通过DNA改组技术获得高活性β-葡萄糖苷酸酶

β 葡萄糖苷酸酶是在植物转基因中广泛应用的报告基因 .以质粒pBI12 1中的GUS基因为基础 ,利用DNA改组方法 ,经DNaseⅠ降解 ,PrimerlessPCR ,PrimerPCR对GUS基因进行了突变和改组 ,然后将改组的GUS基因连接到原核表达载体pG2 5 1中 ,构建了库容为 10 8的突变体库 .经过活性的筛选 ,得到活性提高的克隆 ,再以此为基础 ,经过新的改组、筛选得到活性大幅度提高的克隆GUS2 4 .基因测序显示 ,GUS2 4与GUS基因之间的同源性为 99 7% ,共有 6个核苷酸位点发生了改变 ,分别是 :379位的A突变为G ,396位的T突变为C ,711位的G突变为A ,95 8位T突变为C ,990位的T突变为C ,1649位的A突变为G .核苷酸序列推导的氨基酸序列显示 ,3个氨基酸发生了突变 ,12 7位的Ser突变为Gly ,32 0位的Trp突变为Arg ,5 5 0位的Asn突变为Ser.X gluc染色检测和荧光测活结果显示GUS2 4基因表达的 β 葡萄糖苷酸酶基较GUS基因表达产物活性提高 3倍     

从人血液中克隆过氧化氢酶基因

目的从人血液中克隆过氧化氢酶基因。方法以新鲜的人血液为材料,提取全血RNA,运用RT—PCR方法扩增过氧化氢酶基因,构建pMDl8T／CAT克隆载体,并进行了不同来源过氧化氢酶的氨基酸序列同源分析。结果从人血液中成功扩增出过氧化氢酶基因,获得了重组载体PMD-18T／CAT,氨基酸序列分析的同源性达80％以上。结论从人血液中可以很方便地克隆出过氧化氢酶基因。     

7-木糖紫杉烷糖基水解酶Lxyl—pl-2基因突变库与高通量筛选体系的构建

对7－木糖紫杉烷糖基水解酶Lxyl－p1－2基因进行定向进化的初步研究，确定易错PCR条件、优化克隆连接方法、建立基于48微孔板高通量筛选模型。利用易错PCR技术对Lxyl－pl－2基因进行随机突变，比较不同浓度M聋’进行易错PCR，每组随机挑取10个克隆进行测序。序列分析表明：在M聋’浓度为7mmol／L条件下，碱基平均突变率为0．12％，即对于Lxyl－p1－2基因（2．4kb）来说，每个基因序列平均有3个碱基突变，达到构建突变文库的频率要求，选择该条件作为易错PCR条件。利用in-fusion技术将Lxyl－p1－2突变基因克隆到pPIC3．5K载体中，获得大量重组突变质粒。该研究优化了in-fusion连接反应中基因片段和载体片段的摩尔比，并探讨了基因片段与载体片段问同源序列长度对in—fusion连接效率的影响。试验结果表明：基因片段与载体片段的摩尔比最佳梯度为5：1；同源序列长度的选择成为影响in—fusion连接效率的关键因素之一。当同源序列长度为100bp时连接效率达到最大值，且显著高于同源序列长度为15～50bp的连接效率，此时阳性重组率提高至90％左右。与常规酶切一连接法相比，in-fusion法具有明显优势，同时将克隆周期由3～4d缩短为1～2d。挑取单菌落，在48微孔培养板上进行培养、诱导表达2d后，取菌液进行酶活测定（底物PNP-Xyl）。以野生型为例，β-木糖苷酶的酶活013405均数μ=3．415，其数据组标准差为δ=±0．078，数值符合正态分布的原理，建立统计学野生型均数分布范围和48微孔板高通量筛选，为后续突变体筛选提供基础。     

2,5-DKG还原酶Ⅱ基因的克隆和在大肠杆菌中的表达

参照文献上的2,5-二酮基-D-葡萄糖酸（简称2,5-DKG）还原酶II基因序列,合成两个引物序列并在两端加上EcoRI和BamHI两个酶切位点,抽提棒状杆菌SCB3058菌株的染色体为模板进行PCR反应,克隆得到2,5-DKG还原酶II基因,酶切验证与预期的结果相符合。将此片段克隆到pGEM-T载体上保存．将2,5-DKG还原酶II基因用EcoRI和BamHI内切酶切下,连接到pBV220载体上,构建成表达载体。42℃诱导不能得到稳定的蛋白表达条带和酶活力,测序发现基因的3’末端的原PCR引物外少合了一个碱基,终止密码子发生移码突变而消失。此外在5’端的启始密码子ATG前有三个碱基与pBV220载体上的SD序列发生配对。据此,重新设计和合成了PCR引物,并用pBV220和pBL4载体构建了两个表达载体。42℃诱导表达均得到了稳定的表达条带和较高的酶活力．     

小鼠白细胞介素-33基因的克隆及序列分析

目的:克隆小鼠IL-33基因全长编码区cDNA,并对其进行序列分析。方法:从BALB/c小鼠的脊髓组织中提取总RNA,逆转录为cDNA,用热启动PCR技术,扩增小鼠IL-33基因全长编码区cDNA,经双酶切后,克隆入pcDNA3.1( )载体中,构建真核表达载体pcDNA3.1-mIL-33,然后进行酶切鉴定与序列分析。结果:小鼠IL-33基因的PCR产物和重组载体经凝胶电泳和酶切鉴定、测序分析证实,其序列与GenBank中数据一致。小鼠IL-33基因的全长编码序列为801 bp,编码266个氨基酸。结论:小鼠IL-33基因成功的克隆并构建了其真核表达载体,为进一步进行IL-33的表达与功能研究奠定了基础。     

bax转录调控pGL3-Basic荧光素酶报告基因载体的构建

目的:构建能用来检测bax转录调控作用的报导基因载体.方法:采用PCR方法克隆出bax 5'UTD基因,构建载体后利用酶切与测序进行鉴定.然后将该基因亚克隆至pGL3-Basic报告基因载体上.结果:克隆基因产物酶切结果与理论预测值一致,测序未出现一个碱基突变.PCR鉴定发现,bax5'UTD正确连接到了pGL3-Basic报告基因载体上,构建了pGL3-Basic-Baxregular载体.结论:bax 5'UTD报导基因载体的构建为进一步研究bax转录调控作用提供了载体资源.     

拟南芥生长素受体基因TIRl启动子的克隆及序列分析

以野生型拟南芥（Arabidopsis Columbia）基因组DNA为模板,通过PCR扩增得到拟南芥生长素受体基因TIRI启动子2008片段,将该片断克隆到PGM-T载体上。序列分析表明,该启动子大小为2008bp,RNA聚合酶识别序列TATA-box,TIR1特异表达和增强序列CAAT-box皆完整,与已报道的序列比较仅有3个核苷酸发生改变,同源性为99．85％。将该启动子与GUS基因融合,构建成表达载体后,在拟南芥和烟草叶片中做瞬时表达,结果分析显示：拟南芥和烟草叶片中均有GUS酶活性存在。     

FLT-1启动子荧光素酶报告基因重组体的构建和鉴定

目的：为探索FLT-1启动子靶向调控活性分析,克隆FLT-1启动子基因序列,构建并鉴定肿-1启动子调控的荧光素酶报告基因重组体pGL3-FLT—Basic—luc。方法：应用聚合酶链式反应（PCR）技术扩增FLT-1启动子序列,定向亚克隆至荧光素酶表达载体pGL3-Basic—luc中,构建含有正确目的基因的报告基因重组体pGL3-FIT—Basic—luc,通过限性内切酶酶切、PCR及测序进行鉴定。结果：通过酶切鉴定及基因测定证明,所克隆的基因产物与预期结果-致,序列无碱基突变。结论：成功构建了含有FLT-1启动子基因序列的荧光素酶报告基因真核表达载体,为下-步分析该启动子活性及血管疾病的基因治疗奠定基础。     

紫杉烷13α-羟基化酶基因的克隆及其转化烟草的研究

本研究利用东北红豆杉(Taxus cuspidata )cDNA文库作为模版,通过PCR技术扩增得到我国东北红豆杉紫杉烷13α-羟基化酶(Taxane 13α-hydroxylase,简称13OH)的全长cDNA,将PCR产物克隆到pGM-T载体后测序结果表明该序列长度为1458bp。同源性比较分析结果表明：其碱基序列与已经报道的东北红豆杉(Taxus cuspidata)的13OH基因的一致性为99.38%,其氨基酸序列同与已经报道的东北红豆杉的13OH氨基酸序列的一致性为99.18%。将获得的cDNA全长序列正向插入到含有GUS报告基因的pCambia1305.1后成功地构建出东北红豆杉13OH植物表达载体pC13OH,通过电击法把pC13OH转入根癌农杆菌GV3101中。利用该工程菌株对普通烟草进行了转化,在潮霉素选择压力下获得了完整的再生植株。利用13OH基因特异引物,通过PCR技术筛选到4株阳性再生植株。在这4株再生植株中,有3株植株GUS报告基因的组织化学染色呈现阳性反应,表明该植物载体表达载体中与13OH相融合的GUS基因成功地得到了表达。本研究为今后深入研究13OH基因在烟草中的表达和开展紫杉烷13α-羟基化酶基因对红豆杉细胞的转化以及研究作用于该基因的小RNA调节子打下了基础。     

小鼠Daintain/AIF-1 基因启动子的克隆及其荧光素酶报告基因 载体的构建

目的:对Daintain/AIF-1(大炎肽/同种异体移植炎症因子-1)基因启动子进行克隆并构建荧光素酶报告基因载体,为进一步研究Daintain/AIF-1的转录调控作用提供了质粒资源。方法:提取单核巨噬细胞系RAW264.7基因组DNA,以其为模板采用PCR方法克隆出Daintain/AIF-1基因5’端UTR区1.6 kb DNA序列,将该序列同源重组到pGL3-Basic载体上,转化感受态DH5α并酶切鉴定和测序。结果:PCR产物片段与预期结果一致,Daintain/AIF-1基因5’端UTR区1.6 kb DNA序列连接到pGL3-Basic载体上,构建成pGL3-Basic-Daintain/AIF-1(pGL3-Basic-DT)载体,酶切结果与理论预测值一致,经测序证实无碱基突变。结论:Daintain/AIF-1基因报告基因载体的构建为进一步研究Daintain/AIF-1转录调控作用提供了载体资源。     

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.