禽流感病毒核蛋白在噬菌体表面的展示

利用PCR技术,扩增禽流感病毒（AIV）核蛋白基因片段,将其克隆至pR质粒的T4噬菌体SOC基因C末端获得重组质粒pR—np,以此重组载体转化大肠杆菌Escherichia coli2（E2）,用溶菌酶缺陷噬菌体T4-zl感染重组E2菌,重组载体与缺陷噬菌体T4-zl基因发生同源重组,将np基因整合到噬菌体基因组中,用PCR方法筛选重组噬菌体并命名为T4-zl—np。经Western blot免疫电镜与ELISA检测证实,T4-zl-np表达的NP融合蛋白具有免疫学活性。结果表明,AIV核蛋白成功地在T4噬菌体表面展示,为禽流感防治奠定了基础。     

8株动物源编码Stx2短尾噬菌体的生物学特性

[目的]对8株源自大肠杆菌O157编码Stx2毒素的噬菌体生物学特性进行研究.[方法]丝裂霉素C诱导8株大肠杆菌O157菌株释放噬菌体,采用PCR作初步鉴定,分离、纯化噬菌体基因组,随机引物法地高辛(DIG)标记stx2基因片段作为探针,对纯化的噬菌体采用Southernblot进行Stx2噬菌体再次鉴定,透射电子显微镜观察纯化的8株Stx2噬菌体的形态特征,通过限制性内切酶图谱分析,确定噬菌体的核酸类型和基因组大小、以及限制性内切酶酶切片段多态性,并分析噬菌体的蛋白质组成特征.[结果]Southern blot证实分离的8株噬菌体为Stx2噬菌体,电镜下观察的各株Stx2噬菌体形态一致,头部均为正六边形,尾部很短,属于短尾噬菌体科,各株噬菌体之间存在相同的蛋白结构模式,基因组为双链DNA,限制性内切酶片段长度表现出一定的多态性,噬菌体的基因组大小从48.0-65.3 kb不等.[结论]来源不同菌株的8株编码Stx2噬菌体均为短尾噬菌体,其蛋白结构模式一致,但基因组具有不同组成.     

一株感染铜绿假单胞菌的双链RNA噬菌体PaP6的分离和鉴定

【目的】鉴定一株新分离的铜绿假单胞菌噬菌体PaP6的生物学特性。【方法】利用铜绿假单胞菌临床分离株PA038为宿主,从西南医院污水中分离得到一株裂解性噬菌体PaP6,观察其噬斑特点;氯化铯密度梯度离心纯化噬菌体颗粒后,用透射电子显微镜观察噬菌体形态;提取PaP6基因组,通过DNA酶和RNA酶酶切,做基因组酶切图谱分析;按照感染复数(MOI)分别为10、1、0.1、0.01、0.001和0.000 1加入噬菌体和宿主菌,裂解细菌后,测定噬菌体滴度;以MOI=10的比例加入噬菌体和宿主菌,绘制一步生长曲线;用112株铜绿假单胞菌临床分离株检测PaP6宿主谱。【结果】PaP6的噬斑直径约2 mm-4 mm,圆形透明,边缘清晰;PaP6噬菌体呈多面体立体对称的头部,直径约45 nm;酶切图谱表明PaP6基因组对DNase不敏感,对RNase敏感,未酶切基因组具有3节段双链RNA(dsRNA),长度分别约为9.0、4.5、3.5 kb,共约17 kb;当MOI为0.1时PaP6感染其宿主菌产生的子代噬菌体滴度最高,达到3.4×109 PFU/m L;用一步生长曲线描绘了其生长特性;PaP6可以感染40.1%的临床分离株,是一株比较广谱的噬菌体。【结论】首次报道了一株铜绿假单胞菌的ds RNA分节段噬菌体,分类学上属于囊病毒科,该噬菌体具有较广的宿主谱,在噬菌体治疗领域具有应用前景。     

霍乱弧菌分型噬菌体VP3基因组序列的测定与分析

测定和分析霍乱弧菌分型噬菌体VP3基因组序列,并为ElTor型霍乱弧菌两类菌株的分型方法原理提供研究基础。鸟枪法构建VP3噬菌体全基因组随机文库;测序拼接成最小重叠群,引物步移法填补缝隙序列,拼接后获得VP3全基因组序列。PCR随机扩增噬菌体DNA片段并酶切鉴定;预测可能存在的开放读码框(ORF);对VP3和相关噬菌体的DNA聚合酶基因作进化树分析,协助判定VP3的分类;对预测的部分启动子区利用报道基因进行活性分析。VP3全基因组为环状双链DNA,长度39504bp;酶切鉴定结果与序列一致。确定了49个ORF,注释了27个ORF的编码产物,其中有20个基因产物与T7样噬菌体同源,包括RNA聚合酶(RNAP)、参与DNA复制的蛋白、衣壳蛋白、尾管及尾丝蛋白、DNA包装蛋白等。DNA聚合酶(DNAP)进化树分析表明VP3与T7样噬菌体有同源性。将预测的10个启动子序列克隆到lacZ融合质粒pRS1274上,经检测均具有启动子活性。测定和分析VP3的基因组序列,基因组结构与进化树分析提示VP3属于T7噬菌体家族。     

快速从医院废水中大规模分离铜绿假单胞杆菌噬菌体

目的:从医院废水中快速分离多株不同的铜绿假单胞杆菌噬菌体,研究其生物学特性,为建立铜绿假单胞杆菌噬菌体库做准备。方法:利用噬菌斑法从未经处理的医院污水中分离和鉴定铜绿假单胞杆菌噬菌体,根据感染谱的不同确定它们为不同的铜绿假单胞杆菌噬菌体;重点研究其中一株宿主谱较广的噬菌体的生物学特性,采用负染法电镜观察噬菌体的形态和大小,提取该噬菌体的基因组并进行酶切电泳分析,测定噬菌体感染复数并观察其一步生长曲线。结果:通过噬菌斑法分离出90株铜绿假单胞杆菌噬菌体。电镜观察显示,噬菌体Pa27P1头部呈立体对称,有一长尾;酶切结果显示,噬菌体Pa27P1的基因组为双链DNA;生长曲线表明噬菌体Pa27P1感染宿主菌的潜伏期为25 min,爆发时间为25 min,裂解量为514。结论:90株铜绿假单胞杆菌噬菌体中有5株具有较广的噬菌谱,其组合能裂解所有18株铜绿假单胞杆菌,为深入研究铜绿假单胞杆菌噬菌体的生物学特性及其功能提供了依据。     

人源抗汉坦病毒核衣壳蛋白T7噬菌体抗体库的构建

构建人源T7噬菌体单链抗体(scFv)库筛选抗汉坦病毒核衣壳蛋白(NP)抗体。从肾综合征出血热恢复期患者外周血淋巴细胞中提取总RNA,反转录合成cDNA第一条链,PCR分别扩增抗体重链可变区基因(VH)和轻链可变区基因(VL),经重叠延伸拼接(SOE)PCR组成scFv基因,并将其与T7噬菌体载体的2个臂相连接。体外包装后,在宿主菌BLT5403中,扩增重组噬菌体抗体库。以基因工程表达NP进行4轮“吸附-洗脱-扩增”的筛选,酶免疫实验检测抗体活性。所建抗体库库容为1.35×107,扩增后初级库滴度为2.12×1010pfu/mL。以NP抗原筛选后抗体出现特异性富集,经酶免疫实验鉴定,得到2株与NP抗原特异结合的噬菌体抗体。结果表明,研究成功构建了人源抗NP蛋白T7噬菌体抗体库。     

重组T4噬菌体gp37基因可扩大其在沙门氏菌中的宿主范围

[目的]将T4噬菌体WG01宿主决定区的gp37基因片段,与另一株T4噬菌体QL01的相应基因进行同源重组,从而获得嵌合噬菌体并进行宿主谱分析,为阐明T4噬菌体的宿主谱形成机制以及快速筛选针对特定病原菌的噬菌体奠定了基础。[方法]通过同源重组的方法将WG01 gp37上的8个基因片段分别替换给QL01,用沙门氏菌作为宿主菌筛选嵌合噬菌体,并对嵌合噬菌体进行宿主谱、最佳感染复数、一步生长曲线和遗传稳定性测定。[结果]本研究共获得了5株嵌合噬菌体(QWA、QWC、QWF、QWG、QWFG)。宿主谱试验结果表明,与噬菌体QL01相比,嵌合噬菌体对21株沙门宿主菌分别可以多裂解7、8、4、10和9株菌,即嵌合噬菌体都获得了相对较宽的宿主谱,其中QWG的沙门氏菌宿主菌拓宽最多。生物学特性试验结果表明,嵌合噬菌体QWG生物学特性稳定。嵌合噬菌体QWG经连续传代培养20代,测序分析第1代和第20代嵌合噬菌体尾丝蛋白基因在传代过程中的稳定性,测序结果表明,嵌合噬菌体改造部分的基因能稳定遗传。[结论]用基因改造的方法可以产生宿主谱拓宽且能稳定遗传的嵌合噬菌体,为快速筛选针对特定病原菌的噬菌体提供了可能。     

具超强富集U(Ⅵ)能力工程菌E.coli的构建

以鼠伤寒沙门氏菌基因组DNA为模板,PCR扩增得到非特异性酸性磷酸酶基因（phoN）,将其克隆到pMD18T-Vector中。用Spe I、Nde I限制性内切酶对重组转移载体T-VectorphoN与穿梭载体pRADZ3分别进行双酶切,再将phoN片段和穿梭载体pRADZ3中的大片段通过T4DNA连接酶连接。经PCR与双酶切双重鉴定,证实重组穿梭载体pRADZ3phoN构建成功。转化Escherichia coli DH5α感受态细胞,使其在正常情况下表达PhoN蛋白,经Western blot 证实phoN基因在DH5α中成功表达。利用含pRADZ3phoN的工程菌进行富集U(Ⅵ)实验,结果表明该工程菌对U(Ⅵ)的富集量较宿主菌提高约4倍,达46.16mg/g,去除率为92.32%。     

抗乙型肝炎病毒表面抗原T7噬菌体抗体库的构建

构建T7噬菌体单链抗体(scFv)库筛选抗乙型肝炎病毒表面抗原抗体.从抗-HBs阳性患者外周血淋巴细胞中提取总RNA,反转录合成cDNA第1条链,PCR分别扩增抗体重链可变区基因(VH)和轻链可变区基因(VL),经重叠延伸拼接(SOE)PCR组成scFv基因,并将其与T7噬菌体载体的2个臂相连接.体外包装后,在宿主菌BLT5403中,扩增重组噬菌体抗体库.以乙型肝炎病毒表面抗原进行4轮“吸附-洗脱-扩增”的筛选,酶免疫实验检测抗体活性.所建抗体库库容为1.53×107,扩增后初级库滴度为2.42×1010pfu/mL.以乙型肝炎病毒表面抗原筛选后抗体出现特异性富集,经酶免疫实验鉴定,得到2株与HBsAg抗原特异结合的噬菌体抗体,成功构建了抗HBsAg蛋白T7噬菌体抗体库.     

中华苜蓿根瘤菌噬菌体的分离及主要壳蛋白基因g23的系统进化分析

【目的】揭示苜蓿根瘤菌噬菌体的形态学特征及主要壳蛋白g23基因的分布地位,为根瘤菌噬菌体的生态学研究提供数据支持。【方法】以中华苜蓿根瘤菌(Sinorhizobium meliloti USDA1002T)为宿主,采用双层平板法分离土壤环境中的苜蓿根瘤菌噬菌体,利用电子显微镜观察纯化的噬菌体形态特征;提取噬菌体DNA,PCR扩增编码噬菌体壳蛋白的g23基因,构建系统进化树,以形态学鉴定和分子生物学相结合的方法,明确分离获得的苜蓿根瘤菌噬菌体g23基因的系统进化地位。【结果】分离获得了3株噬菌体,头部均呈二十面体,直径大小为81–86 nm,尾部有收缩尾鞘,长度大约为54–70 nm。克隆测序结果显示,获得的3株噬菌体g23基因株间相似度较高,但与可培养的Exo T-、Schizo T-、T-、Pseudo T-evens相似度较低。系统进化分析表明,获得的3株噬菌体不隶属于目前已划分的不同环境噬菌体g23基因的分类类群中。【结论】3株噬菌体均属于肌尾噬菌体科的裂性噬菌体,与目前获得的所有噬菌体g23基因相似性较低,属于新的侵染中华苜蓿根瘤菌的噬菌体株。     

DNA sequence heterogeneity in the genes of T-even type Escherichia coli phages encoding the receptor recognizing protein of the long tail fibers

Summary Genes (g) 36 and 37 code for the proteins of the distal half of the long tail fibers of phage T4, gene product (gp) 35 links the distal half to the proximal half of this fiber. The receptor, lipopolysaccharide, most likely is recognized by gp37. Using as probe a restriction fragment consisting of most of g36 and g37 of phage T4 the genes corresponding to g35, g36, and g37 of phages T2 and K3 (using the E. coli outer membrane proteins OmpF and OmpA, respectively, as receptors) have been cloned into plasmid pUC8. Partial DNA sequences of g37 of phage K3 have been determined. One area, corresponding to residues 157 to 210 of the 1026 residue gp37 of phage T4, codes for an identical sequence in phage K3. Another area corresponds to residues 767 to 832 of the phage T4 sequence. Amino acid residues 767 to 832 of the phage T4 sequence are almost identical in both phage proteins while the remainder is rather different. DNAs of T2, T4, T6, another T-even type phage using protein Tsx as a receptor, and 10 different T-even type phages using the OmpA protein as a receptor have been hybridized with restriction fragments covering various parts of the g37 area of phage K3. With probably only one exception all of the 13 phages tested possess unique genes 37 and within the majority of these, sequences highly homologous to parts of g37 of K3 are present in a mosaic type fashion. Other regions of these genes 37 did not show any homology with the K3 probes; in case of the OmpA specific phage M1 absence of homology was evident in most of its g37 even including the area that should serve for recognition of the cellular receptor. In sharp contrast to this situation it was found that a major part of the gene (g23) coding for the major capsid protein is rather highly conserved in all phages studied. The extreme variability in sequences existing in genes 37 might be a consequence of phages during evolution being able to more or less drastically change their receptor specifities.     

The capsid of the T4 phage superfamily: the evolution, diversity, and structure of some of the most prevalent proteins in the biosphere

The Escherichia coli bacteriophage T4 has served as a classic system in phage biology for more than 60 years. Only recently have phylogenetic analyses and genomic comparisons demonstrated the existence of a large, diverse, and widespread superfamily of T4-like phages in the environment. We report here on the T4-like major capsid protein (MCP) sequences that were obtained by targeted polymerase chain reaction (PCR) of marine environmental samples. This analysis was then expanded to include 1,000 s of new sequences of T4-like capsid genes from the metagenomic data obtained during the Sorcerer II Global Ocean Sampling (GOS) expedition. This data compilation reveals that the diversity of the major and minor capsid proteins from the GOS metagenome follows the same general patterns as the sequences from cultured phage genomes. Interestingly, the new MCP sequences obtained by PCR targeted to MCP sequences in environmental samples are more divergent (deeper branching) than the vast majority of the MCP sequences coming from the other sources. The marine T4-like phage population appears to be largely dominated by the T4-like cyanophages. Using approximately 1,400 T4-like MCP sequences from various sources, we mapped the degree of sequence conservation on a structural model of the T4-like MCP. The results indicate that within the T4 superfamily there are some clear phylogenetic groups with regard to the more conserved and more variable domains of the MCP. Such differences can be correlated with variations in capsid morphology, the arrangement of the MCP lattice, and the presence of different capsid accessory proteins between the subgroups of the T4 superfamily.     

Genome analysis of phage JS98 defines a fourth major subgroup of T4-like phages in Escherichia coli

Numerous T4-like Escherichia coli phages were isolated from human stool and environmental wastewater samples in Bangladesh and Switzerland. The sequences of the major head gene (g23) revealed that these coliphages could be placed into four subgroups, represented by the phages T4, RB69, RB49, and JS98. Thus, JS98 defines a new major subgroup of E. coli T4-like phages. We conducted an analysis of the 169-kb JS98 genome sequence. Overall, 198 of the 266 JS98 open reading frames (ORFs) shared amino acid sequence identity with the reference T4 phage, 41 shared identity with other T4-like phages, and 27 ORFs lacked any database matches. Genes on the plus strand encoded virion proteins, which showed moderate to high sequence identity with T4 proteins. The right genome half of JS98 showed a higher degree of sequence conservation with T4 and RB69, even for the nonstructural genes, than did the left genome half, containing exclusively nonstructural genes. Most of the JS98-specific genes were found in the left genome half. Two came as a hypervariability cluster, but most represented isolated genes, suggesting that they were acquired separately in multiple acquisition events. No evidence for DNA exchange between JS98 phage and the E. coli host genome or coliphages other than T4 was observed. No undesired genes which could compromise its medical use were detected in the JS98 genome sequence.     

Plasticity of the gene functions for DNA replication in the T4-like phages

We have completely sequenced and annotated the genomes of several relatives of the bacteriophage T4, including three coliphages (RB43, RB49 and RB69), three Aeromonas salmonicida phages (44RR2.8t, 25 and 31) and one Aeromonas hydrophila phage (Aeh1). In addition, we have partially sequenced and annotated the T4-like genomes of coliphage RB16 (a close relative of RB43), A. salmonicida phage 65, Acinetobacter johnsonii phage 133 and Vibrio natriegens phage nt-1. Each of these phage genomes exhibited a unique sequence that distinguished it from its relatives, although there were examples of genomes that are very similar to each other. As a group the phages compared here diverge from one another by several criteria, including (a) host range, (b) genome size in the range between approximately 160 kb and approximately 250 kb, (c) content and genetic organization of their T4-like genes for DNA metabolism, (d) mutational drift of the predicted T4-like gene products and their regulatory sites and (e) content of open-reading frames that have no counterparts in T4 or other known organisms (novel ORFs). We have observed a number of DNA rearrangements of the T4 genome type, some exhibiting proximity to putative homing endonuclease genes. Also, we cite and discuss examples of sequence divergence in the predicted sites for protein-protein and protein-nucleic acid interactions of homologues of the T4 DNA replication proteins, with emphasis on the diversity in sequence, molecular form and regulation of the phage-encoded DNA polymerase, gp43. Five of the sequenced phage genomes are predicted to encode split forms of this polymerase. Our studies suggest that the modular construction and plasticity of the T4 genome type and several of its replication proteins may offer resilience to mutation, including DNA rearrangements, and facilitate the adaptation of T4-like phages to different bacterial hosts in nature.     

Comparative genomics of the T4-Like Escherichia coli phage JS98: implications for the evolution of T4 phages

About 130 kb of sequence information was obtained from the coliphage JS98 isolated from the stool of a pediatric diarrhea patient in Bangladesh. The DNA shared up to 81% base pair identity with phage T4. The most conserved regions between JS98 and T4 were the structural genes, but their degree of conservation was not uniform. The head genes showed the highest sequence conservation, followed by the tail, baseplate, and tail fiber genes. Many tail fiber genes shared only protein sequence identity. Except for the insertion of endonuclease genes in T4 and gene 24 duplication in JS98, the structural gene maps of the two phages were colinear. The receptor-recognizing tail fiber proteins gp37 and gp38 were only distantly related to T4, but shared up to 83% amino acid identity to other T6-like phages, suggesting lateral gene transfer. A greater degree of variability was seen between JS98 and T4 over DNA replication and DNA transaction genes. While most of these genes came in the same order and shared up to 76% protein sequence identity, a few rearrangements, insertions, and replacements of genes were observed. Many putative gene insertions in the DNA replication module of T4 were flanked by intron-related endonuclease genes, suggesting mobile DNA elements. A hotspot of genome diversification was located downstream of the DNA polymerase gene 43 and the DNA binding gene 32. Comparative genomics of 100-kb genome sequence revealed that T4-like phages diversify more by the accumulation of point mutations and occasional gene duplication events than by modular exchanges.     

T4-Like genome organization of the Escherichia coli O157:H7 lytic phage AR1

We report the genome organization and analysis of the first completely sequenced T4-like phage, AR1, of Escherichia coli O157:H7. Unlike most of the other sequenced phages of O157:H7, which belong to the temperate Podoviridae and Siphoviridae families, AR1 is a T4-like phage known to efficiently infect this pathogenic bacterial strain. The 167,435-bp AR1 genome is currently the largest among all the sequenced E. coli O157:H7 phages. It carries a total of 281 potential open reading frames (ORFs) and 10 putative tRNA genes. Of these, 126 predicted proteins could be classified into six viral orthologous group categories, with at least 18 proteins of the structural protein category having been detected by tandem mass spectrometry. Comparative genomic analysis of AR1 and four other completely sequenced T4-like genomes (RB32, RB69, T4, and JS98) indicated that they share a well-organized and highly conserved core genome, particularly in the regions encoding DNA replication and virion structural proteins. The major diverse features between these phages include the modules of distal tail fibers and the types and numbers of internal proteins, tRNA genes, and mobile elements. Codon usage analysis suggested that the presence of AR1-encoded tRNAs may be relevant to the codon usage of structural proteins. Furthermore, protein sequence analysis of AR1 gp37, a potential receptor binding protein, indicated that eight residues in the C terminus are unique to O157:H7 T4-like phages AR1 and PP01. These residues are known to be located in the T4 receptor recognition domain, and they may contribute to specificity for adsorption to the O157:H7 strain.     

Isolation of Escherichia coli bacteriophages from the stool of pediatric diarrhea patients in Bangladesh

A 3-week coliphage survey was conducted in stool samples from 140 Bangladeshi children hospitalized with severe diarrhea. On the Escherichia coli indicator strain K803, all but one phage isolate had 170-kb genomes and the morphology of T4 phage. In spot tests, the individual T4-like phages infected up to 27 out of 40 diarrhea-associated E. coli, representing 22 O serotypes and various virulence factors; only five of them were not infected by any of these new phages. A combination of diagnostic PCR based on g32 (DNA binding) and g23 (major capsid protein) and Southern hybridization revealed that half were T-even phages sensu strictu, while the other half were pseudo-T-even or even more distantly related T4-like phages that failed to cross-hybridize with T4 or between each other. Nineteen percent of the acute stool samples yielded T4-like phages, and the prevalence was lower in convalescent stool samples. T4-like phages were also isolated from environmental and sewage water, but with low frequency and low titers. On the enteropathogenic E. coli strain O127:K63, 14% of the patients yielded phage, all of which were members of the phage family Siphoviridae with 50-kb genomes, showing the morphology of Jersey- and beta-4 like phages and narrow lytic patterns on E. coli O serotypes. Three siphovirus types could be differentiated by lack of cross-hybridization. Only a few stool samples were positive on both indicator strains. Phages with closely related restriction patterns and, in the case of T4-like phages, identical g23 gene sequences were isolated from different patients, suggesting epidemiological links between the patients.