方法设计引物,在基因翻译起始密码子ＡＴＧ 附近加入植物偏爱的碱基组合ＡＡＣＡＡＴＧ．另外,将该突变体基因插入具有双３５ Ｓ（ＣａＭＶ３５Ｓ）强启动子的植物双元表达载体ｐＧＰＴＶｄ３５Ｓ－ＢＡＲ中,获得了带有αα突变体的植物双元表达载体．通过农杆菌介导的叶盘转化法转化烟草ＮＣ８９,获得了抗除草剂的转基因植株．经ＰＣＲ－Ｓｏｕｔｈｅｒｎ 和蛋白Ｄｏｔ－ｂｌｏｔｔｉｎｇ 检测,证明了αα突变体在烟草中的嵌合与表达．抗重金属实验证明转基因烟草可以在Ｃｄ４００（４００ μｍ ｏｌ／Ｌ）中生长．     

一种高效、稳定的分泌型原核表达载体的构建及应用

以本室构建的原核表达载体pTO-T7为基础载体,PCR合成ompT引导序列,插入该载体多克隆位点上游,构建了分泌型原核表达载体pTO—OT。将2个外源基因克隆至pTO—OT,2个重组质粒在大肠杆菌中均得以高效表达,表达量为25％～30％。Western印迹分析证实了重组蛋白在大肠杆菌中表达后可被信号肽酶有效识别,切割后的重组蛋白具有良好的免疫学活性。对重组表达菌株的连续传代实验证实了该表达载体具有良好的遗传稳定性,显示了该原核表达载体在基因工程中的应用价值。     

双表达骨形态发生蛋白2、9重组腺病毒载体的构建和表达

目的构建双表达骨形态发生蛋白（Bone morphogenic protein,BMP）2、9腺病毒重组体并进行鉴定。方法自单一表达的BMP2或BMP9 AdEasy质粒上扩增BMP2和BMP9片段,先后亚克隆至穿梭质粒pASG2,获得双表达穿梭质粒pASG2-BMP2、9。酶切及PCR鉴定确认、测序正确后同源重组获得双表达BMP2、BMP9腺病毒质粒,转染至HEK-293细胞中包装和扩增得到高滴度双表达BMP2、BMP9腺病毒,体外感染C3H10细胞,RT-PCR鉴定并观察其早期诱导成骨情况。结果成功构建双表达BMP2、BMP9的腺病毒,滴度约为1010IU/mL,RT-PCR证实双表达腺病毒在C3H10细胞中表达,其感染的C3H10细胞早期碱性磷酸酶含量较单一表达的BMP2或BMP9腺病毒组增加。结论成功构建双表达BMP2、9的重组腺病毒载体,为进一步研究BMP2和BMP9的协同成骨作用和制备高效的组织工程人工骨提供了有利的工具。     

利用cryparin基因的启动子构建板栗疫病菌高效表达载体

低毒病毒/板栗疫病菌是研究植物病原菌致病机理和病毒与宿主相互作用的一个优秀模式系统.本研究克隆了板栗疫病菌转录水平最高的cryparin基因的启动子,并构建了由该启动子控制的表达载体.构建的载体能成功表达GFP蛋白.利用该载体表达积累量较高的CHV1-Euro7病毒的病毒量控制基因,能提高细胞内CHV1-EP721的积累量,反式互补效率从常用的gpd启动子控制的低于10%提高至67%和80%.高效表达载体的成功构建,为研究板栗疫病菌功能基因以及低毒病毒与宿主板栗疫病菌的相互作用提供了新的工具.     

蝴蝶兰LFY基因克隆及高效植物表达载体的构建

根据NCBI中蝴蝶兰LFY花序分生组织基因序列设计2对引物,用RT-PCR法从蝴蝶兰花芽中扩增出LFY基因,对扩增产物进行克隆和测序.结果表明,获得的蝴蝶兰LFY基因约为1500 bp,与报道序列同源性达98.71％.将LFY基因插入pRI101-ON载体中,经PCR、双酶切及测序鉴定,证实重组表达质粒中含有目的片段,表明成功构建了高效植物表达载体pRI1O1-LFY.     

人FGF21原核表达载体的构建及重组蛋白表达

构建人FGF21(fibroblast growth factor,FGF)cDNA的原核表达载体并诱导其重组蛋白表达。提取人肝脏总RNA后,经RT-PCR扩增获得目的片段,构建其T载体进行保存。再构建重组原核表达载体pET-28a(+)-h FGF21,重组质粒转化至大肠杆菌菌株BL21(DE3)中,在IPTG诱导下得到可溶性表达,采用亲和层析法纯化表达产物后,进行Western blot鉴定。成功构建重组质粒pET-28(+)-hFGF21,对其进行可溶性表达后成功纯化出his-hFGF21,经Western blot鉴定该融合蛋白可与FGF21抗体特异性结合。成功构建pET-28(+)-hFGF21,并可溶性表达his-hFGF21蛋白。     

口蹄疫病毒VP1高效植物表达载体构建及鉴定

采用高保真PCR方法从pGEM-VP1-T质粒扩出VP1基因,定向克隆到含DHA的融合中间载体pUC18-DHA,得到pUC18-VP1-DHA,经测序证实核酸序列正确后,再亚克隆到转化范围广,转化效率高,且含有双增强子的高效植物双元表达载体pGreen0029-GFP上,获得含VP1融合DHA基因的植物双元表达载体pGreen0029-VP1-DHA,采用电击法将含VP1的植物表达载体转入根癌农杆菌G3101中,获得了含VP1基因的双元植物表达载体,为下一步的广范围转基因植物表达研究奠定了基础。     

P.pastoris高效表达外源蛋白的研究进展

Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ表达系统具有原核细胞和哺乳类细胞表达系统特点,已广地应用于表达外源基因。为获得高质量外泊蛋白的表达而进行了大量研究并取得了许多进展,如构建并筛选多拷贝的由ＡＯＸ启动的表达盒,优化培液组分,减少蛋白降低,分泌蛋白的纯化和Ｎ－连接寡糖链的特点。     

Production of recombinant proteins in clonal root cultures using episomal expression vectors

We have developed a fully contained system for expressing recombinant proteins that is based on clonal root cultures and episomal expression vectors. Clonal root lines expressing green fluorescent protein (GFP) or human growth hormone were generated from Nicotiana benthamiana leaves infected with the tobacco mosaic virus-based vector 30B after exposure to Agrobacterium rhizogenes. These lines accumulated GFP at over 50 mg per kg fresh tissue, a level that is comparable with other plant production systems in early stage development. Accumulation of both hGH and GFP in the clonal root lines was sustained over a 3-year period, and in the absence of antibiotic selection. This technology shows promise for commercial production of vaccine antigens and therapeutic proteins in contained facilities.     

Construction of non-invasively constitutive expression vectors using a metagenome-derived promoter for soluble expression of proteins

Expression of soluble and functional proteins has been one of the critical challenges to many aspects of synthetic biology, metabolic and protein engineering. Among the current methods for expression of target proteins, constitutive expression systems offer several advantages over inducible systems, which require a chemical or physical inducer. In a previous study, a G196 DNA fragment containing constitutive promoters was mined from the soil metagenome and evaluated for the expression of target proteins in the functional and soluble state. In this study, we further improved this system by constructing a series of constitutive expression vectors, pCEM (using the CEM promoter trimmed from G196), pCEMT (incorporating rrnB T1 and T2 terminator into the downstream region of MCS in pCEM) and pRCEMT (grafting the cis-acting region of pCEMT into a low-copy-number plasmid). Subsequently, genes encoding GFPuv, esterase 1767 and β-glucosidase were subcloned into the resulting vectors, and their expression level and solubility were compared with those of IPTG-inducible vector systems pQE30 and pTrc99A. The extent of homogeneity and the ratio of the soluble fraction in the pRCEMT vector were relatively higher, without any delay of growth rate, than that of the pQE30 or pTrc99A. These results indicate that new expression vectors with moderate constitutive function could more easily lead to a homogenous population of cells expressing target proteins than those with conventionally inducible promoters.     

Development of anaerobically inducible nar promoter expression vectors for the expression of recombinant proteins in Escherichia coli

Dissolved oxygen (DO)-controlled nar promoter expression vectors were constructed, and their expression efficiency was compared with that of the T7 promoter pET22 expression vector by expressing human growth hormone (hGH), enhanced green fluorescence protein (EGFP), and β-tyrosinase in Escherichia coli cells. The nar promoter expression vector pRBS, which was engineered with a 5′-untranslated region and ribosomal binding site for the T7 promoter, expressed hGH at a rate of up to 32% of the total cellular proteins (TCP) in E. coli W3110narL⁻. The expression level of hGH was further enhanced, up to ∼42% of the TCP, by adding the N-terminal peptide tag of β-galactosidase to hGH, which was comparable to the expression of ∼43% of the TCP in pET-lac:hGH/BL21(DE3). A further engineered expression vector, pRBS(fnr), which coexpressed fumarate/nitrate reductase (fnr), expressed more EGFP than pET22 in BL21(DE3). In addition, recombinant β-tyrosinase was successfully expressed at a rate of up to ∼45% of the TCP in pRBS(fnr) in W3110narL⁻. From these results, the DO-controlled nar promoter system developed in this study can be considered a reliable and cost-effective expression system for protein production, especially in large-scale fermentation, as an alternative to the pET/BL(DE3) system.     

A system using convertible vectors for screening soluble recombinant proteins produced in Escherichia coli from randomly fragmented cDNAs

Protein insolubility is a major problem when producing recombinant proteins (e.g., to be used as antigens) from large cDNAs in Escherichia coli. Here, we describe a system using three convertible plasmid vectors to screen for soluble proteins produced in E. coli. This system experimentally identified any random cDNA fragments producing soluble protein domains. Shotgun fragments introduced into any of our three plasmids, which contain Gateway recombination sites, fused in-frame to the ORF of the protein tag. These plasmids produced N-terminal GST- and C-terminal three-frame-adaptive FLAG-tagged proteins, kanamycin-resistant gene-tagged proteins (which were pre-selected for in-frame fused cDNAs), or GFP-tagged fusion proteins. The latter is useful as a fluorescence indicator of protein folding. The Gateway recombination sites promote smooth conversion for enrichment of in-frame clones and facilitate both protein solubility assays and final production of proteins without the C-terminal tag. This high-throughput screening method is particularly useful for procedures that require the handling of many cDNAs in parallel.     

串联亲和纯化原核表达载体的构建

【目的】构建串联亲和纯化原核表达载体,用于研究细菌中(生理状态或接近生理条件下的)蛋白-蛋白相互作用。【方法】设计并合成两条串联亲和标签序列,分别可以在靶蛋白N端和C端融合Protein G和链亲和素结合肽(Streptavidin binding peptide,SBP)标签;以pUC18载体为骨架,去除原有的阻遏蛋白基因,构建组成型表达载体pNTAP和pCTAP。【结果】成功构建N端和C端标签表达载体pNTAP和pCTAP,它们在大肠杆菌(Escherichia coli)BL21(DE3)、肠出血性大肠杆菌O157:H7和痢疾杆菌福氏5型M90T菌株中都可以实现表达。【结论】本实验构建的两个串联亲和纯化表达载体可以在部分革兰氏阴性细菌中表达,为研究细菌内蛋白-蛋白相互作用及致病菌毒力蛋白的作用机制奠定了基础。     

New vectors for high level expression of recombinant proteins in bacteria.

A system has been developed for synthesis and rapid purification of recombinant polypeptides expressed in frame with glutathione S-transferase (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). Expressed fusion proteins are purified from bacterial extracts by glutathione-agarose affinity chromatography. A thrombin protease cleavage site allowed for proteolysis of the fusion protein. We reported the construction of the vector pGEX-KG (K. Guan and J. E. Dixon, 1991, Anal. Biochem. 192, 262-267) which has a glycine-rich "kinker" immediately after the thrombin cleavage site. This kinker dramatically improved the thrombin cleavage efficiency of several fusion proteins. One potential drawback of expressing proteins in this vector is that, following proteolytic cleavage, unrelated amino acids from the vector remain at the amino terminus of the released protein. These extensions could affect enzymatic activity or protein structure. We have constructed two new vectors, pGEX-KT and pGEX-KN, which have the glycine kinker placed N-terminal to the thrombin cleavage site in order to minimize the unrelated amino acids associated with the cleaved protein. The change in location of the kinker had no effect on the increased thrombin cleavage efficiency. A strategy combining the kinker in the vector pGEX-KN with polymerase chain reaction has also been developed to express fusion proteins which when cleaved with thrombin released a protein having no amino terminal extensions of any kind.     

Epitope-tagging vectors for the expression and detection of recombinant proteins in mycobacteria

New tools are required to study the growing number of uncharacterised genes derived from genome sequence projects that are specific to bacterial pathogens such as Mycobacterium tuberculosis. We have developed a series of vectors that permit the specific detection of recombinant proteins expressed in mycobacterial species. Gene expression in these vectors is driven by the strong hsp60 promoter of Mycobacterium bovis BCG and detection of expressed products is facilitated by C-terminal fusion of residues 409-419 of the human c-myc proto-oncogene. Using the M. tuberculosis Ag85B as a reporter of gene expression, we demonstrate that the vectors permit the specific detection of recombinant products expressed in the host species M. bovis BCG. BCG over-expressing Ag85B was a potent inducer of Ag85B-specific T cells in immunised mice, indicating that the C-terminal c-myc tag did not alter the characteristics of the recombinant protein. The versatility of the epitope-tagging vectors was demonstrated by the efficient secretion and detection of recombinant products in BCG. The vectors described in this study will facilitate the expression of foreign proteins in mycobacterial host systems.     

Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells

Homologous recombination technologies enable high-throughput cloning and the seamless insertion of any DNA fragment into expression vectors. Additionally, retroviral vectors offer a fast and efficient method for transducing and expressing genes in mammalian cells, including lymphocytes. However, homologous recombination cannot be used to insert DNA fragments into retroviral vectors; retroviral vectors contain two homologous regions, the 5′- and 3′-long terminal repeats, between which homologous recombination occurs preferentially. In this study, we have modified a retroviral vector to enable the cloning of DNA fragments through homologous recombination. To this end, we inserted a bacterial selection marker in a region adjacent to the gene insertion site. We used the modified retroviral vector and homologous recombination to clone T-cell receptors (TCRs) from single Epstein Barr virus-specific human T cells in a high-throughput and comprehensive manner and to efficiently evaluate their function by transducing the TCRs into a murine T-cell line through retroviral infection. In conclusion, the modified retroviral vectors, in combination with the homologous recombination method, are powerful tools for the high-throughput cloning of cDNAs and their efficient functional analysis.