健康人血液微生物宏基因组和噬菌体组的深度挖掘分析

目的探索健康人血液中微生物和噬菌体的存在及其可能的相互作用,通过生物信息学深度挖掘探讨血液微生物生态系统的结构和功能。方法收集2012—2018年绵阳市中心血站的1600份献血者血浆标本,通过DNA提取和文库构建,使用Illumina Hiseq 4500平台进行高通量测序后进行生物信息学深度挖掘。采用Bowtie2、Trimmomatic及Kraken等宏基因组分析软件对血液标本中的微生物丰度进行分析;随后进一步使用Megahit、geNomad、CheckV和eggNOG-mapper等软件进行噬菌体组分析,包括序列质控、组装、识别、聚类和功能注释,并通过使用iTOL、BLAST和PhaBOX等软件对发现的血液噬菌体构建系统发育树、物种注释分析以及宿主分析与预测。结果宏基因组测序结果从血浆标本中识别出36个菌门、151个菌目、338个菌科、338个菌属和3757个菌种的微生物。在种的分类水平上,丰度排名前11的菌种分别是炭疽芽孢杆菌(Bacillus cereus)、鼠杆菌属乳杆菌(Lactobacillus murinus)、约翰逊乳杆菌(L.johnsonii)、普劳兹氏梭杆菌(Faecalibacterium prausnitzii)、苏云金芽孢杆菌(B.thuringiensis)、路氏乳杆菌(L.reuteri)、痤疮丙酸杆菌(Cutibacterium acnes)、海迪茨氏菌(Dietzia sp.JS16-p6b)、猪鼻炎支原体(Mycoplasma hyorhinis)、猪肺炎支原体(M.hyopneumoniae)、金黄色葡萄球菌(Staphylococcus aureus)。通过噬菌体组分析鉴定了202个噬菌体操作分类单元(vOTU),物种注释揭示了24种噬菌体类型,通过病毒宿主数据库与宿主分析,完成15个潜在的噬菌体宿主的比对,包括嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia)、湖红胞菌(Rhodoferax lacus)、海洋拟变形菌(Pseudoalteromonas marina)、Thalassotalea loyana、海藻糖弧菌(Vibrio alginolyticus)、塔斯马尼亚弧菌(V.tasmaniensis)、溶血弧菌(V.vulnificus)、假单胞菌(Pseudomonas sp.)、农杆菌(Agrobacterium sp.ST15.13/040)、鸡肠球菌(Enterococcus gallinarum)、黄杆菌属(Flavobacterium sp.)、嗜油热袍菌(Thermotoga naphthophila)、赤杆菌属RU33C(Chryseobacterium sp.RU33C)、酸鱼乳杆菌(L.acidipiscis)和粘液奈瑟菌(Neisseria mucosa)。结论对健康人血液微生物宏基因组和噬菌体组的挖掘与分析研究,可揭示血液中的微生物和噬菌体的存在,可能对人类健康有深远的影响。

肠道噬菌体组生物信息学分析方法的研究进展

噬菌体是感染细菌的病毒,广泛存在于各类环境中。由于传统实验研究的局限性及噬菌体基因的特异性,导致对肠道噬菌体的研究很少。随着宏基因组测序技术的发展和各种生物信息分析软件的开发,可以通过噬菌体组学,加深对肠道噬菌体的认识。噬菌体组分析流程主要包括原始数据质量控制和预处理,病毒基因组序列的拼接组装,类病毒颗粒的筛选和系统分类注释以及进化分析和预测相应宿主细菌。本文对噬菌体组分析流程和其中所需要的常用生物信息分析工具和数据库进行详细的介绍,可以为肠道噬菌体研究以及相关的研究人员提供参考。

肠道噬菌体组与人体健康

噬菌体作为肠道微生物群落的重要组成部分,自生命初期即定植于肠道,在不同生命时期均以较为稳定的状态存在于人体。目前已知的肠道噬菌体主要是DNA噬菌体,且大多以前噬菌体形式存在。肠道噬菌体可通过多种机制优化其宿主微生物群的结构和组成,并以多种方式直接或间接影响人体的生理状态,其结构变化与多种疾病相关。利用测序方法,能够直接获取噬菌体组信息。本文基于噬菌体组的相关研究,综述了肠道噬菌体组与人体健康相关的研究进展,并对该领域的研究方法和发展方向做出展望。

噬菌体培养与鉴定方法研究进展

噬菌体是地球上数量最多的生命体,不仅广泛存在于自然界,也大量存在于人体之中。但是,目前分离、鉴定的噬菌体数量却仅有数万个。虽然宏基因组技术的出现为探究噬菌体提供了高通量的方法,但是宏基因组测序结果中,大量噬菌体序列功能未知,也不知道其来源于何宿主菌,是一个亟须研究的“黑匣子”。微生物的分离培养是研究微生物的起点,噬菌体的研究亦是如此。本文首先对噬菌体分离、培养与鉴定研究进行总结,然后对裂解性和溶原性噬菌体分离方法进行归纳和分析,为分离新的噬菌体提供较为全面的方法学综述。

Immunotherapy of EGFR-positive Tumor Based on Recombinant EGFR Phage Vaccine

Objective： To construct the recombined phage which was inserted with the gene of extracellular domain of chicken EGFR as a new type vaccine and to evaluate the efficiency of the phage vaccine against the EGFR positive tumor. Methods： The T7 phage display system was used to display 5 fragments of the extracellular domain of chicken EGFR. The EGFR was expressed as a fused protein on the surface of the T7 phage 10B capsid protein. The EGFR expression of the phage vaccine was verified by the Western blot analysis. The anti-EGFR antibody was detected by ELISA. The splenic lymphocytes of the immunized mice were separated and used to determine the cellular immunotoxic effect against A431 cells. The phage vaccines were injected into the C57 mice 4 times before lewis lung cancer cells were inoculated. The tumor volume was recorded to evaluate the anti-tumor effect of each vaccine. Results： Five phage vaccines inserted with the chicken EGFR gene were constructed successfully. Western blot assay showed that the extracellular domain of chicken EGFR proteins was displayed on the surface of the phage. The specific antibody was induced in the immunized mice compared with the control group. The splenic lymphocytes of the immunized mice were shown to be immunotoxic against A431 cells. The killing rates of the experimental groups were higher than in the control group （P〈0.001, by t test）. The highest killing rate was （45.74±7.21）%. The tumor growth was inhibited in the experimental groups as compared with the control group （P〈0.05 in C1, C2, C3 and C4 groups, P〉0.05 in C5 group）. Conclusion： The phage vaccine could induce both the protective and therapeutic antitumor immunity against EGFR-positive tumor.

浅谈DNA发现之路

从选择研究材料步步逼近生命遗传物质的本质DNA分子。伴随研究重心在西欧和北美来回变迁,吸引来众多不同学科的代表人物。围绕DNA分子的探索和模型的建立,显现各路研究者不同反响的研究格调和学养,或许我们能从他们身上吸取到某些有益的东西。

Construction and quality identification of T7 recombination expression cDNA library form human lung cancer

Objective:Currently,only a limited numbers of tumor markers for non small lung cancer(NSCLC) diagnosis,new biomarker,such as serum autoantibodies may improve the early detection of lung cancer.Our objective is construction human lung squamous carcinoma and adenocarcinoma T7 phage display cDNA library from the tissues of NSCLC patients.Methods:mRNA was isolated from a pool of total RNA extract from NSCLC tissues obtained from 5 adenocarcinomas and 5 squamous carcinomas,and then mRNA was reverse transcribed into double stranded cDNA.After digestion,the cDNA was inserted into T7Select 10-3 vector.The phage display cDNA library was constructed by package reaction in vitro and plate proliferation.Plaque assay and PCR were used to evaluate the library.Results:Two T7 phage display cDNA library were established.Plaque assay show the titer of lung squamas carcinoma library was 1.8 × 106 pfu,and the adenocarcinoma library was 5 × 106 pfu.The phage titer of the amplified library were 3.2 × 1010 pfu/mL and 2.5 × 1010 pfu/mL.PCR amplifica-tion of random plaque show insert ratio were 100%(24/24) in adenocarcinoma library and 95.8% in human lung squamas carcinoma library(23/24).Insert range from 300 bp to 1 500 bp.Conclusion:Two phage display cDNA library from NSCLC were constructed.

肠道噬菌体作为健康与疾病的标志物的研究进展

人类的肠道内寄居着数量庞大、种类繁多的噬菌体,统称为肠道噬菌体组。肠道正常噬菌体组在维持肠道微生物群落的丰度和构成方面具有重要作用。个体肠道噬菌体组的丰度和多样性主要取决于年龄和健康状况,因此肠道噬菌体可以作为人类健康与疾病的生物标志物,是一种潜在的针对肠道菌群的干预手段。本文旨在总结健康人和病人肠道噬菌体组的组成及进化,并综述肠道噬菌体作为健康与疾病的标志物的研究进展。