肠道分节丝状菌(SFB)与宿主间相互作用关系研究进展

SFB作为一种肠道共生菌受到越来越多的关注。研究显示其可能在宿主的免疫系统成熟过程中扮演着重要角色,包括调控T细胞的分化与平衡,增加Th17细胞的比例,刺激机体内分泌型免疫球蛋白IgA的分泌等。随着研究的不断深入,人们发现肠道内SFB的定植有利于宿主抵抗外来致病菌,如大肠杆菌和梭菌等的感染。同时也有研究表明,SFB的定殖能促进T细胞向Th17的分化,有利于自身免疫性疾病,如脑脊髓炎和类风湿性关节炎等的发生。另一方面,SFB的分布与定植也受到宿主因子的影响,不同物种来源的SFB存在着宿主特异性,肠道不同解剖部位SFB的形态及定殖数量也存在着差异等。     

Generation and characterization of C305, a murine neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA

B-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) family and a key regulator of B cell response. Neutralizing single-chain fragment variable (scFv) antibody against BLyS binding to its receptor BCMA has the potential to play a prominent role in autoimmune disease therapy. A phage display scFv library constructed on pill protein of MI 3 filamentous phage was screened using BLyS.After five rounds of panning, their binding activity was characterized by phage-ELISA. Nucleotide sequencing revealed that at least two different scFv gene fragments (C305 and D416) were obtained. The two different scFv gene fragments were expressed to obtain the soluble scFv antibodies, then the soluble scFv antibodies were characterized by means of competitive ELISA and in vitro neutralization assay. The results indicated that C305 is the neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA.     

A new combination of mutated loxPs in a vector for construction of phage antibody libraries

In the construction of large antibody libraries by in vivo recombination, two non-homogeneous loxP sites are required for the exchange of Vgenes between phagemids to create many new VH-VL combinations.The mutated loxP511 was designed not to recombine with the wild-type loxP (loxPwt) in early studies and a combination of the two has been used to construct antibody libraries. But recent reports have shown that recombination occurs between loxPwt and loxP511. This suggests that the combinational use of loxP511 and loxPwt might lead to the loss of the V gene diversity of antibody libraries. Therefore, it is necessary to find a new combination of loxPs to avoid the excision recombination in the antibody library. In this study,we found that the excision recombination between loxP511 and loxP2272, another mutated loxP sequence,was undetectable within one phagemid, while the excision recombination between loxP511 and loxPwt occurred at a frequency of 40%, higher than that reported previously. Furthermore, the in vivo recombination of different phagemids with loxP511 and loxP2272 showed that the V gene exchange was efficiently mediated to produce new VH-VL combinations. It was concluded that the loxP511 and loxP2272 combination was more favorable for reducing the excision recombination and constructing large phage antibody libraries with high diversity.     

抗体工程研究进展

噬菌体抗体库技术的出现为抗体工程的研究带来了革命性的变化,本文总结了当前国内外抗体工程,特别是单链抗体方面的研究进展。     

CglI基因在大肠杆菌中的功能活性分析

通过PCR技术从谷氨酸棒杆菌基因组中扩增CglⅠ基因,克隆到载体pMD18-T Simple后测序.将CglⅠ基因亚克隆到表达载体pJL23,构建重组质粒pJL23-GglⅠ,转化大肠杆菌HB101菌株,通过PCR反应筛选鉴定阳性克隆.通过噬菌体感染实验,初步分析了CglⅠ基因在大肠杆菌中的功能活性.     

利用噬菌体展示技术筛选胰岛素模拟肽

为了寻找能够模拟胰岛素生物活性的小肽,以胰岛素多克隆抗体为靶标,筛选噬菌体展示随机C7C环肽库.3轮筛选后,通过ELISA方法挑取与靶分子特异性结合的15个阳性克隆,测序获得两条序列,分析所得序列并合成相应短肽.通过细胞生物学活性检测,小肽CPTSQANSC（ZJ1）能够竞争性的抑制胰岛素与其受体的结合,并对正常小鼠和四氧嘧啶诱导的糖尿病小鼠,都有明显的降血糖作用.上述结果表明,小肽CPTSQANSC具有胰岛素样生物学活性.而小肽CVQPSHSSC（ZJ2）表现出胰岛素拮抗活性,能引起正常小鼠血糖升高.这表明筛选到了能够模拟胰岛素表位的短肽CPTSQANSC,可能为治疗胰岛素依赖性糖尿病提供了新线索.     

肌球蛋白X在细胞运动中的作用

肌球蛋白X(myosin X,Myo X)是一类尾部具有MyTH4(myosin tail homology 4,MyTH4)和FERM(band4.1/ezrin/radixin/moesin,FERM)结构域的非传统型肌球蛋白,广泛分布于脊椎动物细胞中.近几年的研究表明,Myo X尾部结构域能够与众多的信号分子相互作用,参与调节从丝状伪足形成到胞质分裂等多种细胞运动过程,但其具体的调控机制目前尚不完全清楚.Myo X作为一类连接细胞膜与细胞骨架的动力蛋白分子,它的研究越来越受到人们的关注,其功能的揭示将为进一步阐明细胞多种运动机制提供理论依据.     

噬菌体抗体库技术及其应用研究进展

噬菌体呈现抗体库是近年发展的一项分子生物学新技术,它的建立是抗体技术领域中的一次革命性进展。它以其独特的构建和筛选系统,彻底改变了抗体制备的传统途径,使抗体工程技术进入了一个新的发展阶段,并对生物学领域中许多技术的发展起到了巨大的推动作用。该技术是迄今发展最成熟、应用最广泛的制备抗体技术。我们简要综述此项技术的研究应用进展。     

从大容量噬菌体抗体库中筛选抗Acr蛋白人源单链抗体

目的:从天然的大容量噬菌体抗体库中筛选特异的抗结核分枝杆菌晶体蛋白( alpha-crystallin Acr)的人源抗体.方法:以结核分枝杆菌Acr蛋白包被免疫管,通过对噬菌体抗体库进行4轮“吸附-洗脱-扩增”的过程从大容量抗体库中筛选特异性抗结核分枝杆菌Acr蛋白的抗体,并对可变区序列进行了测序分析.将特异性的噬菌体抗体感染HB2151菌,经IPTG诱导表达,制备了抗结核分枝杆菌Acr蛋白的可溶性单链抗体;对其序列和抗原结合活性进行分析鉴定.结果:经过4轮筛选,获得了43个与结核分枝杆菌Acr蛋白结合的阳性克隆,其中29个特异结合的克隆;测序分析有26不同的可变区片段;通过可溶性单链抗体(scFv)表达筛选到14株特异性结合Acr蛋白的可溶性单链抗体克隆;经过基因测序,分析了可变区基因的亚群.成功制备了可溶性单链抗体.Westren blotting分析证实筛选的人源单链抗体能与天然蛋白结合.结论:利用单链大容量抗体库获得抗结核分枝杆菌Acr蛋白的噬菌体抗体并且成功制备抗结核分枝杆菌Acr天然蛋白的可溶性单链抗体,为今后的研究和应用奠定基础.     

嗜盐古菌噬菌体研究进展

嗜盐古菌噬菌体是噬菌体的一个重要分支,在群体生态学以及生命的起源与进化历程中扮演重要的角色.综述了嗜盐古菌噬菌体的形态多样性、研究方法、起源与进化几方面的研究报道,指出目前研究中存在的问题,对未来研究进行了展望.