pNK289衍生质粒在芽孢杆菌中的分离稳定性*

报道关于一系列ｐＮＫ２８９衍生质粒分离稳定性研究结果。这些起源相同的质粒在Ｂａｃｉ－ｌｌｕｓ．ｓｕｂｔｉｌｉｓＡＳＩ．１１７６中的分离稳定性存在差异，这种差异与质粒的大小和复制方式无关，而与质粒的拷贝数有一定的关系。由于不稳定质粒ｐＮＫ２１９在Ｂ．Ｓｕｂｔｉｌｉｓ ＢＤ２２４宿主中能稳定遗传，所以推测宿主的遗传背景可能影响质粒的分离稳定性。这些研究不仅为进一步寻找与ＰＮＫ２８９衍生质粒稳定性相关的基因奠定了基础，而且为在芽孢杆中构建稳定的重组质粒提供了理论依据。     

芽孢杆菌基因工程新载体的构建

利用从Bacillus pumilus 289中分离出的隐秘质粒pNK289(7.2kb)的复制起始调控区及启动区DNA片段和质粒pPL 601上的氯霉素乙酰基转移酶结构基因(cat-86),构建了能在B.subtilis和B. pumilus中稳定传代的质粒pNQ216(4.1kb)和pNQ402(2.8kb)。在非诱导条件下,其在含Cm的LB平皿上的外显率都比pPL600高约30％,可以用作芽孢杆菌基因克隆的质粒载体。     

从Staphylococcus aureus 307株分离的三个质粒在Bacillus subtilis Ki-2和168株中的表达和特性

Staphylococcus aureus 307株是临床分离的一多重耐药株,其中包括抗氯霉素(Cm~R),抗卡那霉素(Km~R)和抗四环素(Tc~R)。实验结果表明,这三种抗药性是分别为三个质粒所决定的。我们把它们分别称为pC307、pK307和pT307。通过转化把三个质粒引入Bacillus subtilis Ki-2和168株,获得Cm~R Km~R Tc~R转化体。本文研究了这些质粒所携带的抗药基因在Ki-2和168株中的表达,以及质粒在新宿主中的相容性和稳定性。     

短小芽孢杆菌作为芽孢杆菌属基因工程受体菌的研究

以质粒pUB110 DNA转化B. pumilus 289原生质体,转化频率为10~(-3)—10~(-9)与B.tubtilis 168系统相当;但B.pumilus 289原生质体的再生频率(0.3—12.0％)略低于B.subtilis 168(1.53—24.16％);在无选择压力条件下质粒pUB110在B.pumilus 289中经过45个世代周期,自发丢失率小于3％,同于B.subtilis 168系统。外源基因在B.pumilus 289中经25个世代周期丢失率低于5％,而在B.subtilis 168系统中则高达24％;外源基因的表达水平亦高于B.subtilis 168系统。因此,B.pumilus 289是一个值得进一步开发的基因工程受体系统。     

产腈水合酶重组大肠杆菌的质粒稳定性研究

成功构建了腈水合酶(nitrile hydratase,NHase)高表达的重组大肠杆菌E.coliBL21(DE3)/pETNH^M(Kan^r),研究了重组质粒pETNH^M在重组菌株中的质粒稳定性。结果表明,pETNH^M具有较好的结构稳定性,连续传代60代后质粒的基因序列没有明显缺失,且能够正常表达腈水合酶。pETNH^M具有分离不稳定性,在无抗生素选择压力下,连续传代48代后质粒丢失的无质粒细胞开始出现。琼脂糖凝胶电泳定量分析表明,2/3的质粒pETNH^M以二聚体形式存在,导致质粒拷贝数的下降。进一步研究表明,重组细胞的连续高速分裂及腈水合酶的高表达也会造成质粒拷贝数的下降,从而降低其分离稳定性。反之,重组菌株相对于宿主菌株的较高比生长速率有利于保持含质粒细胞的生长优势,卡那霉素的选择压力则能够保证质粒的稳定遗传。     

外源质粒在枯草芽孢杆菌BF7658中的稳定性及其基因表达

通过B.subtilis噬菌体PBSI转导,已将携带热稳定α-淀粉酶基因的质粒pAmy411引进了B.subtilis BF7658.转导频率为10_(-9)转导子/PFU。尽管pAmy 411的诲贝数在B.subtilis BF7658中较在B.subtilis AS 1.1176中高1倍,但其传代稳定性却较后者低。质粒携带的热稳定α-淀粉酶基因的表达水平在B.subtilis BF 7658中较在B.subtilisAS1.1176中高6倍。     

PUC衍生质粒的一个低拷贝数突变型

本文报道大肠杆菌的ColE1类似质粒的一个低拷贝数突变型。从载体质粒pUC4衍生的重组质粒pPGVT3在大肠杆菌宿主DF2145中是不稳定的,以pPGVT3转化DF2145时在4o℃培养得不到转化子。用诱发点突变的羟胺体外处理pPGVF3质粒DNA,得到一个稳定性提高了的突变质粒pPGVT3HA,突变的位置被确定在质粒的pUC部分,突变降低了pUC及其衍生质粒的拷贝数。文中对质粒的稳定性与拷贝数的关系作了讨论。     

表达NM23-H1/NDPK-A工程菌的遗传稳定性研究

目的:研究重组工程菌的遗传稳定性。方法:利用重组表达质粒pBVNM-H1转化宿主菌E.coli DH5α,筛选重组工程菌DH5α-pBVNM-H1。将新构建好的重组工程菌在无选择压力的条件下进行连续传代培养,比较菌落在LB(-)和LB(+)培养基上的生长状况,并对传代菌株目标蛋白的表达情况以及质粒数量和目的基因DNA进行电泳鉴定。结果:重组工程菌连续传代50次中,在LB(-)和LB(+)培养基上的生长状况相同,目标蛋白表达量无显著差异,质粒数量及目的基因DNA结构稳定。结论:重组工程菌DH5α-pBVNM-H1具有良好的遗传稳定性。     

热稳定α—淀粉酶稳定性工程菌的构建

本文以质粒pE194为载体亚克隆B.licheniformis热稳定α-淀粉酶基因,构建成重组质粒pNW102,通过噬菌体PBS1将它转导进入中温α-淀粉酶生产菌B.subtilis BF7658。B.subtilis BF7658(pNW102)经过长时间非许可温度处理,筛选得到2株热稳定α-淀粉酶稳定性表达的工程菌株。酶学分析显示同源重组具有热点,2株重组菌株B.subtilis BFNW产生的热稳定α-淀粉酶符合B.licheniformis产生的淀粉酶特性。     

海参溶菌酶基因在枯草芽孢杆菌WB600中的整合及表达

采用枯草芽孢杆菌WB600(Bacillus subtilis WB600)在体外表达海刺参溶菌酶(Stichopus japonicus lysozyme,SjLys)蛋白。通过构建克隆质粒pMD18-P43-SjLys,将B.subtilis自身强启动子P43序列和已分离得到的SjLys基因(GenBank登录号EF036468)连接(P43-SjLys)。经BamH I/EcoR I酶切,将P43-SjLys片段连接到B.subtilis整合载体pDG1730上,构建整合重组质粒pDG-P43-SjLys。经Xho I酶切处理,线性化的pDG-P43-SjLys质粒转化B.subtilis WB600细胞。P43-SjLys片段通过同源双交换重组整合到B.subtilis WB600染色体上,成功得到具有稳定遗传的基因工程菌B.subtilis WB600/P43-SjLys。经SDS-PAGE和抑菌试验分析表明,培养60 h后,B.subtilis WB600/P43-SjLys能够表达可溶的,对常见的海洋细菌溶壁微球菌和金黄色葡萄球菌具有较强的抑菌活性的SjLys蛋白。首次在B.subtilis表达系统中得到可溶且具有酶活功能的SjLys蛋白,为SjLys的生产提出了一种具有可行性的和潜力的新方法。     

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     

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.     

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.     

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.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

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).     

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.