表达人α-1，2-岩藻糖苷转移酶、衰变加速因子和CD59基因的转基因小鼠的建立及其抗异种移植排斥反应的研究

在进行非协调性异种器官移植时,目前已经证实表达人α-1,2-岩藻糖苷转移酶（HT）或者补体调节蛋白的供体动物器官均可以部分克服超急性排斥反应．本文的研究目的是探讨是否共表达人HT和补体调节蛋白（衰变加速因子与CD59）的转基因小鼠的外周血单核细胞能更有效的克服异种移植排斥反应．利用受精卵显微注射技术建立转人HT,DAF和／或CD59基因的小鼠动物模型,流式细胞技术筛选表达不同基因的转基因小鼠．将小鼠的外周血单核细胞与15％的人血清共孵育,检测细胞表面天然抗体的沉积、补体的激活以及黏附分子的表达等．三种目的基因均可以在转基因小鼠体内表达,并且HT基因的表达显著减少了引起异种移植排斥反应的主要抗原半乳糖α-1,3-半乳糖（α—Gal）的表达．功能实验表明,与单一目的基因表达的转基因小鼠相比,共表达HT／DAF或HT／CD59的转基因小鼠的外周血单核细胞对抗人血清介导溶破细胞的能力显著增强．而且三基因共表达的外周血单核细胞对抗人血清介导溶破细胞的能力最强,可以克服超急性排斥反应并且可以减少黏附分子的表达．高水平表达人HT,DAF和CD59基因的转基因小鼠可以完全克服异种器官移植超急性排斥反应并且可以部分克服急性血管排斥反应．本研究表明,表达三种基因的转基因小鼠的器官异种移植时存活时间可以显著延长,对异种移植来说也许是更合适的选择．     

可调控肝脏特异性表达HCV全基因转基因小鼠模型的建立

目的:构建一种可调控肝细胞特异性表达丙型肝炎病毒(HCV)全基因的小动物模型。方法:将9.6 kb的HCV全长基因JFH1插入Tet-on系统效应表达载体p TRE2中,并在HCV全基因3'端插入丁型肝炎病毒(HDV)核酶序列,从而构建转基因载体p TRE2-JFH1(HCV);将该转基因载体线性化后显微注射获得整合有HCV全基因的TRE2-HCV首建鼠;将该阳性鼠与本室保存的Alb-rt TA转基因小鼠杂交,获得肝细胞白蛋白启动子调控HCV全基因表达的Tet-on-Alb-HCV双转基因小鼠;在强力霉素(Dox)诱导后通过PCR、Western印迹、免疫组化等方法鉴定转基因小鼠HCV基因整合及HCV蛋白在肝组织中的表达;实时定量PCR检测小鼠血清及肝组织中的HCV滴度;用HCV反义小分子药物anti-mi R122评价该模型在药物评价中的应用。结果:获得了整合rt TA基因和HCV全长基因的双转基因小鼠;Dox诱导下,该转基因小鼠可在肝组织长期特异性表达HCV全长基因,小鼠血清中可检测到HCV的RNA;分子药物anti-mi R122可降低血清中的HCV滴度。结论:构建了可调控肝组织特异性表达HCV全基因的转基因小鼠模型,该小鼠模型可应用于HCV药物筛选和评价研究。     

foxp3转染的CD4~+ CD25~- T细胞治疗急性肺损伤的研究

目的通过基因转染技术将foxp3基因导入CD4+CD25-T淋巴细胞,生成CD4+CD25-Foxp3+T细胞,通过输入急性肺损伤小鼠模型体内,促进炎症消退,为体外定向操纵调节T细胞生成和细胞过继治疗急性肺损伤奠定基础。方法采用RT-PCR法从小鼠胸腺来源cDNA文库中扩增获得foxp3 cDNA,亚克隆至构建pIRES2-EGFP质粒中,构建并筛选出重组质粒pmFoxp3-IRES2-EGFP;采用免疫磁珠法分离CD4+CD25-T淋巴细胞,穿孔法转染Foxp3基因至CD4+CD25-T淋巴细胞,流式细胞仪检测CD4+和EGFP共表达的细胞比例;将foxp3基因转染的小鼠CD4+CD25-T细胞通过尾静脉输注急性小鼠体内,通过ELISA法检测小鼠肺泡灌洗液中TNF-α以及IL-1β细胞因子的表达,通过肺组织病理研究肺部炎症的消退状况。结果成功构建双基因共表达重组质粒pmFoxp3-IRES2-EGFP;免疫磁珠法较高纯度分离CD4+CD25+、CD4+CD25-T淋巴细胞,电穿孔法成功将重组质粒pmFoxp3-IRES2-EGFP转染致CD4+CD25-T细胞,CD4+和EGFP共表达的细胞比例为35.5%;过继Foxp3基因转染的小鼠CD4+CD25-T细胞以及Treg均能抑制急性肺损伤小鼠肺泡灌洗液中TNF-α以及IL-1β炎性细胞因子的表达,促进炎症的消退。结论转染Foxp3基因的CD4+CD25-细胞同Treg(CD4+CD25-)细胞一样可以通过细胞过继治疗急性肺损伤。     

乙型肝炎病毒蛋白和绿色荧光蛋白的共表达研究

目的：构建增强型绿色荧光蛋白（EGFP）标记的乙型肝炎病毒（HBV）真核表达载体,并研究其在真核细胞和小鼠体内的共表达。方法：以质粒pBR322-HBVadr2．0和pCX-EGFP为基础,构建含有双拷贝HBV全基因组DNA和EGFP基因的真核表达载体pCX-EGFP-HBVadr2．0,分别转染真核细胞和小鼠肝组织,建立体外、体内表达系统,研究GFP和HBV基因的表达。结果：构建了真核表达载体pCX-EGFP-HBVadr2．0,EGFP和HBV病毒蛋白在体内和体外均可表达。结论：构建的pCX-EGFP-HBVadr2．0真核表达载体可以GFP作为HBV存在与否的报告基因,提高了培育检测转基因小鼠的效率,为转基因小鼠的制备及后续研究奠定了基础。     

HCV全长基因真核表达载体的构建及其细胞内表达

本实验成功构建了HCV全长基因的真核表达载体pCI-HCVFL,转染HepG2细胞后经免疫荧光和免疫组化法分别检测到结构基因区(C)和非结构基因区(NS3)病毒蛋白的表达,该载体可用于建立HCV转基因细胞模型以及进一步开展有关HCV复制与表达的深入研究。     

2型腺相关病毒载体介导正常人β珠蛋白基因在重型地贫流产胎儿造血细胞中的表达

目的:探讨2型重组腺相关病毒(recombinant adeno-associated virus 2,rAAV2)载体介导人β-珠蛋白基因转染地贫患者造血细胞治疗β-地中海贫血的可行性.方法:分离β 41-42/β 654杂合子型重型β-地贫流产胎儿造血细胞,经rAAV2-β-globin病毒转染(MOI=50)后移植入经X射线照射的BALB/c裸小鼠体内,分别于移植后14d、21d处死受体小鼠,RT-PCR及等位基因特异性PCR法检测人珠蛋白基因在受体小鼠体内的表达.结果:RT-PCR方法于3只受体小鼠骨髓样本中成功检测到人β-actin和人β-珠蛋白基因的表达,而21d处死的转染与对照小鼠外周血样本中未检测到人β-珠蛋白基因表达;等位基因特异性PCR方法在所有受体鼠体内同时检测到β 41-42和β 654突变基因,以及正常β-珠蛋白基因的表达,但rAAV2-β-globin转染组小鼠体内正常人β-珠蛋白基因表达水平明显高于对照组.结论:rAAV2可有效转染β 41-42/β 654杂合子型重型地中海贫血患者造血细胞,并通过exvivo途径介导β-珠蛋白转基因的体内表达,提高红系细胞内正常β-珠蛋白基因的表达水平.     

HCV全长基因真核表达载体的构建及其细胞内表达

本实验成功构建了HCV全长基因的真核表达载体pCI-HCVFL,转染HepG2细胞后经免疫荧光和免疫组化法分别检测到结构基因区(C)和非结构基因区(NS3)病毒蛋白的表达,该栽体可用于建立HCV转基因细胞模型以及进一步开展有关HCV复制与表达的深入研究.     

应用绿色荧光蛋白研究外源基因在造血细胞的表达调控

以增强型绿色荧光蛋白为报道分子,研究双基因逆转录病毒载体介导小鼠骨髓细胞的基因转移中,SV(simianvirus)40启动子和内部核糖体进入位点(internalribosomeentrysite,IRES)对双基因共表达的影响。构建双基因中间序列分别为SV40启动子和IRES的载体pLESN和pLEIN。经包装获得较高滴度的病毒上清,以共培养的方式感染5-氟尿嘧啶预刺激的小鼠骨髓细胞。流式细胞仪检测表明转染效率约25%,PCR证明EGFP基因整合至骨髓细胞基因组。半固体培养转基因细胞7d,LEIN组获得具有G418抗性的集落中98%表达绿色荧光,而LESN组54%。结果表明:在双基因逆转录病毒载体介导的小鼠骨髓细胞的基因转导中,IRES与内部SV40启动子相比,更能保证双基因的共同表达。     

带有小鼠CD40L基因的汉滩病毒糖蛋白表达载体的构建

目的:构建汉滩病毒包膜糖蛋白基因的真核表达载体,并加入可增强免疫应答效应的细胞因子CD40L基因,检测其可否在真核细胞中表达。方法与结果:参照GenBank中汉滩病毒M基因和小鼠CD40L的全基因序列设计引物,通过聚合酶链反应(PCR)获得M和CD40L基因片段,将其与pCI-neo载体相连,测序证实该载体构建成功后,将此真核表达载体以脂质体转染法转染至哺乳动物细胞CHO-K1中,利用间接免疫荧光法(IFA)检测发现M基因和CD40L基因可以同时表达于CHO-K1细胞中。结论:构建了带有CD40L基因的汉滩病毒包膜糖蛋白重组质粒并获得表达,为深入研究汉滩病毒感染后包膜糖蛋白引起的特异性免疫应答规律奠定了实验基础。     

三种丙型肝炎病毒转基因小鼠系的同时制备

为研究丙型肝炎病毒的致病致瘤机理及结构基因与非结构基因3区(NS3)的功能及其在HCV感染致病中的作用,建立一个HCV分子治疗的动物模型,构建了含金属硫蛋白启动子和HCV结构基因或NS3基因的质粒,将两者等量混合后用显微注射法接种于昆明白小鼠受精卵内制备转基因小鼠.通过PCR筛选获得三种整合HCV结构基因或/和NS3基因的首建鼠.结果表明:a.注射后卵存活率与仔鼠出生率分别为81%、30%;b.检测60只G0代小鼠,结构基因整合鼠6只(10%),NS3基因整合鼠4只(6.7%),双基因整合鼠9只(15%),总整合率为31.7%;c.RT-PCR法检测阳性鼠肝中有靶基因mRNA的转录;d.4只首建鼠与正常鼠回交获得38只G1小鼠,其中20只为整合鼠,整合率为52.6%;e.转基因鼠表型迄今无明显异常.表明一次显微注射同时获得了三种整合HCV结构基因或/和NS3基因的转基因小鼠.     

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.