SA-hIL2双功能融合蛋白的研制及其生物学鉴定

目的：制备链亲和素标记的人白细胞介素-2(SA-hIL2)融合蛋白,并研究其生物学功能。方法：构建SA-L-IL2-pET24重组表达质粒,在大肠杆菌中表达SA-hIL2融合蛋白,对表达的SA-hIL2融合蛋白采用镍金属螯合（Ni-NTA）层析柱进行纯化,透析复性。CCK-8法检测SA-hIL2融合蛋白对PHA刺激的人外周血淋巴细胞的增值活性,流式细胞仪分析SA-hIL2融合蛋白对生物素化的B16.F10肿瘤细胞表面锚定修饰效率。结果：SA-hIL2在大肠杆菌中实现了高效表达,约占菌体总蛋白的20％,制备的SA-hIL2融合蛋白纯度达到95％,并具有双重活性,即hIL-2促进PHA刺激的人外周血淋巴细胞的增值活性和SA介导的高效结合至已生物素化的B16.F10肿瘤细胞表面的功能（表面锚定修饰效率约95％）。结论：研制的SA-hIL2融合蛋白具有双重活性,可为研制表面修饰的新型肿瘤细胞疫苗提供基础。     

SA/TNFα双功能融合蛋白的克隆、表达及活性研究

目的：制备链亲合素标记的TNFα双功能融合蛋白,并对其活性进行研究。方法：构建原核表达质粒pET24a-SA-TNFα和pET21a-TNFα-SA;转化大肠杆菌BL21（DE3）,IPTG诱导表达,Ni-NTA亲合层析纯化后进行透析复性;流式细胞仪检测融合蛋白对生物素化MB49细胞的锚定活性,L929细胞杀伤实验检测融合蛋白的TNFα活性。结果：成功制备了两种链亲合素标记的TNFα双功能融合蛋白SA-TNFα和TNFα-SA;其表达量分别约占总蛋白的30%和23%,纯化效率均达90%以上;两种融合蛋白均能有效地锚定于生物素化的MB49细胞表面,其锚定效率分别为95%和92%;L929细胞杀伤实验显示SA-TNFα具备TNFα活性,但TNFα-SA不具备TNFα活性。结论：成功制备了具备双功能活性的链亲合素标记的TNFα融合蛋白,TNFα在该融合蛋白中的位置与其活性有重要的关系。     

生物素化荧光素酶的克隆表达及其固定化研究

为了在体内实现萤火虫荧光素酶的生物素酰化修饰,我们将大肠杆菌中编码生物素羧基载体蛋白（biotin carboxyl carrier protein,BCCP）C端87个氨基酸的功能域基因融合到萤火虫(Pyrocoelia pectoralis)荧光素酶cDNA的末端。经大肠杆菌生物素合成酶（biotin holoenzyme synthetase）的催化,生物素与BCCP上特定的赖氨酸（Lys）残基共价结合,由此与BCCP融合的萤火虫荧光素酶间接实现了生物素化的修饰。利用生物素与配体亲和素或链霉亲和素的特异性耦合,可以将荧光素酶固定到亲和素或链霉亲和素包被的磁珠上,从而使荧光检测的应用更加灵活和方便。本文将就生物素化荧光素酶的克隆、表达以及功能检测进行具体讨论。     

生物素化ATP硫酸化酶的表达、固定化与应用

现代大规模焦测序技术的产生是DNA测序技术的一次革命,其关键技术之一是得到高活性的、固定于磁性微球表面的ATP硫酸化酶．生物素化的ATP硫酸化酶可以通过生物素与亲和素之间的特异结合特性固定在包被亲和素的磁性微球表面,但是利用化学修饰法将ATP硫酸化酶进行生物素化修饰很可能会影响酶的活性．利用融合表达策略,将大肠杆菌生物素酰基载体蛋白C端87个氨基酸肽段(BCCP87)与ATP硫酸化酶在大肠杆菌内融合表达,经SDS-PAGE和Western blot分析,表达的融合蛋白分子质量约为64 ku,并且能够在大肠杆菌内被生物素化．生物素化的ATP硫酸化酶能够与亲和素包被的磁珠结合,固定后的ATP硫酸化酶具有活性,并且能够用于定量检测焦磷酸盐(PPi)和焦测序,为今后建立高通量大规模焦测序系统提供了一个有效的工具酶．     

抗CEA单链抗体与链亲和素融合基因的表达

克隆分泌CEA杂交瘤细胞重链可变区（V_H）和轻链可变区（V_L）,以Linker连接V_H及V_L构建抗CEA单链抗体.同时以Spacer连接单链抗体和链亲和素,构建成功单链抗体和链亲和素融合基因,克隆该融合基因至原核表达载体,pET21a(+),经IPTG诱导表达出该双特异性融合蛋白.活性鉴定表明该融合蛋白具有结合CEA及生物素的双特异性.该融合蛋白在生物领域中有较广阔的应用前景.     

一种基于三顺反子表达载体的生物素诱导表达系统的建立

目的：建立一种以无毒的生物素为诱导剂的哺乳动物细胞诱导表达系统。方法：通过基因组PCR或重叠延伸PCR获得该系统的三个基本构件：大肠杆菌生物素连接酶（BirA）、链亲和素-四环素依赖的抑制因子融合蛋白（SA-TetR）和生物素化信号-VP16转录激活结构域融合蛋白（Avitag-VP16）。将上述基因连入三顺反子表达载体,与响应载体一起共转染293f细胞,以EGFP为报告基因,检测EGFP荧光强度随培养体系中生物素浓度变化的情况。结果：随着生物素浓度的增加,目的基因表达出现OFF-ON-OFF的变化,诱导状态下的EGFP荧光强度约为抑制状态下的3倍。结论：可通过调节生物素浓度对目的基因的表达进行可逆的调节,该系统是一种有应用前景的诱导表达系统。     

乙型肝炎病毒e抗原基因与绿色荧光蛋白基因的融合及其表达

质粒pS65T含有T7启动子驱动的绿色荧光蛋白突变型gfpS65T基因,经修饰后,在其中插入乙型肝炎病毒e抗原（HBeAg）基因,使两基因同框成为融合基因。在大肠杆菌BL21中由于T7启动子的控制,高效表达了具有双功能（抗原性和发光性）的融合蛋白（GFP－HBeAg）。融合蛋白MW为52kDa,N端有6个组氨酸残基,因而用Ni金属螯合层析柱对融合蛋白进行了分离纯化,利用诊断HBV的ELISA试剂盒检测了融合蛋白的抗原性,在荧光显微镜下观察到了融合蛋白的绿色荧光。并探讨了用其组装成新型免疫诊断试剂的可能性。     

水蛭素融合蛋白的表达及其与靶向适配子的偶联

目的:优化表达并纯化水蛭素融合蛋白SA-H-RGD,检测其生物学活性,获得能够与生物素标记的纤维蛋白适配子G81-2结合的偶联物。方法:将序列正确的质粒p ET-44b-SA-H-RGD进行原核表达,采用不同浓度的IPTG及时间优化融合蛋白表达条件,镍亲和凝胶层析柱纯化融合蛋白,Western-blot鉴定蛋白。通过凝血酶原时间(PT)和抗血小板聚集实验检测融合蛋白活性;之后按照生物素-G81-2:SA-H-RGD摩尔比为4:1的比例制备纤维蛋白特异性的偶联物,用凝胶迁移阻滞实验(EMSA)验证二者的偶联。结果:融合蛋白SA-H-RGD在IPTG 0.9 mmol/L、5 h时在大肠杆菌中获得可溶性高效表达,纯化的融合蛋白具有延长PT的作用和抑制血小板聚集的活性,EMSA表明SA-H-RGD具有结合生物素标记的G81-2适配子的功能。结论:本研究成功地优化表达了具有抗凝血和抗血小板聚集功能的融合蛋白SA-H-RGD,获得了水蛭素融合蛋白与生物素-G81-2适配子组成的靶向偶联物。     

基于Avi-tag技术的人胚肾细胞内增强型绿色荧光蛋白的生物素化、纯化和检测方法

目的:建立并优化基于Avi-tag标签技术的人胚肾细胞增强型绿色荧光蛋白(eGFP)的定点生物素化标记、纯化和检测方法。方法:分别构建具有Avi-tag标签的eGFP真核表达载体plenti-Avi-eGFP和BirA酶真核过表达载体pQCXIH-BirA,将plenti-Avi-eGFP和pQCXIH-BirA共转染人胚肾293T细胞,12 h后观察Avi-tag标签对eGFP蛋白在细胞内定位的影响;48 h后裂解细胞,用链霉亲和素珠子纯化生物素标记的eGFP,SDS-PAGE观察eGFP纯化和富集情况,并优化基于Western印迹的生物素化eGFP检测方法。结果:Avi-tag标签对eGFP在细胞内的定位无影响,同时BirA酶在293T细胞内可将带Avi-tag标签的eGFP标记上生物素;生物素化的eGFP可特异性地被链霉亲和素珠子纯化和富集,纯度可达95%;Western印迹检测生物素化蛋白的最终条件为5%的BSA作为封闭液和终浓度为100 ng/mL的链霉亲和素-HRP。结论:建立了基于Avi-tag技术的人胚肾细胞内增强型绿色荧光蛋白的生物素化标记、纯化与检测方法,为该方法的广泛应用奠定了前期技术基础。     

链霉亲和素-藻胆蛋白的融合表达及其在液相芯片检测中应用

[目的]重组表达制备链霉亲和素-藻胆蛋白融合蛋白,并应用于致病性弧菌液相芯片的检测体系中.[方法]在大肠杆菌中构建链霉亲和素-藻胆蛋白融合蛋白的生物合成途径,通过发酵和亲和层析纯化,制备融合蛋白;设计副溶血弧菌、霍乱弧菌、创伤弧菌和溶藻弧菌等四种致病性弧菌特异性检测引物和核苷酸探针,以融合蛋白为荧光标志物,建立四种致病...     

Construction of bifunctional fusion proteins consisting of duck BAFF and EGFP

We constructed fusion proteins consisting of fluorescence-enhanced green fluorescent protein (EGFP) and soluble domain of duck B-cell-activating factor of the TNF family (dsBAFF). The soluble EGFP/dsBAFF was efficiently expressed in Escherichia coli BL 21 (DE3) and was purified in milligram amounts using metal chellate affinity chromatography. The fusion protein exhibited similar fluorescence spectra with free EGFP and promoted the survival of duck bursal B cells in vitro as well as dsBAFF. EGFP/dsBAFF has shown specific binding to duck BAFF receptors positive-cells and the stained cells could be analyzed with flow cytometry. Thus, the fusion protein represents a readily obtainable source of biologically active dsBAFF that may prove useful in further studies on duck BAFF and its receptors.     

TAT-PTD融合蛋白可能存在的跨膜递送作用机制

为探讨TAT-PTD融合蛋白的跨膜递送作用机制,采用DNA重组技术构建pGEX-TAT-GFP-表达质粒.在E.coli- BL21表达GST-TAT-GFP,并用谷胱甘肽(glutathione) Sepharose-4B亲和柱进行纯化.GST-TAT-GFP在不同条件下与细胞的作用结果表明,GST-TAT-GFP能有效进入HeLa、SMMC-7721、L-02和BEL-7402细胞,GST-TAT-GFP在递送时对时间和浓度有依赖关系.同时,温度对GST-TAT-GFP跨膜作用具有明显的影响,GST-TAT-GFP的跨膜作用受代谢抑制剂的影响很小,肝素的存在能明显抑制GST-TAT-GFP跨膜进入细胞的能力,GST-TAT-GFP对细胞活性没有影响.这些结果说明,TAT-PTD可能是通过与细胞表面的硫酸乙酰肝素等受体相结合介导融合蛋白跨膜递送进入细胞的.     

The construction and characterization of a bifunctional EGFP/sAPRIL fusion protein

A fusion protein of enhanced green fluorescent protein (EGFP) and soluble domain of human a proliferation-inducing ligand (sAPRIL) was efficiently expressed in Escherichia coli BL 21 (DE3). The soluble EGFP/sAPRIL, around 43 kDa, was purified in milligram amounts using metal chellate affinity chromatography and detected with anti-His₆ and anti-hsAPRIL monoclonal antibody. The chimeric protein exhibited similar fluorescence spectra with free EGFP. In vitro, purified EGFP/sAPRIL specifically bound receptor B cell maturation antigen (BCMA) detected by enzyme linked immunosorbent assay (ELISA) and receptors [including heparan sulfate proteoglycan (HSPGs)]-positive cell lines analyzed by fluorescence-activated cell sorting (FACS). Confocal laser microscopy images visibly showed the HSPGs’-dependent binding of EGFP/sAPRIL to NIH-3T3 cell. In addition, the chimera retained the bioactivity to stimulate/co-stimulate proliferation of NIH-3T3 and Jurkat cell/human B cell in vitro. Therefore, the fusion protein shows a readily obtainable source of biologically active sAPRIL which has considerable potential for single-step fluorescence detection assay in the study of APRIL and its receptors.     

A genetically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>, expressing the fusion protein of green fluorescent protein and carboxylesterase B1, can be easily detected in the environment following degradation of pesticide residues

Genetically engineered Escherichia coli, expressing the fusion protein of enhanced green fluorescent protein (EGFP) and carboxylesterase B1 (CarE B1), was successfully constructed by cloning the genes into the pET-28b vector and then transforming E. coli BL21 (DE3). Expression of the fusion protein was induced in E. coli BL21 (DE3) which could then degrade environmental pesticides and could be easily detected using fluorescence spectrophotometry or by the naked eye in daylight.     

Expression of recombinant EARLI1, a hybrid proline-rich protein of Arabidopsis, in Escherichia coli and its inhibition effect to the growth of fungal pathogens and Saccharomyces cerevisiae

EARLI1 is an Arabidopsis gene with pleiotropic effects previously shown to have auxiliary functions in protecting plants against freezing-induced cellular damage and promoting germinability under low-temperature and salinity stresses. Here we determined whether recombinant EARLI1 protein has anti-fungal activity. Recombinant EARLI1 protein lacking its signal peptide was produced in Escherichia coli BL21(DE3) using isopropyl β-d-1-thiogalactopyranoside (IPTG) induction and the prokaryotic expression vector pET28a. Expression of EARLI1 was analyzed by Western blotting and the protein was purified using affinity chromatography. Recombinant EARLI1 protein was applied to fungal cultures of Saccharomyces cerevisiae, Botrytis cinerea and Fusarium oxysporum, and membrane permeability was determined using SYTOX green. Full-length EARLI1 was expressed in S. cerevisiae from the GAL1 promoter using 2% galactose and yeast cell viability was compared to control cells. Our results indicated that application of recombinant EARLI1 protein to B. cinerea and F. oxysporum could inhibit the growth of the necrotrophic fungi. Besides, addition of the recombinant protein to liquid cultures of S. cerevisiae significantly suppressed yeast growth and cell viability by increasing membrane permeability, and in vivo expression of the secreted form of EARLI1 in S. cerevisiae also had a remarkable inhibition effect on the growth of yeast cells.     

厚壳贻贝粘附蛋白十肽重复序列的表达及应用

 利用PCR扩增 Mcfp-1（M. coruscus foot protein-1）基因的12个十肽重复序列粘附功能片段（Mcfp~1 1~12）并连接到pGEX-4T1表达载体中,在大肠杆菌BL21(DE3)中诱导表达.纯化的多肽产物经凝血酶处理切除GST标签,获得Mcfp-1 1~12功能肽段,最后用酪氨酸酶对该产物进行修饰.通过材料表面包被、石英晶体微天平（QCM, quartz crystal microbalance）分析、细胞粘附和细胞毒性等实验,研究了该表达产物作为生物粘合剂的粘附特性.结果显示,重组表达产物Mcfp-1 1~12在多种材料表面的包被能力与Cell-TakTM（天然提取的贻贝粘附蛋白混合物）相当,甚至更佳;对细胞的粘附能力与Cell-TakTM相当;用HeLa细胞和293T细胞进行的MTT实验未发现细胞毒性.上述结果表明,Mcfp-1 1~12作为生物医用粘合剂具有潜在应用价值;同时,基因重组技术可以为制备新型海洋贻贝粘附蛋白防水生物粘合剂提供有效的手段.本研究为临床使用更优质的生物医用粘合剂提供了理论依据.     

PhaP介导EGFR靶向多肽修饰的肿瘤靶向PHBHHx纳米药物递送载体的构建

聚羟基脂肪酸(PHA)颗粒表面结合蛋白Pha P具有与疏水性高分子材料表面紧密结合的能力,本研究将EGFR靶向多肽(ETP)与PhaP进行融合表达,构建了ETP-PhaP融合蛋白表达的重组工程菌Escherichia coli BL21(DE3)(pPI-ETP-P)。经对工程菌株的诱导表达及ETP-PhaP融合蛋白的纯化后,通过PhaP蛋白介导能够有效地将ETP-PhaP融合蛋白修饰于3-羟基丁酸-3-羟基己酸共聚酯(PHBHHx)纳米微球表面,构建成为具有EGFR靶向作用的药物递送载体。分别检测宫颈癌细胞系SiHa(EGFR高表达)和CaSKi(EGFR低表达)对ETP-PhaP修饰的PHBHHx纳米药物载体和未经修饰的纳米药物载体的吞噬情况。结果显示,纯化的ETP-PhaP融合蛋白能够很好地吸附于PHBHHx颗粒的表面,经ETP-PhaP融合蛋白修饰的PHBHHx纳米药物载体对EGFR高表达的宫颈癌Si Ha细胞的靶向效果强于EGFR低表达的CaSKi细胞系。这一结果表明了PhaP介导的PHBHHx纳米微球表面EGFR靶向多肽修饰具有简便、高效的优势,为疏水性纳米药物载体表面功能多肽修饰提供了一种新策略。     

Lentivirus‐mediated bifunctional cell labeling for in vivo melanoma study

Lentiviral vectors (LVs) are capable of labeling a broad spectrum of cell types, achieving stable expression of transgenes. However, for in vivo studies, the duration of marker gene expression has been highly variable. We have developed a series of LVs harboring different promoters for expressing reporter gene in mouse cells. Long‐term culture and colony formation of several LV‐labeled mouse melanoma cells showed that promoters derived from mammalian house‐keeping genes, especially those encoding RNA polymerase II (Pol2) and ferritin (FerH), provided the highest consistency for reporter expression. For in vivo studies, primary B16BL6 mouse melanoma were infected with LVs whose luciferase–green fluorescence protein fusion gene (Luc/GFP) was driven by either Pol2 or FerH promoters. When transplanted into syngeneic C57BL/6 mice, Luc/GFP‐labeled B16BL6 mouse melanoma cells can be monitored by bioluminescence imaging in vivo, and GFP‐positive cells can be isolated from the tumors by fluorescence‐activated cell sorter. Pol2‐Luc/GFP labeling, while lower in activity, was more sustainable than FerH‐Luc/GFP labeling in B16BL6 over consecutive passages into mice. We conclude that Pol‐2‐Luc/GFP labeling allows long‐term in vivo monitoring and tumor cell isolation in immunocompetent mouse melanoma models.     

炭疽芽孢杆菌EA1蛋白的融合表达和纯化

目的：原核表达重组炭疽芽孢杆菌EA1蛋白。方法：用PCR方法从炭疽芽孢杆菌A16R疫苗株染色体中扩增编码EA1蛋白的eag基因序列,经过纯化、酶切后克隆到含有GST标签的原核表达载体pGEX-6P-2中,构建重组载体pGEX-EA1;将空载体（作为对照）、重组载体转化大肠杆菌BL21（DE3）菌株获得表达工程菌株,对其表达和纯化条件进行优化;利用Western印迹检测融合蛋白的表达。结果：构建了EA1蛋白的融合表达载体,并在大肠杆菌中获得高效表达;经Glutathione Sepharose 4B纯化获得了EA1蛋白;Western印迹表明,此蛋白可与GST标签抗体反应。结论：在原核表达系统中表达并纯化得到EA1融合蛋白,为进一步对其进行功能研究奠定了基础。