亚麻中雄性不育基因同源序列MS2-F的克隆和表达分析

用同源序列克隆法从亚麻中克隆了雄性不育基因同源序列MS2-F cDNA(登陆号:EU363493).该cDNA全长1 91lbp,包含一个1 608 bp的ORF,编码535个氨基酸.推导的蛋白质序列中包含2个雄性不育保守区:NAD结合区域和雄性不育C-末端区域.该基因与油菜和拟南芥雄性不育基因的一致性分别为59.65%和59.16%,为花蕾特异表达基因,推测在亚麻花粉发育过程中与脂酰辅酶A还原酶有相似功能.MS2-F cDNA对应的gDNA大小为2 696 bp(登陆号:EU365361),含有8个内含子和9个外显子.     

装载RNA"病毒样"纳米颗粒表面巯基化表达载体的构建及荧光修饰

以本实验室构建的能够装载外源RNA片段的MS2噬菌体“病毒样”颗粒表达载体为基础,利用定点突变技术将衣壳蛋白基因的第15位密码子由编码苏氨酸突变为胱氨酸。表达产物在35％蔗糖密度梯度处有一目的产物,目的产物在透射电镜下呈球形,直径大小约为27nm。用马来酰亚胺-5’-荧光素对表达产物进行化学修饰,经光谱分析、SDS-PAGE分析和MALDI-TOF MS鉴定,证明巯基在表达产物的外表面,并能与马来酰亚胺-5＇-荧光素进行反应。这种携带外源RNA片段的荧光修饰的天然纳米颗粒为制备各种功能性纳米材料提供了新途径。     

大肠杆菌0157噬菌体中vt2基因A、B亚单位的克隆与序列分析

根据GenBank中毒素基因vt1、vt2序列设计合成4对引物,以大肠杆菌O157菌株DNA为模板,扩增vt1、vt2,从只含有vt2的菌株中诱导释放噬菌体,以噬菌体DNA为模板进行PCR扩增,获得vt2、vt2-A、vt2-B3条特异性DNA带;将vt2-A、vt2-B扩增产物纯化后,分别插入pMD18-T载体,测序结果与相应序列比较,vt2-A和vt2-B亚单位的基因序列与GenBank中编码VT2毒素的A、B亚单位的核苷酸序列(X07865,NC_002655,：BA000007,AF291819)的同源性分别为98％～99％、96％～100％,确定vt2位于噬菌体,并为进一步研究大肠杆菌O157中VT噬菌体的毒力转导、VT2毒素的表达和应用奠定基础。     

基于MS2噬菌体病毒样颗粒的RT-PCR检测新型冠状病毒(SARS-CoV-2)质控品制备*

目的: 制备热稳定性好、耐RNase攻击及可全程监控操作的核酸检测新型冠状病毒阳性质控品。方法: 分别扩增MS2噬菌体外壳蛋白CP(含PAC位点)基因以及成熟酶蛋白A基因序列(含核糖体结合位点),先后插入质粒pET28a多克隆位点不同位置,构建通用重组载体pET28a/CP-A。合成包含ORF1ab基因、N基因和E基因三个靶标的特定核酸序列,插入到重组载体pET28a/CP-A中PAC位点的下游,构建包含靶序列的重组载体pET28a/CP-A/S。通过原核表达系统表达目的蛋白,采用硫酸铵和凝胶过滤层析进行纯化,利用透射电镜和动态光散射对蛋白质进行物理表征。全能核酸酶消化形成的盔甲RNA,通过RT-PCR检测其残余核酸和热稳定性。结果: 成功构建包含MS2噬菌体外壳蛋白CP基因、成熟酶蛋白A基因和外源靶核酸的重组载体,目的蛋白在25℃、IPTG 0.3mmol /L、诱导14h时以可溶性形式得到高效表达,纯化后,得到了大小均一、直径为23~28nm的病毒样颗粒,经核酸酶消化后RT-PCR检测,颗粒溶液中几乎无核酸残余且形成了包封靶基因的盔甲RNA。加速破坏试验表明该盔甲RNA无菌过滤后可在37℃稳定保持10天。结论: 在体外,利用MS2噬菌体外壳蛋白和成熟酶蛋白自组装包封外源靶序列制备的盔甲RNA,其热稳定性好,可全程监控整个检测过程,可作为核酸检测SARS-CoV-2的定性或定量质控品。     

基于MS2噬菌体病毒样颗粒的RT-PCR检测新型冠状病毒(SARS-CoV-2)质控品制备*

目的: 制备热稳定性好、耐RNase攻击及可全程监控操作的核酸检测新型冠状病毒阳性质控品。方法: 分别扩增MS2噬菌体外壳蛋白CP(含PAC位点)基因以及成熟酶蛋白A基因序列(含核糖体结合位点),先后插入质粒pET28a多克隆位点不同位置,构建通用重组载体pET28a/CP-A。合成包含ORF1ab基因、N基因和E基因三个靶标的特定核酸序列,插入到重组载体pET28a/CP-A中PAC位点的下游,构建包含靶序列的重组载体pET28a/CP-A/S。通过原核表达系统表达目的蛋白,采用硫酸铵和凝胶过滤层析进行纯化,利用透射电镜和动态光散射对蛋白质进行物理表征。全能核酸酶消化形成的盔甲RNA,通过RT-PCR检测其残余核酸和热稳定性。结果: 成功构建包含MS2噬菌体外壳蛋白CP基因、成熟酶蛋白A基因和外源靶核酸的重组载体,目的蛋白在25℃、IPTG 0.3mmol /L、诱导14h时以可溶性形式得到高效表达,纯化后,得到了大小均一、直径为23~28nm的病毒样颗粒,经核酸酶消化后RT-PCR检测,颗粒溶液中几乎无核酸残余且形成了包封靶基因的盔甲RNA。加速破坏试验表明该盔甲RNA无菌过滤后可在37℃稳定保持10天。结论: 在体外,利用MS2噬菌体外壳蛋白和成熟酶蛋白自组装包封外源靶序列制备的盔甲RNA,其热稳定性好,可全程监控整个检测过程,可作为核酸检测SARS-CoV-2的定性或定量质控品。     

含口蹄疫病毒IRES RNA病毒样颗粒表达载体的构建

利用PCR技术扩增大肠杆菌MS2噬菌体的外壳蛋白和成熟酶蛋白基因,将其克隆到pET32a中构建中间载体pET32a-CP。将FMDV的内部核糖体结合位点（IRES）保守序列连接到中间载体噬菌体基因的下游,构建原核表达载体pCPES。将重组质粒pCPES转化宿主菌BL21(DE3),1 mmol/L IPTG诱导表达。蔗糖密度梯度离心纯化表达产物。透射电镜观察到直径大约26 nm的圆形病毒样颗粒。检测病毒样颗粒的稳定性并进行RT-PCR鉴定。结果表明该病毒样颗粒含口蹄疫病毒IRES RNA序列,并且稳定性良好,本研究构建的病毒样颗粒可以作为RNA病毒检测时的标准品和质控品使用。     

噬菌体Qβ病毒样颗粒的制备、纯化及鉴定

目的: 利用大肠杆菌原核表达系统制备噬菌体Qβ VLPs,验证其自组装能力,纯化后免疫动物以确定其免疫原性,同时制备兔多克隆抗体验证其在哺乳动物细胞中内化能力。方法: 合成pET-28-Qβ-CP质粒,利用大肠杆菌原核表达系统制备Qβ VLPs,通过蔗糖密度梯度离心纯化VLPs,将经凝胶层析柱Sephacryl S-400纯化的Qβ VLPs用透射电镜观察颗粒形态;纯化后的Qβ VLPs加入或不加入佐剂分别免疫新西兰兔,获得血清后用Protein G纯化得到兔多克隆抗体,通过Western blot确定制备的兔多克隆抗体特异性,并利用间接免疫荧光法对Qβ VLPs的细胞内化能力进行鉴定。结果: 制备并获得纯度较高的Qβ VLPs,通过透射电镜观察到大量直径约为28 nm的颗粒;Western blot结果表明制备的兔多克隆抗体能特异性识别Qβ VLPs,且在免疫实验中加入佐剂与不加入佐剂分别免疫动物,对抗体水平的影响不显著。间接免疫荧光法结果表明Qβ VLPs在哺乳动物细胞中具有内化能力。结论: 成功制备Qβ VLPs为后续研发以噬菌体Qβ VLPs为载体的相关疫苗奠定基础。     

含RGD修饰的病毒样颗粒递送ICG靶向肿瘤的研究

目的:获取含RGD靶向肽的乙肝核心病毒样颗粒,为药物靶向纳米递送系统提供一种新型载体。方法:将实验室前期构建测序正确的含RGD修饰的乙肝核心病毒重组质粒转化入大肠杆菌BL21(DE3)中,单因素分析及正交试验探究重组蛋白最适表达条件。在最适表达条件下扩培,收集菌体超声破碎后离心,采用凝胶过滤层析、离子交换和蔗糖密度梯度离心进行纯化,利用透射电镜对形成的RGD-HBc VLPs的形态及稳定性进行鉴定。纯化的RGD-HBc VLPs利用其体外自组装的特性,将光敏剂ICG装载到颗粒的内部,通过静脉注射到4T1乳腺癌荷瘤小鼠,探究重组RGD-HBc VLPs作为纳米递送系统的靶向性。结果:RGD-HBc VLPs在温度32℃、IPTG0.5mmol/L、诱导4h时以可溶性蛋白的形式得到高效表达。经蔗糖密度梯度离心纯化后纯度到达95%以上。透射电镜下观察纯化的RGD-HBc VLPs形态、大小均一,直径约为32nm,通过近红外荧光活体成像证实了RGD-HBc作为纳米载体的靶向性。结论:经表达和纯化后,RGD-HBc VLPs具有较高的表达量和大小均一的形态外貌,近红外荧光活体成像证实具有较好的靶向性,这不仅为肿瘤的可视化诊断提供一种快速、精准、方便的方法,而且为今后靶向免疫治疗提供一种新型载体。     

大肠杆菌O157噬菌体中vt2基因A、B亚单位的克隆与序列分析

根据GenBank中毒素基因 vt1、vt2序列设计合成 4 对引物,以大肠杆菌 O157 菌株 DNA为模板,扩增 vt1、vt2,从只含有vt2的菌株中诱导释放噬菌体,以噬菌体DNA为模板进行PCR扩增,获得vt2、vt2 A、vt2 B 3条特异性DNA带;将 vt2 A、vt2 B扩增产物纯化后,分别插入 pMD18 T载体,测序结果与相应序列比较,vt2 A和 vt2 B亚单位的基因序列与 GenBank中编码 VT2 毒素的 A、B亚单位的核苷酸序列(X07865,NC_002655, BA000007,AF291819)的同源性分别为98%~99%、96%~100%,确定 vt2 位于噬菌体,并为进一步研究大肠杆菌 O157 中VT噬菌体的毒力转导、VT2毒素的表达和应用奠定基础。     

大肠杆菌O157噬菌体中vt2基因A、B亚单位的克隆与序列分析

根据GenBank中毒素基因vt1、vt2序列设计合成4对引物,以大肠杆菌O157菌株DNA为模板,扩增vt1、vt2,从只含有vt2的菌株中诱导释放噬菌体,以噬菌体DNA为模板进行PCR扩增,获得vt2、vt2-A、vt2-B 3条特异性DNA带;将vt2-A、vt2-B扩增产物纯化后,分别插入pMD18-T载体,测序结果与相应序列比较,vt2-A和vt2-B亚单位的基因序列与GenBank中编码VT2毒素的A、B亚单位的核苷酸序列(X07865,NC_002655,BA000007,AF291819)的同源性分别为98%～99%、96%～100%,确定vt2位于噬菌体,并为进一步研究大肠杆菌O157中VT噬菌体的毒力转导、VT2毒素的表达和应用奠定基础.     

猪细小病毒vp2基因的表达和类病毒颗粒的构建

利用PCR方法从病毒基因组中获得了vp2完整的编码区并经测序后克隆到原核表达载体pET-32(a)上.在大肠杆菌中诱导表达了VP2与Trx-His-S Tag的融合蛋白,通过免疫家兔获得了高特异性的兔抗血清.另外,将vp2克隆到pFastBacDUAL载体上,利用昆虫表达系统,即AcMNPV的Bac-to-Bac系统对vp2进行了表达,经SDS-PAGE和Western blot分析,表达产物为64kD,并且该蛋白能在昆虫细胞中形成类病毒颗粒.     

猪细小病毒vp2基因的表达和类病毒颗粒的构建

利用PCR方法从病毒基因组中获得了vp2完整的编码区并经测序后克隆到原核表达载体pET-32(a)上。在大肠杆菌中诱导表达了VP2与Trx-His-STag的融合蛋白,通过免疫家兔获得了高特异性的兔抗血清。另外,将vp2克隆到pFastBacDUAL载体上,利用昆虫表达系统,即AcMNPV的Bac-to-Bac系统对vp2进行了表达,经SDS-PAGE和Westernblot分析,表达产物为64kD,并且该蛋白能在昆虫细胞中形成类病毒颗粒。     

Effect of Fluorophenylalanine on Bacteriophage MS2 Replication

A concentration of 10 mug of fluorophenylalanine per ml added to a chemically defined medium reduced by 100-fold the number of bacteriophage MS2 produced on Escherichia coli C3000 and increased the latent period. Fluorophenylalanine was most effective when added concurrent with infection. Addition of a 10-fold greater concentration of phenylalanine reversed the inhibition caused by fluorophenylalanine. Radioactive fluorophenylalanine was incorporated into the coat protein. The four phenylalanine-containing chymotryptic peptides are not equally accessible to fluorophenylalanine. Only two of the peptides are highly labeled by fluorophenylalanine. Incorporation of fluorophenylalanine decreased the specific infectivity and the rate of adsorption but did not increase the sensitivity of the whole virus to ribonuclease. MS2 ribonucleic acid (RNA) functioned as messenger RNA for the incorporation of both phenylalanine and fluorophenylalanine in a cell-free incorporating system from E. coli.     

Intermediates of Bacteriophage MS2 Assembly In Vivo

The in vivo process of virion assembly was studied in rifampin-treated, MS2-infected Escherichia coli during late times of infection-after 18 min postinfection. Differential sucrose gradient sedimentation of infected-cell lysates taken at various times after radioactive labeling indicated a definite temporal order of appearance of phage-specific protein in assembly-related structures. Labeled MS2 protein appears first as a low-molecular-weight peak at the tops of gradients, then as a peak at 40S and as a large number of almost unseparable structures between 40 and 80S, and finally as 80S mature phage particles. During the chase of a short labeling period, radioactive phage protein was found to disappear from gradients in the same temporal order as it appeared; the soluble peak disappears first, followed by the 40 to 70S region. The chased label appears quantitatively in the 80S phage peak. Labeled phage RNA was found to appear first in the 40S peak, then in the structures between 40 and 70S, and finally in 80S phage particles. The order of disappearance of labeled phage RNA during a chase is the same as its appearance. Resedimentation of the 40 to 70S region indicated the presence of distinct structures at 60 and 70S and many indistinct ones between 40 and 60S. The smaller intermediates exhibit separable maturation protein-rich and coat protein-rich segments, indicating nonrandom binding of the two proteins during the initial steps of assembly. Larger, discrete intermediates appear at 60 and 70S. Treatment of the various structures with pancreatic RNase results in destruction of those from 40 through 60S; treatment of the 70S structure results in the conversion of some of it to a 45S peak, presumably the complete capsid. A small fraction of the 80S phage peak is also sensitive to RNase, resulting in a similar 45S peak. Pulse-chase experiments indicate that structures from 40 through 60S as well as the RNase-sensitive 70S structure are assembly intermediates, but that the RNase-insensitive 70S and the RNase-sensitive 80S structures are not.     

Whole Genome Sequence Analysis of Lactiplantibacillus Plantarum Bacteriophage P2

Phage P2 was isolated from failed fermentation broth carried out by Lactiplantibacillus plantarum IMAU10120. A previous study in our laboratory showed that this phage belonged to the Siphoviridae family. In this study, this phage’s genomic characteristics were analyzed using whole-genome sequencing. It was revealed that phage P2 was 77.9 kb in length and had 39.28% G + C content. Its genome included 96 coding sequences (CDS) and two tRNA genes involved in the function of the structure, DNA replication, packaging, and regulation. Phage P2 had higher host specificity; many tested strains were not infected. Cell wall adsorption experiments showed that the adsorption receptor component of phage P2 might be a part of the cell wall peptidoglycan. This research might enrich the knowledge about genomic information of lactobacillus phages and provide some primary data to establish phage control measures.     

Low-Frequency Electromagnetic Fields Alter the Replication Cycle of MS2 Bacteriophage

The effect of exposure to 60-Hz electromagnetic fields (EMFs) on RNA coliphage MS2 replication was studied. EMF exposure commenced when the bacterial cultures were inoculated with the phage (t = 0). In 12 experiments in which the strength of the field was 5 G, a significant delay in phage yield was found in the EMF-exposed cultures 45–65 min after inoculation, compared with control cultures. However, the EMF did not alter the final phage concentration. Experiments at 25 G (N = 5) suggested that the stronger field resulted in both impeded phage replication and increased phage yield. No differences between test groups were found in experiments involving sham-EMF exposure, thereby indicating that the results obtained with the EMFs were not due to systematic error. It appears that MS2, which codes for only four proteins, is the simplest biological system in which an EMF-induced effect has been demonstrated. The MS2 system is, therefore, conducive to follow-up studies aimed at understanding the level and nature of the underlying interaction process, and perhaps to biophysical modeling of the interaction process. Received: 26 August 1997 / Accepted: 17 November 1997     

Male-Specific Bacteriophage MS2 Propagation in Fluorophenylalanine-Resistant Escherichia coli K12

Mutation of Escherichia coli K12 HfrH to resistance to fluorophenylalanine resulted in changes in the plaque morphology of bacteriophage MS2 on this strain and led to an increased efficiency of propagation of the phage in liquid cultures. Evidence was obtained that the mutation resulted in inhibition of early lysis in infected cells and that lysis involved the production of a lysozyme. Genetic studies suggested that the observed pleiotropy of the resistance mutation was due to informational suppression.     

Mimicking Somatic Hypermutation: Affinity Maturation of Antibodies Displayed on Bacteriophage Using a Bacterial Mutator Strain

Human antibodies can now be isolated from antibody repertoires displayed on the surface of filamentous bacteriophage in a process that mimics the primary immune response. Here we have attempted to mimic the secondary response, the natural process of affinity maturation of antibodies occurring in germinal centres, by multiple cycles of random mutation and selection. Phage displaying a human antibody fragment recognising the hapten 2-phenyl-5-oxazolone were grown in a mutator strain of bacteria (Escherichia coli: mutD5) to generate a large repertoire of antibodies that should include the majority of possible single nucleotide point mutations. The repertoire of phage antibody mutants was then selected by binding to hapten. By multiple rounds of growth in the mutator strain, and increasingly stringent selection, we succeeded in isolating mutants with improved binding affinities; furthermore, the distribution of mutations and nucleotide substitution preferences strongly resembled those of somatic hypermutation. We then constructed a genealogical tree from the sequences of mutants taken at different rounds, and identified four sequentially acquired mutations that together improve the binding affinity of the antibody by a factor of 100-fold (fromK_d320 nM to 3.2 nM).