铁蛋白重链亚铁氧化酶活性突变体的构建及鉴定

[目的]构建小鼠铁蛋白重链(ferritin heavy chain,FTH)亚铁氧化酶活性突变基因的真核表达载体。[方法]针对小鼠FTH基因的亚铁氧化酶活性关键位点(K86、E62和H65),设计并合成6条PCR引物。利用重叠延伸PCR技术,获得小鼠FTH基因的突变体m FTH(K86Q、E62K和H65G),简写为m FTH(3M)。然后将m FTH(3M)插入到实验室已有的C端带Flag标签的真核表达载体pc DNA3中,插入位置为多克隆位点区域中限制性内切酶Bam HⅠ和HindⅢ之间,从而得到亚铁氧化酶活性缺失的FTH真核表达载体pc DNA3-m FTH(3M)-Flag。重组质粒经酶切和测序鉴定正确后,用脂质体lipofectamineTM2000将其转染到RAW264.7细胞中,检测细胞中带有Flag标签的m FTH(3M)蛋白的表达。[结果]重组质粒酶切得到预期片段,测序显示所构建的m FTH(3M)真核表达载体序列正确,细胞内检测到带Flag标签的m FTH(3M)蛋白信号的强烈表达。[结论]实验成功构建了小鼠FTH亚铁氧化酶活性位点基因突变的真核表达载体pc DNA3-m FTH(3M)-Flag。     

抗黄曲霉毒素B1单链抗体在Sf9昆虫细胞中的表达与性质分析

目的:在原核表达抗黄曲霉毒素B1(aflatoxin B1,AFB1)单链抗体(single chain Fv fragment,scFv)研究的基础上,为进一步了解和提高抗AFB1 scFv的活性,利用Sf9昆虫细胞表达抗AFB1 scFv,并对其活性进行探索研究。方法:构建pFastBac 1-scFv2E6VHVL重组质粒,将重组质粒转化Escherichia coli (E. coli) DH10Bac细胞,进行蓝白斑筛选,挑取阳性克隆。提取相应的重组杆状病毒穿梭载体Bacmid侵染Sf9昆虫细胞,表达scFv,利用镍亲和层析法纯化scFv,并以ELISA检测scFv活性。结果:蓝白斑筛选后,经菌落PCR和测序验证挑取的白斑阳性单克隆含有正确的单链抗体基因。提取相应的重组杆状病毒穿梭载体Bacmid侵染Sf9昆虫细胞,通过Western blot检测得知抗AFB1 scFv在Sf9昆虫细胞中成功表达。AFB1对scFv的抑制中浓度(IC₅₀)为30μg/ml。结论:与E. coli BL21(DE3)表达系统相比,scFv灵敏度转好,但仍有较大提升空间。     

FTH1基因多态性与湖羊和小尾寒羊产羔数的关联分析

为了解FTH1基因在2个绵羊品种中的遗传变异及其与产羔数的关系,本研究以具有详细产羔记录的湖羊(n=424)和小尾寒羊(n=432)为研究对象,采用DNA-pooling结合PCR-RFLP的方法进行单核苷酸多态性(single nucleotide polymorphism, SNP)扫描,同时分析SNP位点与绵羊产羔数的相关性。结果表明,在FTH1基因第一内含子上发现一个g.1635GA的SNP位点,并在小尾寒羊和湖羊群体中均有AA、AG和GG三种基因型,其基因型频率分别为0.08、0.45、0.47和0.10、0.43、0.47,A和G等位基因频率分别为0.305、0.695和0.315、0.685,表明GG为优势基因型,G为优势等位基因;湖羊群体的基因He、Ho、Ne和PIC分别为0.431、0.569、1.759和0.338;在小尾寒羊中则分别为0.424、0.576、1.737和0.334。χ~2适合性检验表明,在此位点湖羊和小尾寒羊群体均不处于Hardy-Weinberg平衡状态(p0.05)。与产羔数的关联分析发现,在湖羊群体中,GG基因型个体的产羔数显著高于AA基因型(p=0.029),而在小尾寒羊群体中各基因型个体的产羔数差异不显著。综上所述,FTH1基因可以作为提高湖羊产羔数的分子辅助标记。     

细菌生产人铁蛋白的新功能及应用

铁蛋白(Ferritin)是一种广泛存在于生物体中的笼状蛋白,由24个亚基自组装形成的蛋白质外壳和铁内核两部分组成,是维持机体铁代谢平衡的重要蛋白。最新发现,人血清铁蛋白含量的变化与某些疾病相关,特别是发现利用大肠杆菌重组表达、仿生合成的磁性人铁蛋白具有双功能特性,即识别肿瘤并使其可视化。此外,铁蛋白独特的结构及理化性质使其成为理想的纳米载体,用于构筑多功能肿瘤成像和药物输送的平台。本文重点介绍人铁蛋白的新功能及其在疾病诊断和肿瘤靶向治疗中的应用前景。     

高效构建卵清白蛋白scFv噬菌体文库及其筛选

目的:建立一种高效噬菌体文库构建方法,获得抗鸡卵清蛋白(ovalbumin,OVA)的单链抗体(scFv)噬菌体展示文库,筛选鉴定获得OVA单链抗体。方法:用OVA蛋白免疫Balb/C小鼠,选取血清抗体效价高的小鼠提取脾脏RNA,利用RT-PCR方法扩增获得小鼠重链和小鼠轻链基因。通过无缝连接酶一步将小鼠重链基因、轻链基因和linker DNA连接起来,插入噬菌体表达载体中,构建OVA scFv噬菌体展示文库。测定文库容量,对文库进行富集筛选,ELISA鉴定阳性克隆,测序后构建真核表达载体,转入Expi-CHO悬浮细胞进行真核表达,利用Western blot进行鉴定。结果:成功获得库容量为1. 2×10~7cfu的OVA scFv噬菌体展示文库,并从中筛选出8个阳性克隆,选取效价最高的2号克隆,在Expi-CHO悬浮细胞中表达获得可溶性抗体。结论:建立了一种高效构建scFv噬菌体文库的方法,筛选获得高结合活性的OVA单链抗体,并成功进行了真核表达,为OVA ELISA检测试剂盒的研制奠定了基础。     

丁苯酞对哮喘小鼠气道黏液高分泌及IL-13、TNF-α的影响*

目的: 研究丁苯酞对哮喘小鼠气道黏液高分泌及白介素-13(IL-13)、肿瘤坏死因子-α(TNF-α)的影响。方法: 小鼠随机分为空白对照组、模型对照组、阳性对照组和丁苯酞高、中、低剂量(100、50、25 mg/kg)组(n=12)。实验第1日、8日、15日通过注射卵清白蛋白(OVA)致敏,第22日吸入OVA连续激发5周复制哮喘模型,同时在激发前给予丁苯酞20 mg/kg进行干预,观测哮喘行为学、气道杯状细胞及黏蛋白5ac(Muc5ac)的分泌,测定支气管肺泡灌洗液(BALF)粘度,采用ELISA法测定BALF中Muc5ac、IL-13及TNF-α的水平。结果: 与空白对照组比较,模型对照组小鼠打喷嚏、抓鼻及哮喘的程度显著加重(P＜0.01),小鼠气道上皮杯状细胞增生及Muc5ac的分泌显著增加(P＜0.01),BALF的粘度及其中的Muc5ac、IL-13和TNF-α的含量显著升高(P＜0.01);与模型对照组比较,25、50、100 mg/kg丁苯酞干预后哮喘行为学评分明显下降(P＜0.01);小鼠气道上皮杯状细胞增生、Muc5ac的分泌、BALF的粘度及其中的Muc5ac、IL-13和TNF-α的含量均明显降低(P＜0.05, 0.01)。结论: 丁苯酞具有抑制哮喘气道黏液高分泌而平喘的作用,缓解IL-13、TNF-α异常高表达是其抑制气道黏液高分泌的作用机制之一。     

血红素对大肠杆菌铁蛋白稳定性及自组装的影响

大肠杆菌铁蛋白(bacterioferritin,BFR)是由同源亚基24聚体组成的高度对称的八面体壳状结构,其中处在C2对称轴的每两个亚基之间即存在一个血红素分子。为了探讨血红素对细菌铁蛋白结构稳定性及在蛋白质自组装过程中的影响,通过定点突变将第52位蛋氨酸突变为丙氨酸以去除血红素分子,并应用体积排阻色谱、透射电镜、紫外-可见光吸收光谱及圆二色谱等手段对突变蛋白M52A的聚合态、二级结构及热力学性质等进行分析。结果表明:去除血红素虽然对铁蛋白的自组装没有明显影响,但是对其热稳定性产生较大影响,突变后蛋白质的T_m值为54.3℃,远小于野生型铁蛋白T_m值69.9℃,且经过氯化血红素与同浓度下该突变体M52A化学诱导结合后,发现其T_m值重新恢复至67.7℃。由此可知,血红素对大肠杆菌铁蛋白稳定性影响很大,且具有调控铁蛋白稳定性的作用。     

幽门螺旋杆菌重组尿素酶B亚基的纯化及其免疫学性质的研究

目的:幽门螺旋杆菌(Hp)尿素酶是Hp重要的定制因子和致病因子,Hp尿素酶活性位点位于Hp尿素酶B亚基(UreB),研发基于UreB的Hp疫苗是一种很有前景的防治Hp感染的策略。方法:主要利用基因克隆技术从幽门螺旋杆菌标准菌株SS1(Hp SS1)获得Hp尿素酶B亚基基因,并构建含有重组Hp尿素酶B亚基(rUreB)基因的重组表达载体pET-rUreB及其重组菌株;重组菌株经蛋白表达和优化后,利用Ni-NTP镍离子亲和层析和DEAE Sepharose FF阴离子交换层析纯化重组尿素酶B亚基(rUreB),并进一步通过腹腔注射免疫BALB/c小鼠,研究rUreB的免疫学性质。结果:通过基因克隆技术成功获得了Hp尿素酶B亚基基因,并成功构建了重组表达载体pET-rUreB及其重组菌株BL21(DE3)/pET-rUreB,经蛋白表达优化及纯化,可获得高纯度(96.5%)的重组蛋白rUreB。重组蛋白rUreB辅以弗氏佐剂腹腔注射免疫BALB/c小鼠,经间接ELISA鉴定小鼠能够产生针对天然Hp尿素酶和UreB的高滴度特异性抗体,且能够显著性抑制Hp尿素酶的活性。结论:重组Hp尿素酶B亚基能够在大肠杆菌表达系统中获得较高水平的表达,具有较高的免疫学特异性,其抗体能够有效抑制Hp尿素酶活性。为研究基于尿素酶的防治Hp感染的Hp疫苗奠定了一定的实验基础。     

铁蛋白与口蹄疫病毒VP1在大肠杆菌中融合表达及纳米颗粒自组装

口蹄疫是由口蹄疫病毒引起的世界上最重要的畜牧疾病之一,严重影响世界畜牧业的发展,而疫苗免疫仍然是对疫情预防和控制的最有效手段.铁蛋白具有自组装和生物修饰的特性,在纳米疫苗等领域具有广阔的应用前景.选用O型口蹄疫病毒的vp1基因和幽门螺杆菌铁蛋白基因,通过融合PCR将vp1基因构建到铁蛋白亚基基因前端,在大肠杆菌中表达后...     

抗人胎盘酸性铁蛋白单链抗体的构建、表达与鉴定

目的:制备人胎盘酸性铁蛋白(PAF),构建、表达并鉴定抗人PAF单链可变区抗体片段(scFv)。方法:设计以SfiⅠ、NotⅠ为酶切位点、以(Gly4Ser)3为linker的2对引物,从抗人PAF单克隆抗体可变区基因的克隆载体中扩增VH和VL基因,用重叠延伸PCR在VH和VL基因间引入连接短肽,构建VH-linker-VL的scFv基因。经SfiⅠ、NotⅠ酶切后克隆到原核分泌型表达载体pUC19/119上,转化大肠杆菌TG1和筛选后测序验证,IPTG诱导表达,SDS-PAGE鉴定其相对分子质量(Mr)。以人PAF为抗原,scFv为一抗,抗His单克隆抗体为二抗,间接ELISA鉴定其抗体活性。结果:重叠延伸PCR扩增产物经凝胶电泳可见约700bp的条带,DNA序列分析证明2株抗体具有完整的scFv序列,并含有c-myc和His。IPTG诱导阳性菌表达产物经SDS-PAGE鉴定有Mr约27000的显示条带,符合scFv与表达标签融合蛋白的理论值;间接ELISA证明表达产物可与PAF结合。结论:成功构建并表达了抗人PAF单链抗体,为进一步建立检测PAF的间接ELISA奠定了基础。     

The use of highly expressed FTH1 as carrier protein for cytosolic targeting in Hansenula polymorpha

The iron storage protein ferritin is a member of the non-heme iron protein family. It can store and release iron, therefore it prevents the cell from damage caused by iron-dioxygen reactions as well as it provides iron for biological processing. To study whether the human ferritin heavy chain (FTH1) can be expressed in Hansenula polymorpha, we integrated an expression cassette for FTH1 and analyzed the protein expression. We found very efficient expression of FTH1 and obtained yields up to 1.9 g/L under non-optimized conditions. Based on this result we designed a FTH1-PTH fusion protein to successfully express the parathyroid hormone fragment 1-34 (PTH) for the first time intracellular in H. polymorpha.     

Long non‐coding RNA FTH1P3 activates paclitaxel resistance in breast cancer through miR‐206/ABCB1

Emerging evidence has indicated the important function of long non‐coding RNAs (lncRNAs) in tumour chemotherapy resistance. However, the underlying mechanism is still ambiguous. In this study, we investigate the physiopathologic role of lncRNA ferritin heavy chain 1 pseudogene 3 (FTH1P3) on the paclitaxel (PTX) resistance in breast cancer. Results showed that lncRNA FTH1P3 was up‐regulated in paclitaxel‐resistant breast cancer tissue and cells (MCF‐7/PTX and MDA‐MB‐231/PTX cells) compared with paclitaxel‐sensitive tissue and parental cell lines (MCF‐7, MDA‐MB‐231). Gain‐ and loss‐of‐function experiments revealed that FTH1P3 silencing decreased the 50% inhibitory concentration (IC50) value of paclitaxel and induced cell cycle arrest at G2/M phase, while FTH1P3‐enhanced expression exerted the opposite effects. In vivo, xenograft mice assay showed that FTH1P3 silencing suppressed the tumour growth of paclitaxel‐resistant breast cancer cells and ABCB1 protein expression. Bioinformatics tools and luciferase reporter assay validated that FTH1P3 promoted ABCB1 protein expression through targeting miR‐206, acting as a miRNA “sponge.” In summary, our results reveal the potential regulatory mechanism of FTH1P3 on breast cancer paclitaxel resistance through miR‐206/ABCB1, providing a novel insight for the breast cancer chemoresistance.     

Iron loading-induced aggregation and reduction of iron incorporation in heteropolymeric ferritin containing a mutant light chain that causes neurodegeneration

Hereditary ferritinopathy (HF) is a neurodegenerative disease characterized by intracellular ferritin inclusion bodies (IBs) and iron accumulation throughout the central nervous system. Ferritin IBs are composed of mutant ferritin light chain as well as wild-type light (Wt-FTL) and heavy chain (FTH1) polypeptides. In vitro studies have shown that the mutant light chain polypeptide p.Phe167SerfsX26 (Mt-FTL) forms soluble ferritin 24-mer homopolymers having a specific structural disruption that explains its functional problems of reduced ability to incorporate iron and aggregation during iron loading. However, because ferritins are usually 24-mer heteropolymers and all three polypeptides are found in IBs, we investigated the properties of Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymeric ferritins. We show here the facile assembly of Mt-FTL and FTH1 subunits into soluble ferritin heteropolymers, but their ability to incorporate iron was significantly reduced relative to Wt-FTL/FTH1 heteropolymers. In addition, Mt-FTL/FTH1 heteropolymers formed aggregates during iron loading, contrasting Wt-FTL/FTH1 heteropolymers and similar to what was seen for Mt-FTL homopolymers. The resulting precipitate contained both Mt-FTL and FTH1 polypeptides as do ferritin IBs in patients with HF. The presence of Mt-FTL subunits in Mt-FTL/Wt-FTL heteropolymers also caused iron loading-induced aggregation relative to Wt-FTL homopolymers, with the precipitate containing Mt- and Wt-FTL polypeptides again paralleling HF. Our data demonstrate that co-assembly with wild-type subunits does not circumvent the functional problems caused by mutant subunits. Furthermore, the functional problems characterized here in heteropolymers that contain mutant subunits parallel those problems previously reported in homopolymers composed exclusively of mutant subunits, which strongly suggests that the structural disruption characterized previously in Mt-FTL homopolymers occurs in a similar manner and to a significant extent in both Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymers.     

TNFα‐induced abnormal activation of TNFR/NF‐κB/FTH1 in endometrium is involved in the pathogenesis of early spontaneous abortion

Early spontaneous abortion (ESA) is one of the most common complications during pregnancy and the inflammation condition in uterine environment such as long‐term exposure to high TNFα plays an essential role in the aetiology. Ferritin heavy chain (FTH1) is considered to be closely associated with inflammation and very important in normal pregnancy, yet the underlying mechanism of how TNFα induced abortion and its relationship with FTH1 remain elusive. In this study, we found that TNFα and FTH1 were positively expressed in decidual stromal cells and increased significantly in the ESA group compared with the normal pregnancy group (NP group). Besides, TNFα expression was positively correlated with FTH1 expression. Furthermore, in vitro cell model demonstrated that high TNFα could induce the abnormal signals of TNFR/NF‐κB/FTH1 and activate apoptosis both in human endometrium stromal cells (hESCs) and in local decidual tissues. Taken together, the present findings suggest that the excessive apoptosis in response to TNFα‐induced upregulation of FTH1 may be responsible for the occurrence of ESA, and thus provide a possible therapeutic target for the treatment of ESA.     

Impact of single-chain Fv antibody fragment affinity on nanoparticle targeting of epidermal growth factor receptor-expressing tumor cells

To determine the importance of single-chain Fv (scFv) affinity on binding, uptake, and cytotoxicity of tumor-targeting nanoparticles, the affinity of the epidermal growth factor receptor (EGFR) scFv antibody C10 was increased using molecular evolution and yeast display. A library containing scFv mutants was created by error-prone PCR, displayed on the surface of yeast, and higher affinity clones selected by fluorescence activated cell sorting. Ten mutant scFv were identified that had a 3-18-fold improvement in affinity (KD=15-88 nM) for EGFR-expressing A431 tumor cells compared to C10 scFv (KD=264 nM). By combining mutations, higher affinity scFv were generated with KD ranging from 0.9 nM to 10 nM. The highest affinity scFv had a 280-fold higher affinity compared to that of the parental C10 scFv. Immunoliposome nanoparticles (ILs) were prepared using EGFR scFv with a 280-fold range of affinities, and their binding and uptake into EGFR-expressing tumor cells was quantified. At scFv densities greater than 148 scFv/IL, there was no effect of scFv affinity on IL binding and uptake into tumor cells, or on cytotoxicity. At lower scFv densities, there was less uptake and binding for ILs constructed from the very low affinity C10 scFv. The results show the importance of antibody fragment density on nanoparticle uptake, and suggest that engineering ultrahigh affinity scFv may be unnecessary for optimal nanoparticle targeting.     

一株针对人EGFR的单链抗体克隆与哺乳细胞表达

目的:制备特异性抗人表皮生长因子受体(EGFR)的单链抗体(sc Fv),鉴定其生物学活性,为进一步研究基于单链抗体的免疫治疗奠定基础。方法:从分泌抗人EGFR单克隆抗体的杂交瘤细胞系提取总RNA,利用5'RACE技术扩增轻链和重链可变区(VL、VH)基因,构建具有VL、VH基因的单链抗体基因,并将构建的单链抗体基因克隆到真核细胞表达载体pc DNA3.1中进行表达和鉴定。ELISA鉴定单链抗体对抗原的特异性;Fortebio检测抗原抗体间的亲和力,流式细胞术检测单链抗体结合肺癌细胞系天然EGFR的功能活性。结果:获得唯一的轻重链可变区序列VL、VH,成功构建EGFR-sc Fv,特异性与天然EGFR蛋白结合,亲和力达3.22×10-9mol/L。结论:成功构建了抗人EGFR单链抗体,为肺癌免疫导向治疗研究奠定了基础。     

Simultaneous inhibition of epidermal growth factor receptor (EGFR) signaling and enhanced activation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-mediated apoptosis induction by an scFv:sTRAIL fusion protein with specificity for human EGFR

Epidermal growth factor receptor (EGFR) signaling inhibition by monoclonal antibodies and EGFR-specific tyrosine kinase inhibitors has shown clinical efficacy in cancer by restoring susceptibility of tumor cells to therapeutic apoptosis induction. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent with tumor-selective apoptotic activity. Here we present a novel approach that combines EGFR-signaling inhibition with target cell-restricted apoptosis induction using a TRAIL fusion protein with engineered specificity for EGFR. This fusion protein, scFv425:sTRAIL, comprises the EGFR-blocking antibody fragment scFv425 genetically fused to soluble TRAIL (sTRAIL). Treatment with scFv425:sTRAIL resulted in the specific accretion to the cell surface of EGFR-positive cells only. EGFR-specific binding rapidly induced a dephosphorylation of EGFR and down-stream mitogenic signaling, which was accompanied by cFLIP(L) down-regulation and Bad dephosphorylation. EGFR-specific binding converted soluble scFv425:sTRAIL into a membrane-bound form of TRAIL that cross-linked agonistic TRAIL receptors in a paracrine manner, resulting in potent apoptosis induction in a series of EGFR-positive tumor cell lines. Co-treatment of EGFR-positive tumor cells with the EGFR-tyrosine kinase inhibitor Iressa resulted in a potent synergistic pro-apoptotic effect, caused by the specific down-regulation of c-FLIP. Furthermore, in mixed culture experiments binding (L)of scFv425:sTRAIL to EGFR-positive target cells conveyed a potent apoptotic effect toward EGFR-negative bystander tumor cells. The favorable characteristics of scFv425:sTRAIL, alone and in combination with Iressa, as well as its potent anti-tumor bystander activity indicate its potential value for treatment of EGFR-expressing cancers.