蚕豆叶绿体DNA BamH I克隆库的构建及含16S-25S rRNA基因克隆的分析

蚕豆叶绿体DNA(ct—DNA)经BamH I酶切产生26个片段,最大的为14．00kb,最小的为0．42kb。本文以pBR322为载体,E．Coli HB101为受体菌,采用标准分子克隆法构建了蚕豆ct—DNA BamH I克隆库,并从库中分离得到含叶绿体rRNA基因的克隆。32P标记的E．Coil 16S、23S rRNA能和蚕豆ct—DNA BamH I第6(B₆,5．65kb)和第9(B₉,4．70kb)个片段杂交,含有这二个片段的克隆分别命名为pVFB32和pVFBl6。利用几种限制性内切酶酶切和Southern印迹法构建了pVFBl6的物理图谱。pVFBl6电镜下观察到有一变性环(A—T丰富区),经Hind I酶切,电镜观察定位此A—T丰富区位于16S和23S rRNA基因的间隔顺序内,推测该环可能与DNA复制有关。     

Gibson组装技术简化腺病毒载体反向遗传改造

重组腺病毒是常用的基因转移载体,本文介绍一种对腺病毒基因组进行反向遗传改造的策略。拟在维持基因编码蛋白氨基酸序列不变的前提下,突变去除重组人5型腺病毒Ad5GFP基因组的PmeI酶切位点。软件分析腺病毒质粒pAd5GFP序列,选择限制性内切酶BamHI将pAd5GFP切割为大小11.7和24.6kb两个片段,24.6kb大片段自身环化形成一个质粒pAd5GB,PmeI位于其上。PmeI/AscI双酶切pAd5GB质粒,产生2.4kb和22.2kb两个片段;在引物部位引入突变的PmeI位点(由gtttaaac突变为gtttaaaT),PCR扩增得到两端各延长30bp的上述2.4kb片段,与22.2kb片段进行Gibson组装,转化E.coli TOP10感受态细胞,得到pAd5GBXP质粒。BamHI酶切pAd5GBXP,碱性磷酸酶处理,与11.7kb片段连接,还原得到腺病毒质粒pAd5GXP。PacI线性化pAd5GXP质粒,转染293细胞,拯救得到Ad5GXP病毒;酶切分析证明Ad5GXP基因组不含有PmeI位点。研究结果说明将酶切连接与DNA组装技术相结合,能够方便灵活地对腺病毒基因组进行突变改造。     

青霉素酰化酶基因的克隆与表达Ⅱ 质粒pPAl的限制性酶切图及青霉素酰化酶基因的定位

前文报道重组质粒pPAl中9．1kb的EcoRI片段上带青霉酰化酶基因。用16种限制性内切酶消化pPAl,其中ApaI,KpnI,SacI,SacI,SmaI及XhoI等六种内切酶在pPAl上无切口; BamHI,ClaI,Sph I,BglI为单切口;Sal为双切口,AvaI,HindI及PvuI为三切口,EcoRV为五切口。经交叉双酶解法测定各片段的大小,作出质粒pPAl的限制性酶切图。在包含青霉素酰化酶基因的9．1kbEcoRI片段上,BglI有一个切口,AvaI,HindI 及PvuI都有两个切口,而EcoRV有四个切口,SalI,BamHI,ClaIKSphI不切9．1kb的EeoR I片段。HindI切9．1kb EcoR I片段为A(3．5kb),B(2．7kb)及C(2,9kb)等三个片段。经Hind I部分水解后连接,转化大肠杆菌HB101得到一系列带不同Hind 1片段的质粒的转化子,青霉素酰化酶活性测定证明其基因位于Hin d II—A片段上。合成青霉素酰化酶仍需苯乙酸诱导,并被葡萄糖阻遏,Hind I—B片段的存在能增加青霉素酰化酶基因的表达,而c片段无显著影响。     

龙葵叶绿体ATP合酶α-亚单位基因的克隆

本研究以菠菜叶绿体DNA 2.45kb的SalI片段(含有ATP合酶α-亚单位基因)为探针,从龙英叶绿体DNA BamHI片段文库中筛选出含龙葵叶绿体atpA基因的克隆。通过Southrcn吸印与探针杂交,证明了重组质粒pSB 132的插入片段含有atpA基因。同时将atpA基因定位在龙葵叶绿体DNA SalI、BglI、XhoI和BamHI 4种酶切图谱的限制性片段上。     

水稻线粒体26S rRNA基因在大肠杆菌JM101中的克隆及分子鉴定

用BT型水稻喜峰A黄化苗为材料,按本实验过去报道经修改后的方法提取线粒体DNA,经EcoR1完全酶切后,随机克隆到pUC19载体上,转化大肠杆菌,在含氨苄青霉素(50μg/m1)和X-gal的LB固体平板上筛选白色转化子。随机提取重组子DNA,以玉米26S rRNA基因为探针,经Southern分子杂交鉴定,一个插入1.3kb水稻线粒体DNA片段的重组质粒杂交结果为阳性,并将这个含有26S rRNA基因片段的重组质粒命名为pXMT1。     

蓖麻蚕(Attacus ricini)核糖体RNA基因片段在大肠杆菌中的无性繁殖

利用质粒pBR322作运载体,获得了蓖麻蚕(Attacus ricini)核糖体rRNA 基因(rDNA)的部分片段在E.coli 中的无性繁殖株。酶切图谱分析及Southern 法分子杂交鉴定证明,重组质粒pARI 含有1.95MdrDNA EcoRI-BamHI 双酶切片段;pARⅡ含有2.6Md 的rDNABamHI片段。并测定了BamHI 片段与pBR322连接方向。     

A型产气荚膜梭菌α毒素基因的高效表达

用NooI/EcoRI酶切含α毒素基因质粒pXCPA02,回收1.2kb的α毒素基因片段,通过T4 DNA连接酶,将回收的α毒素基因片段与经NcoI/Eco RI酶切的表达载体pET-28c连接,转化至受体菌BI21(DE3)中,经NcoI/EcoRI,BamHI/Eco RI和NcoI/BamHI/Eco RI酶切反应鉴定和核苷酸序列分析证实,获得的表达质粒aXETA02含有α毒素基因,而且阅读框架是正确的.重组菌株BL21(DE3),(pXETA02)经IPTG诱导后,其表达产物经ELISA检测和SDS-PAGE分析,结果表明重组菌株可以高效表达α毒素蛋白,该蛋白占菌体总蛋白相对含量的36.83%.     

C型产气荚膜梭菌α毒素基因的克隆与表达

利用PCR技术,从C型产气荚膜梭菌染色体基因组中扩增了1．2kb的α毒素基因,将纯化的PCR产物与载体pGEM-T连接,转化至受体菌JM109中,经NcoI／EcoRI和BamHI／EcoRI酶切鉴定及核苷酸序列测定证实,重组质粒pXCPAl中含有α毒素全基因。随后用NcoI／EcoRI酶切质粒pXCPAl,回收α毒素基因片段,插入到事先经同样酶切处理的载体pET-28c中相应酶切位点,构建了表达质粒pETXAl,经NcoI／EcoRI和BamHI／EcoRI酶切鉴定及核苷酸序列测定证实,表达质粒含有α毒素基因且基因序列和阅读框架正确。重组菌株BL21(DE3)(pETXAl)表达产物经ELISA检测和SDS-PAGE分析,重组菌株表达的α毒素蛋白能够被α毒素单抗识别,其表达量占菌体总蛋白相对含量的16．28％。     

基于同尾酶技术构建CCL3L1 基因串联重组质粒的方法

目的:利用同尾酶技术将CCL3L1基因重复连续插入pcDNA6.2-GW/miR载体,构建含有CCL3L1基因串联体的重组质粒,实现小片段CCL3L1有效延长。方法:PCR扩增CCL3L1基因并在引物的两端设有同尾酶BamHI和BglII限制性内切酶位点,纯化PCR产物插入pMD18-T载体,阳性克隆命名为pMD18T-CCL3L1。BamHI和BglII双酶切pMD18T-CCL3L1和pcDNA6.2-GW/miR载体后将第一个CCL3L1片段插入pcDNA6.2-GW/miR载体命名为pcDNA6.2-CCL3L1-1。由于载体本身在BglII位点后带有XhoI酶切位点利用BamHI和XhoI切割pcDNA6.2-CCL3L1-1回收CCL3L1片段,BglII和XhoI切割pcDNA6.2-CCL3L1-1回收大片段做载体重组形成含有两个连续CCL3L1片段的质粒命名为pcDNA6.2-CCL3L1-2,重复此步骤可得到含有N个CCL3L1基因串联体的重组质粒pcDNA6.2-CCL3L1-X。结果:经酶切和测序证实成功构建含有4个CCL3L1基因串联体的重组质粒pcDNA6.2-CCL3L1-4,并同时产生含有1个和2个CCL3L1基因串联体的重组质粒。结论:利用同尾酶技术可以快速有效地构建CCL3L1基因串联重组质粒,实现目的片段的无限扩大,为小片段基因表达的研究奠定基础。     

北京鸭肝脏线粒体DNA 2.17kb的EcoRI/BamHI片段的分子克隆

本文以质粒pAT153为载体,以E.coli SK1592为受体菌,将北京鸭肝脏mtDNA的2.17kb的EcoRI/BamHI限制片段进行了克隆,并对重组质粒作了限制性内切酶分析、Southern吸印与杂交分析。此外,还对重组体的一些性质作了初步研究,并对鸭肝mtDNA的另一个EeoRI/BamHI片段(2.81kb)不能被克隆的原因进行了讨论。     

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.     

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.