中国春小麦puroindoline a基因的克隆及其真核表达载体的构建

目的:puroindoline(pin)基因在控制麦类作物的籽粒硬度中起着重要作用。构建真核表达载体pcDNA3.1( )-pina-gfp,为pina基因在哺乳细胞中的表达提供基础。方法:利用PCR方法从中国春小麦基因组中克隆到了pina基因,将其插入真核表达载体pcDNA3.1( )-gfp,用PCR和酶切鉴定重组子。结果:PCR和酶切鉴定表明,所构建的真核表达重组质粒为pcDNA3.1( )-pina-gfp;将该片段克隆到pCF-T载体中,经测序验证,表明其为目的基因。结论:构建的pina基因真核表达载体pcDNA3.1( )-pina-gfp为pina基因在哺乳动物细胞中的表达提供了基础。     

pcDNA3.1(-)-Bcr-Abl及pcDNA 3.1(-)-Bcr-Abl T3151突变质粒真核表达载体的构建

目的:将Bcr-Abl及Bcr-Abl T3151突变克隆入pcDNA3.1(-)真核表达载体,为研究靶向降解受体型酪氨酸激酶BCR-Abl,抑制肿瘤细胞生长提供研究基础.方法:pcDNA3.1 (-)-Bcr-Abl质粒构建:分别设计引物,通过分段PCR将BCR-ABL克隆入pcDNA3.1(-).首先通过PCR扩增出Bcr-a片段,将其克隆入pcDNA3.1(-)的NheⅠ/XhoⅠ之间;接着将PCR扩增出的Abl-c片段克隆入KpnⅠ/ HindⅢ之间,最后XhoⅠ/KpnⅠ双酶切pGD210,将酶切下片段插入pcDNA3.1(-)的相应位点即可.酶切鉴定及测序正确后,转染293T细胞,Western blot验证质粒的表达.pcDNA 3.1(-)-Bcr-Abl T3151的突变质粒:首先设计引物,第一步以pcDNA3.1(-)-BCR/ABL为模板,以Abl-c-u和ba-M1为引物扩增出A-1:560 bp.第二步,相同模板,以ba-M2和ba-M-down为引物扩增出A-2:870 bp.第三步,以扩增出的A-1和A-2为模板,以Abl-c-u和ba-M-down为引物,扩增出1434 bp的片段A-l+2,以Bcl和Kpn Ⅰ分别酶切pcDNA3.1 (-)-BCR/ABL以及A-1+2,将突变后的A-l+2置换入pcDNA3.1 (-)-BCR/ABL.结果:PCR结果显示3.1(-)-Bcr-Abl及3.1 (-)-Bcr-Abl T3151突变质粒条带大小符合,重组质粒经酶切鉴定和测序结果正确,转染后可见融合蛋白的表达.结论:成功构建pcDNA3.1 (-)-Bcr-Abl及pcDNA 3.1(-)-Bcr-Abl T3151的真核表达载体,并且转染293T细胞后证实其能够正确表达,为后续研究奠定了基础.     

表达血管内皮生长因子-siRNA及双自杀基因yCDglyTK的联合基因载体构建及其应用研究

构建了新型联合基因载体pcDNA3.1(-)VEGF-siRNA/yCDglyTK,研究其在人胃癌细胞系SGC7901细胞中的表达和杀伤作用.构建靶向血管内皮生长因子(VEGF)的干扰质粒pGenesil-VEGF-siRNA,采用PCR法从中扩增siRNA表达框(含U6启动子),亚克隆至双自杀基因载体pcDNA3.1(-)CV-yCDglyTK,构建联合基因质粒pcDNA3.1(-)VEGF-siRNA/yCDglyTK;通过酶切、测序等鉴定重组质粒;以磷酸钙纳米颗粒为载体,将干扰质粒、双自杀基因质粒及联合基因质粒转染SGC7901细胞,RT-PCR、Western-blot验证目的基因表达;MTT法检测转染细胞对5-氟胞嘧啶(5-FC)的敏感性.结果表明:酶切及测序证实联合基因载体pcDNA3.1(-)VEGF-siRNA/yCDglyTK构建成功;SGC7901细胞转染联合基因质粒后,RT-PCR、Western-blot证实融合自杀基因表达,而VEGF基因表达下调;在前体药物5-FC作用下,转染联合基因组细胞存活率最低,与其他组比较有统计学差异.成功构建联合基因载体pcDNA3.1(-)VEGF-si...     

pcDNA3.1/CXCR7真核细胞表达载体的构建及其在内皮细胞中的表达鉴定

目的:CXCR7是基质衍生因子1(stroma derived factor-1,SDF-1)的新受体,且该受体在血管新生部位的内皮细胞中表达上调,故本研究拟构建CXCR7的真核表达载体pcDNA3.1/CXCR7,并检测其在人脐静脉内皮细胞中的表达.方法:采用RT-PCR法从人肝癌细胞HepG2的cDNA中扩增出约1100 bp的CXCR7基因片段.采用KpnI、XbaI将目的基因和载体pcDNA3.1进行双酶切,将酶切产物加入T4 DNA连接酶16℃连接过夜.将连接产物转化到感受态大肠杆菌中.挑取阳性克隆、提质粒,用双酶切、质粒DNA PCR扩增及DNA序列分析鉴定正确后,采用阳离子脂质体LipofectamineTM 2000将其转染人脐静脉内皮细胞(HUwc),通过western-blot检测目的基因在内皮细胞中的表达.结果:阳性克隆经双酶切法鉴定含有CXCR7基因片段,质粒DNAPCR扩增出与CXCR7同等大小的基因片段,基因测序结果与GenBank中序列相同.转染HUVEC后,细胞中CXCR7的表达水平显著上升.结论:成功构建了CXCR7的真核表达栽体,可在内皮细胞中正常表达并.为进一步研究其作用机制奠定了基础.     

Cpn0308基因真核载体构建及鉴定

目的:构建Cpn0308基因真核表达重组质粒,为肺炎衣原体(Chlamydia pneumoniae,Cpn)核酸疫苗的研制做准备。方法:用PCR技术从Cpn AR39株基因组DNA中扩增Cpn 0308基因,经双酶切、连接等反应,重组入pcDNA3.1/HisA真核表达载体,转化到感受态细胞,再经含氨苄青霉素的LB培养基筛选,酶切、PCR扩增及测序鉴定。结果:从Cpn AR39株基因组DNA中扩增出特异的Cpn 0308基因,约400bp;酶切、重组、转化、筛选鉴定出pcDNA3.1/HisA-Cpn0308重组质粒;序列测定证实与GenBank登录的肺炎衣原体Cpn AR39株Cpn0308基因一致。结论:功地构建了pcDNA3.1/HisA-Cpn0308重组质粒,为肺炎衣原体核酸疫苗的研制奠定了基础。     

SUSD2基因真核表达载体构建及其在H322细胞中表达

[目的]构建SUSD2基因真核表达载体,并观察其在真核细胞H322细胞中的表达。[方法]从H322细胞中提取总RNA并逆转录为c DNA,采用PCR技术扩增出含有EcoRⅠ和HindⅢ酶切位点的人SUSD2基因片段,以质粒pc DNA3.1为表达载体,构建重组质粒pc DNA3.1-SUSD2。采用PCR、双酶切鉴定以及测序验证c DNA片段大小和序列正确。将重组载体pc DNA3.1-SUSD2转染H322细胞,Western Blot法检测SUSD2蛋白的表达。[结果]PCR、双酶切鉴定以及测序结果显示pc DNA3.1-SUSD2包含大小、序列正确的SUSD2片段;Western Blot结果显示SUSD2蛋白在转染pc DNA3.1-SUSD2的H322细胞中表达高于转染pc DNA3.1的H322细胞。[结论]成功获得SUSD2基因全长序列,并成功构建SUSD2基因真核表达载体,有效地在非小细胞肺癌细胞株H322中表达,为进一步研究该基因功能及机制奠定了基础。     

小鼠EVL 基因的克隆和真核表达载体的构建

目的:构建小鼠EVL(Ena/VASP like)基因的真核表达载体,为深入研究EVL的功能奠定基础.方法:采用PCR方法,从小鼠cDNA文库中,扩增出1245bp的EVL编码区片段,经电泳、胶回收后连接入pMD- 18T载体中,测序鉴定正确.用BamHI和HincⅡ双酶切,定向克隆EVL编码区片段到真核表达载体pcDNA3.1中,用限制性内切酶酶切鉴定重组质粒正确后.将重组质粒转染入HELA细胞中,以RT-PCR检测EVL的mRNA的表达,以Western Blot检测EVL蛋白的表达.结果:酶切鉴定结果显示小鼠EVL编码区基因被成功克隆入真核表达载体pcDNA3.1中;RT-PCR和Western Blot结果以及免疫荧光染色显示Hela细胞中有EVL的mRNA和蛋白的表达.结论:成功获得pcDNA3.1 -EVL的真核表达载体,为进一步深入研究EVL蛋白的功能奠定了基础.     

甲型流感病毒M1基因真核表达载体的构建及其表达

研究旨在构建含有甲型流感病毒M1基因的真核表达载体,并探讨其在真核细胞中的表达.提取流感病毒Influenza A/FM/1/47 (H1N1) RNA,RT-PCR扩增M1基因并将目的基因插入真核表达载体pcDNA3.1(+).经酶切及PCR鉴定后用PolyFect脂质体将其转染到Vero细胞,免疫荧光技术鉴定其表达.甲型流感病毒M1蛋白重组质粒pcDNA3.1-M1的成功构建,为开发研制流感病毒保守蛋白DNA疫苗奠定了基础.     

人snail真核表达载体构建与鉴定

目的:构建人snail基因真核表达载体并鉴定。方法:使用RT-PCR法获取人snail基因全长c DNA,经Bam H I、Eco R I双酶切、连接,插入pc DNA3.1(+)真核表达载体,转化TOP10感受态细胞,用含氨苄青霉素的LB培养基筛选阳性克隆,提取质粒双酶切电泳及测序鉴定,瞬时转染siha细胞Western-blot从蛋白水平鉴定重组质粒在真核细胞内的表达。结果:pc DNA3.1-snail重组质粒经酶切电泳符合预期片段,测序鉴定插入片段与NCBI Gen Bank文库中人snail序列一致,重组质粒瞬时转染后snail蛋白表达量明显增高。结论:成功构建pc DNA3.1-snail重组质粒载体,为进一步探讨snail基因生物学功能奠定了基础。     

甲状腺转录因子-1融合表达载体的构建及体外活性鉴定

目的:构建人甲状腺转录因子-1(TTF1)编码基因的真核表达载体pcDNA3.1/myc-His(-)C-TTF1,并观察其蛋白质体外转录翻译情况。方法:据已知的TTF1基因序列,应用巢式PCR技术,从人肺腺癌细胞株SPC-A1中扩增出TTF1基因,通过TA连接将其克隆入pGEM-T-easy载体,经测序鉴定后,双酶切插入真核表达质粒pcDNA3.1/myc-His(-)C中;重组质粒经XhoⅠ和BamHⅠ双酶切鉴定后,进行蛋白质体外翻译观察TTF1体外表达情况,用电泳迁移率变动分析(EMSA)验证获得的体外翻译蛋白TTF1是否具有与下游靶基因UGRP1启动子结合的能力。结果:成功构建了含TTF1编码基因的真核表达载体pcDNA3.1/myc-His(-)C-TTF1,并能在体外表达TTF1蛋白。结论:能方便地获得TTF1体外翻译蛋白,为进一步研究TTF1蛋白与相应的DNA反应元件及其他转录因子的相互作用奠定了基础。     

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.