遗传工程微生物细胞间发生的自然遗传转化

将两株具有不同遗传标记的枯草芽孢杆菌在基本培养基中分别培养至对数生长后期后进行短时间混合静置培养,经选择平板筛选、DNaseI敏感性试验、质粒检测和产蛋白酶活性检测,发现两菌株之间可通过自然遗传转化进行染色体DNA和质粒DNA的交换。研究结果表明,自然遗传转化可在细胞间进行,这对揭示微生物群居的自然环境中可能存在的细胞间的DNA转移,以及正确评估遗传工程微生物(GEMs)的安全使用具有重要意义。 Abstract：The culture fluids of two genetically distinct Bacillus subtilis strains were mixed and coincubated for a short time after they reached post-exponentially growth phase in minimal media.The steadily bidirectional gene transfer involving chromosomal DNA and plasmid DNA by natural genetic transformation between these two strains has been demonstrated by the methods of selective medium screening,DNaseI sensitivity test,plasmid detection and the detection of the capability of producing protease.This result indicates that natural genetic transformation occurs not only between“naked”DNA and cells but also between cells.This conclusion is significant in the assessment of both the possibility of intercelluar DNA transfer in natural habitats of microorganisms and the risk of the application of genetically engineered microorganisms (GEMs).     

细菌自然转化的分子机制研究进展

自然环境中,具备自然转化能力的细菌可以自发地从外界获取DNA,从而获得新的遗传性状。为能够自然地被转化,细菌需首先建立一个被称作感受态的生理状态并在此状态下表达DNA摄取和加工相关的基因。DNA摄取基因的表达产物可组装一个能将外源DNA摄入细胞质的蛋白复合物。在细胞质中,进入的DNA可同基因组DNA发生同源重组或建立成一个独立的质粒。一般DNA摄入细胞的过程可分为两个阶段,即从外部基质到细胞周质和跨细胞内膜的转运。近年来,包括作者在内的研究人员发现大肠杆菌中存在新的自然质粒转化模式。本文将首先综述近年来细菌自然转化的分子机制,随后简要介绍大肠杆菌中独特的自然质粒转化模式。     

微生物及其质粒在环境中的传播

抗菌素抗性基因可作为阐明环境微生物间遗传物质自然传播的一种模型。这些基因因为易于识别,并且在人、畜和农业病害的防治上具有医学意义而引人注目。分子生物学与流行病学数据已揭示,这些基因在自然界中惊人地以多种形式发生交流。微生物间的遗传交换细菌可通过质粒和转座子进行部分DNA交换。质粒本身即使不能在新的寄主中存活,也能将它所编码的一个或多个基因引入该寄主。这些基因可由转座子携带,从进入的质粒跳跃到新寄主的染色体上或者该寄主本身具有的质粒上。这样,该固有质粒或者染色体就获得     

用凝胶电泳纯化具有生物学活性的质粒DNA

采用一般柱电泳装置,在琼脂糖凝胶上将质粒ColEl、pBR322、pSC101、pCRI的DNA与染色体DNA及小分子核酸杂质分开,切出含有质粒的凝胶薄片,电洗脱回收质粒DNA,产物可被限制酶Eco RI酶解;将pBR 322、PSC 101、pCRI DNA转化大肠杆菌C_(600),每微克DNA可产生10~4个转化子;从pSC 101、pCRI转化子细胞中再抽提出相应质粒,它们同样具有亲本质粒的遗传特性和分子特性。     

不同培养时期的大肠杆菌和枯草芽孢杆菌间发生的跨属自然遗传转化

携带穿梭质粒的大肠杆菌与作为受体的枯草芽孢杆菌分别培养至不同生长阶段混合均匀后静置40min,涂布选择性平板,37℃培养30h后得到一定数目的转化子,DNaseⅠ敏感实验证实质粒是通过自然遗传转化而非其它形式发生转移。实验发现大肠杆菌可以在特定生长时期向胞外分泌DNA,并且在对数期具有最高的提供质粒的能力,而生长后期的细胞因为体系中DNase量的增加转化频率下降。进一步的研究发现枯草芽孢杆菌在营养丰富的LB培养基中也具有与基本培养基中相当的转化能力,并且在对数生长前期具有较高的转化频率。     

柯萨奇病毒B3 cDNA生物素探针的制备研究

本文将柯萨奇B组3型病毒(CVB3)cDNA的重组质粒DNA(pGP51B)转化到E.coli HB101菌株中.筛选转化阳性菌株,经培养扩增后,提取重组质粒DNA,用缺口求移法制备生物素标记探针,通过原位杂交技术检测CVB1.CVB3感染的Hela细胞及正常Hela细胞对照.结果该探针只与CVB杂交,而不与细胞对照杂交,且可检测出病毒感染5h尚未出现病变细胞中的病毒核酸.表明该探针具有良好的特异性和敏感性.     

转化条件对质粒DNA转化大肠杆菌的影响

研究了质粒DNA大小、质粒DNA浓度、CaCl2 浓度、热休克时间及感受态细胞保藏时间等因素对大肠杆菌HB1 0 1和JM1 0 5转化频率的影响 ,并对转化子中质粒DNA进行了分离、酶切、琼脂糖凝胶电泳检测。结果表明 ,CaCl2 浓度、质粒大小和浓度 ,以及感受态细胞的活力对转化频率有重要影响 ,42℃热休克处理可以提高转化频率。     

大质粒DNA疫苗pSVK-CAVA高稳定性宿主菌的筛选与鉴定

目的:从多种大肠杆菌感受态细胞中筛选出适合该研究大分子质粒DNA疫苗的宿主菌,鉴定其达到中试要求。方法:将疫苗质粒pSVK-CAVA(14.7kb)转化4种大肠杆菌化学感受态细胞并提取质粒,通过琼脂糖凝胶电泳实验检测质粒的形态结构。对基因工程菌进行生化检测,并通过连续传代法和酶切鉴定进行稳定性检测,同时将质粒DNA瞬时转染至293T细胞中检测质粒表达能力。选取质粒含量最高和稳定性最好的宿主菌作为原始种子分装冻存,将原始种子库扩大培养,逐级建立好主种子库和工作种子库即三级种子库。通过摇瓶培养实验在4种常用基础培养基中挑选出最适合质粒生产的培养基。结果:确定了XL-10 Gold作为质粒DNA疫苗pSVK-CAVA的宿主菌,基因工程菌传代稳定性和结构稳定性良好,质粒能在293T细胞中体外表达。筛选出TB培养基为基础培养基,质粒容积产量达到9.9mg/L,比LB培养基提高了接近1倍。结论:该研究筛选出大肠杆菌XL-10 Gold作为质粒DNA疫苗的宿主菌,解决了大质粒在常用宿主菌中不稳定的难题,并对基础培养基进行了初步优化。     

质粒pBR322在recA基因缺陷的大肠杆菌细胞内的遗传重组

使用琼脂糖凝胶电泳、DNA限制性内切酶水解以及电子显微镜等分析手段,证明在两株recA~-的大肠杆菌质粒pBR322转化子中,pBR 322 DNA的多倍周长环形寡聚物被大量合成。这个事实说明质粒pBR322 DNA在大肠杆菌细胞中的遗传重组似有独立于rec A基因的途径。本文介绍一个改进的大肠杆菌“清亮裂解液”制备法。按照这个方法制备的细菌清亮裂解液可排除染色体DNA的污染。     

超声波介导的微生物细胞转化

随着分子生物学的发展, 微生物遗传改造越来越广泛地应用在微生物育种、临床医学、环境保护等方面。其中, DNA转化技术经常是高效遗传改造的瓶颈之一。应用超声波将目的基因导入微生物细胞的技术具有原位、多尺度、活体、高通量、低成本等优点, 因此发展较为迅速。其原理是超声波可以通过声学气穴现象产生一系列的非热能效应, 而声学气穴微泡可产生短暂的细胞膜透化作用。本文综述了超声波转化的基本原理及其在微生物细胞转化中的发展现状, 并结合本实验室应用超声波转化法转化革兰氏阳性菌等研究进展, 分析了其特色、优势及现存挑战。     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

鸡传染性法氏囊病病毒研究进展

传染性法氏囊病(Infection bursal disease, IBD)是由鸡传染性法氏囊病毒(Infectious bursal disease virus, IBDV)引起的鸡和火鸡的一种高度接触性传染病,给世界各国的禽养殖业带来了巨大损失.自IBDV发现至今新的变异株不断出现,分子结构的改变导致病毒致病力的改变及宿主对疫苗应答的改变,使得传统的疫苗已不能控制其流行,因此各国学者对其基因组结构和功能进行了广泛深入的研究,并积极研制新型有效的疫苗以达到防治的目的.     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.