大鼠VDAC1腺病毒过表达载体构建及功能鉴定

目的构建电压依赖性阴离子通道蛋白1(VDAC1)腺病毒过表达载体,并观察其在H9C2细胞中的表达情况,为探究VDAC1在心肌细胞缺血再灌注损伤中的作用机制奠定基础。方法构建ADV6-NC腺病毒及ADV6-VDAC1腺病毒,取第6代H9c2心肌样细胞用于转染。转染细胞分为3组:空白对照组、阴性对照组(转染ADV6-NC腺病毒)和实验组(转染ADV6-VDAC1腺病毒)。通过qPCR和蛋白免疫印迹实验检测各组细胞VDAC1的表达情况。结果成功构建ADV6-NC及ADV6-VDAC1,实验组的VDAC1表达量明显高于空白对照组和阴性对照组,差异具有统计学意义(P<0.05)。结论利用腺病毒载体可使H9c2细胞在体外持续高效表达VDAC1。

Construction of Plant Expression Vector for Hand,Foot and Mouth Virus EV71-VP1 Gene and Its Expression in Tomato

EV71-type virus is one of the main pathogens causing the occurrence of hand,foot and mouth disease(HFMD),and VP1 protein,a factor that directly determines the antigenicity of the virus,has been isolated.The tomato was selected as a bioreactor for the production of an edible EV71 vaccine designed for the VP1 capsid protein.Using molecular biology techniques,the fusion gene EV71-VP1 was cut from vector PGEX-4T-2,a vector containing the p2300-EV71 gene with CaMV35S promoter and TL regulatory elements was constructed,and the hypocotyl and cotyledons of tomato were transformed using Agrobacterium(EHA105)-mediated method,screened,elongated and rooted,and finally 20 resistant tomato plants were obtained.Five transgenic positive seedlings were obtained by digestion and PCR assay,among which three plants were detected by RT-PCR to be capable of transcriptional translation at the RNA level.The experimental results aimed to explore new material support for the preparation of transgenic plant oral vaccines against EV71 infection and provide a theoretical basis for accelerating the development of transgenic plant vaccines in the future.

Immune response to inactivated bacterial vector carrying the recombinant K39 antigen of Leishmania infantum in mice

Objective:To evaluate the immunological response elicited by an inactivated bacterial vector carrying the K39 antigen of Leishmania infantum,and a purified antigen.Methods:Mice were subjected to the following treatments:(1)Purified recombinant K39(rK39)protein at a 20μg dose with complete Freund’s adjuvant;(2)Inactivated Escherichia coli(BL21 DE3)carrying the K39 protein at an equivalent total protein content of 200μg;(3)Inactivated bacteria lacking the K39 protein;(4)Non-immunized control animals.Serological monitoring was performed.All groups were challenged by intraperitoneal injection of 10^(7) Leishmania infantum promastigotes.After euthanasia,the liver and spleen were collected to analyze the levels of TNF,IFN-γ,IL-12,IL-4,and IL-10.Results:Mice immunized with purified rK39 or the inactivated bacterial vector carrying the K39 antigen of Leishmania infantum showed a long-lasting immune response with high levels of polyclonal antibodies specifically recognizing the recombinant proteins.The IgG1 subclass was the predominant immunoglobulin;however,the induction of IgG2a and the profile of cytokines produced were indicative of the induction of a mixed-type response.Conclusions:The inactivated bacterial vector carrying the K39 antigen,as well as the purified antigen can induce a long-lasting immune response in immunized mice,predominantly favouring a Th2 profile response.

Polo-like kinase 1 as a biomarker predicts the prognosis and immunotherapy of breast invasive carcinoma patients

Invasive breast carcinoma(BRCA)is associated with poor prognosis and high risk of mortality.Therefore,it is critical to identify novel biomarkers for the prognostic assessment of BRCA.Methods:The expression data of polo-like kinase 1(PLK1)in BRCA and the corresponding clinical information were extracted from TCGA and GEO databases.PLK1 expression was validated in diverse breast cancer cell lines by quantitative real-time polymerase chain reaction(qRT-PCR)and western blotting.Single sample gene set enrichment analysis(ssGSEA)was performed to evaluate immune infiltration in the BRCA microenvironment,and the random forest(RF)and support vector machine(SVM)algorithms were used to screen for the hub infiltrating cells and calculate the immunophenoscore(IPS).The RF algorithm and COX regression model were applied to calculate survival risk scores based on the PLK1 expression and immune cell infiltration.Finally,a prognostic nomogram was constructed with the risk score and pathological stage,and its clinical potential was evaluated by plotting calibration charts and DCA curves.The application of the nomogram was further validated in an immunotherapy cohort.Results:PLK1 expression was significantly higher in the tumor samples in TCGA-BRCA cohort.Furthermore,PLK1 expression level,age and stage were identified as independent prognostic factors of BRCA.While the IPS was unaffected by PLK1 expression,the TMB and MATH scores were higher in the PLK1-high group,and the TIDE scores were higher for the PLK1-low patients.We also identified 6 immune cell types with high infiltration,along with 11 immune cell types with low infiltration in the PLK1-high tumors.A risk score was devised using PLK1 expression and hub immune cells,which predicted the prognosis of BRCA patients.In addition,a nomogram was constructed based on the risk score and pathological staging,and showed good predictive performance.Conclusions:PLK1 expression and immune cell infiltration can predict post-immunotherapy prognosis of BRCA patients.

Understanding the role of transmembrane 9 superfamily member 1 in bladder cancer pathogenesis

In this editorial we comment on the article by Wei et al,published in the recent issue of the World Journal of Clinical Oncology.The authors investigated the role of Transmembrane 9 superfamily member 1(TM9SF1)protein in bladder cancer(BC)carcinogenesis.Lentiviral vectors were used to achieve silencing or overexpression of TM9SF1 gene in three BC cell lines.These cell lines were then subject to cell counting kit 8,wound-healing assay,transwell assay,and flow cytometry.Proliferation,migration,and invasion of BC cells were increased in cell lines subjected to TM9SF1 overexpression.TM9SF1 silencing inhibited proliferation,migration and invasion of BC cells.The authors conclude that TM9SF1 may be an oncogene in bladder cancer pathogenesis.

水牛SFRP 1基因序列分析、真核表达载体构建及组织表达分析

【目的】获取水牛分泌型卷曲相关蛋白1(SFRP1)基因CDS区序列,并预测其编码蛋白的结构功能,构建SFRP 1基因真核表达载体,检测SFRP 1基因在水牛不同组织中的表达情况,为探索SFRP 1基因在水牛生长发育中的作用奠定基础。【方法】以水牛卵巢组织cDNA为模板,通过RT-PCR对SFRP 1基因CDS区序列进行扩增并测序,利用生物信息学在线分析软件对水牛SFRP 1基因与不同物种进行比对和系统进化树构建,并预测SFRP1蛋白理化性质、信号肽、跨膜结构等。将获得的目的基因连接至pCMV-HAhyPBase-mcheery载体并转染至水牛颗粒细胞,检测转染后荧光和基因表达情况。通过实时荧光定量PCR检测水牛不同组织中SFRP 1基因表达情况。【结果】水牛SFRP 1基因CDS区长927 bp,共编码308个氨基酸。多重序列比对结果显示,水牛SFRP 1基因氨基酸序列与黄牛、牦牛、山羊、虎鲸、黑猩猩、北极狐、猫、人、小鼠的相似性分别为100%、100%、99.65%、98.58%、98.23%、98.23%、98.58%、98.23%、96.45%,存在CRD_FZ、NTR_like和DUF3367结构域。水牛SFRP 1基因核苷酸序列与黄牛、绵羊、山羊、牦牛、野牛、马鹿、野骆驼、猪、人的相似性分别为97.3%、95.9%、95.8%、94.5%、94.2%、93.7%、80.4%、78.5%和77.3%。系统进化树结果显示,水牛与黄牛、牦牛、野牛聚为一支。生物信息学分析结果显示,SFRP1蛋白呈碱性,为不稳定蛋白;第1—15位氨基酸处存在信号肽,为分泌型蛋白,存在跨膜结构,主要定位于细胞外;存在21个磷酸化位点和8个O-糖基化修饰位点。二级结构主要由α-螺旋、延伸链和无规则卷曲构成;水牛、黄牛、人的SFRP1蛋白三级结构高度相似。成功构建pCMV-mcheery-SFRP1真核表达载体并转染水牛颗粒细胞,转染72 h后pCMV-mcheery-SFRP1重组质粒组细胞内SFRP 1基因表达量极显著高于对照组(P<0.01)。实时荧光定量PCR结果显示,SFRP 1基因在水牛不同组织中均有表达,且在脾脏中表达量最高,极显著高于其他组织(P<0.01)。【结论】SFRP 1基因在不同物种以及遗传进化过程中具有高保守性,在水牛不同组织中广泛表达。研究结果为今后探究SFRP 1基因在水牛生长发育中的功能及分子机制奠定基础。

NS特异性干扰载体pGenesil-1-NS的构建及鉴定

目的核干细胞因子(nucleostemin,NS)是维持干细胞和癌细胞增殖所必需的蛋白质,可能成为肿瘤基因治疗的潜在靶点.本文旨在构建靶向NS干扰载体p Genesil-1-NS,为后续实验奠定基础.方法基于已发布的NS mRNA序列(NM_206825),选取5'-AAGCCTA GGAAAGACCCAGG-3'(397-416)作为候选靶序列,按shRNA载体设计原则,设计合成两条互补DNA链,退火后插入载体,并以酶切和测序鉴定重组转化子.结果经酶切及测序鉴定证实合成序列正确插入载体.结论成功构建NS特异性干扰载体p Genesil-1-NS,可用于NS在肿瘤中的功能研究.

利用pGenesil-1.0构建人Dna2真核表达干扰载体

目的:构建人基因Dna2干扰(RNAi)的真核表达载体.方法:根据GenBank上人Dna2的cDNA序列设计并合成两条单链寡核苷酸,退火以后,插入质粒pGenesil-1.0多克隆位点的BamH I和Hind III之间进行重组,构建重组干扰载体,转化DH5α后测序鉴定出阳性菌株.重组质粒以脂质体法转染HeLa细胞,提取Dna2蛋白以免疫印迹法检验干扰效果.结果:设计的目的干扰序列5’-catagccagtagtattcgatg-3’可明显抑制Dna2在HeLa细胞的表达.结论:利用pGenesil-1.0构建的人Dna2干扰载体适用于该基因功能研究.

Pgenesil-1质粒载体介导的表达ERCC1shRNA的质粒构建和鉴定

目的:设计构建Pgenesil-1质粒载体介导的表达ERCC1shRNA的重组体质粒。方法:以ERCC1为靶基因,以Pgenesil-1质粒为载体,设计构建重组体,根据ERCC1基因cDNA序列,设计有小发卡结构的2条寡核苷酸序列,克隆到空载体Pgenesil-1中,转化DH5а菌株,提取质粒,进行酶切鉴定和测序分析。结果:经酶切鉴定筛出的重组体测序结果和目的序列相同,重组体载体构建成功。结论:成功构建了能表达ERCC1shRNA的质粒载体Pgenesil-1/ERCC11和Pgenesil-1/ERCC12,为下一步研究ERCC1基因在卵巢癌细胞株顺铂耐药中的作用奠定了实验基础。

Protection of rat islet viability following heme oxygenase-1 gene transfection via adenoviral vector in vitro

Objective： To investigate the effect of Heme oxygenase-1 （HO-1） gene transfection on the viability of cultured rat islets, and to explore the potential value of HO-1 gene in islet transplantation. Methods：Recombinant adenovirus vector containing human HO-1 gene（Ad-HO-1 ） or enhanced green fluorescent protein gene（Ad-EGFP） was generated by using AdEasy system respectively. The rat islets were transfected with Ad-HO-1, Ad-EGFP or blank vector and then cultured for 7 days. Transfection was confirmed by expression of EGFP and human HO-1 protein detected by fluorescence photographs and western blot, respectively. The insulin release upon different concentration of glucose stimulation was detected using insulin radioimmunoassay kit, and stimulation index（SI） was calculated. Glucose-stimulated insulin release was used ＇to assess islet viability. Results：Adenovirus vector successfully transferred HO-1 gene to rat islet cells in vitro, and the insulin release upon high level of glucose stimulation and stimulation index （SI） of Ad-HO-1-infected islets were significantly higher than those of Ad-EGFP-infected islets and control islets （P 〈 0.05）. Conclusion： Adenovirus-mediated HO-1 gene transfection is a feasible strategy to confer cytoprotection and therefore protect the viability of cultured rat islets.

Constructing recombinant replication-defective adenoviral vectors that express glucose transporter-1 through in vitro ligation

BACKGROUND： We constructed a homologous recombination bacterial method based on the pAdEasy system, a widely used system, for generating recombinant adenoviral vectors that express glucose transporter- 1 （GLUT 1） in rats, OBJECTIVE： This study was designed to investigate the feasibility of generating recombinant replication-defective adenoviral vectors that express GLUT1 in rats by in vitro ligation based on the Adeno-X^TM system. DESIGN： An in vitro cell-based experiment. SETTING： This study was performed at the Linbaixin Medical Research Center of the Second Hospital Affiliated to Sun Yat-sen University and Central Laboratory for Prevention and Treatment of Tumor, Sun Yat-sen University between January and August 2004. MATERIALS： Male, adult, Sprague Dawley rats were used to extract total RNA from brain tissue. E. coli DH5 a and human embryonic kidney 293 cells （HEK293 cells） used in the present study were cryo-preserved by the Second Hospital Affiliated to Sun Yat-sen University. Rabbit anti-rat GLUT1 polyclonal antibody （Chemicon, U.S.A.） and primers （Shanghai Boya Bioengineering Co., Ltd） were also used. METHODS： E1/E3-deleted replication-defective adenoviral vectors were used. Using in vitro ligation, the target gene was first sub-cloned into a shuttle vector plasmid to obtain the fragment containing target gene expression cassettes by enzyme digestion. Subsequently, the fragment was co-transformed with linearized adenoviral backbone vector into the E. coli strain. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly recombinant adenoviral vectors with replication capabilities. The procedure was repeated several times for recombinant adenoviral vectors amplification. MAIN OUTCOME MEASURES： Efficiency of recombinant adenoviral vectors to express the target gene was measured by gene and protein expression through polymerase chain reaction and Western Blot assays, respectively. RESULTS： Results demonstrated that recombinant adenoviral vectors successfully expressed GLUT1 protein, with a relative molecular mass of 55000 in HEK293 cells. These results suggest that recombinant adenoviral vectors obtained by homologous bacterial recombination feature high efficiency, rapidness, and simplicity. CONCLUSION： We successfully amplified the rat GLUT1 gene and constructed replication-defective adenoviral vectors expressing GLUT1. The replication-defective adenoviral vectors proved to successfully express the target gene in HEK293 cells.

Construction of Tobacco Bax Inhibitor-1 ihpRNA Gene Silencing Vector and Transformation into Agrobacterium tumefaciens EHA105

The plasmid pGSA1285 was first modified by substituting its GUS sequence with the Chalcone synthase intron fragment from vector pFGC5941 to get the plant silencing expression vector that contained Kanamycin resistance site and was named as pGSA2285. Using PCR-based amplification, two different restriction sites at both ends of tobacco Bax inhibitor-1 （NtBI-I） gene were created, respectively, which made the construction of ihpRNA gene silencing vector more efficiently. Then, NtBI-1 genes were inserted into Multiple Cloning Site （MCS） of pGSA2285 respectively to form Bax inhibitor-1 ihpRNA gene silencing vector, named as pGSA4285, containing sense and anti-sense BI-1 sequence which was spliced by chalcone synthase intron. Combined PCR identification and enzyme restriction analyses, the results showed that Bax inhibitor-1 ihpRNA gene silencing vector had been constructed and transferred into Agrobacterium tumefaciens EHA105 successfully, which laid a foundation for the further study on the function of BI-1 in plant PCD regulation.

A New Vector Markov Process for M/G/1 Queue

In this paper, by considering the stochastic proces s of the busy period and the idle period, and introducing the unfinished work as a supplementary variable, a new vector Markov process was presented to study th e M/G/1 queue again. Through establishing and solving the density evolution equa tions, the busy-period distribution, and the stationary distributions of waitin g time and queue length were obtained. In addition, the stability condition of th is queue system was given by means of an imbedded renewal process.

Effect of adenoviral vector-mediated rat antisense AT1B gene transfer on neointima proliferation after rat carotid injury

Objective: Angiotensin Ⅱ is a growth-promoting factor for vascular smooth muscle cells in culture andin the intact animal. The biological effects of angiotensin Ⅱ are manifested only by binding to specific receptors oncell membranes. In the study, we observed that the effect of rat antisense AT1B gene transfer mediated by adenoviral vector-on neointimal proliferation following rat carotid injury. Methods: Antisense AT1B gene was transductedinto the carotid by adenoviral vector after carotid bal1oon injury and the restenosis model was established in SD rat.We measured neointima/media area ratio in local artery at day 21 after gene transfer. Results: Rat antisense AT1Bgene was successfully transducted into local carotid after the carotid balloon injury. Neointima/media area ratiowas significantly reduced (47 %, P<0. 01) at day 21 after gene transfer compared with the control group. Conclusion: The results suggest it is possible that antisense AT1B gene transfer as a potential therapeutic approach prevent neointimal hyperplasia.

Construction of Double Right-Border Binary Vector Carrying Non-Host Gene Rxo1 Resistant to Bacterial Leaf Streak of Rice

Rxol cloned from maize is a non-host gene resistant to bacterial leaf streak of rice. pCAMBIA1305-1 with Rxo1 was digested with Sca I and NgoM IV and the double right-border binary vector pMNDRBBin6 was digested with Hpa I and Xma I. pMNDRBBin6 carrying the gene Rxo1 was acquired by ligation of blunt-end and cohesive end. The results of PCR, restriction enzyme analysis and sequencing indicated that the Rxo1 gene had been cloned into pMNDRBBin6. This double right-border binary vector, named as pMNDRBBin6-Rxol, will play a role in breeding marker-free plants resistant to bacterial leaf streak of rice by genetic transformation.

Construction of Plant Expression Vector of Heat Shock Factor Gene AtHsfA1a from Arabidopsis

[ Objective ] Heat shock factors （HSFs） are the major transcription factors of eukaryotic heat shock responses. This study aims to investigate the adversity stress tolerance functions of Arabidopsis heat shock factor AtHsfAla, which has important significance for in-depth understanding of adversity stress tolerance mechanisms of plants and further utilization of heat shock factor genes. [Method] Genomic DNA of Arabidopsis was extracted with CTAB method and purified to obtain Arabidopsis DNA samples for in vitro site-specific recombination cloning （ Gateway cloning） to construct plant expression vector of heat shock factor AtHs- fAla. Firstly, donor vector pDONR 201/AtHsfAla was constructed based on attB and attP site-specific recombination method （BP reaction）, to identify E. coli transformants harboring correct sequence of AtHsfAla by sequencing; secondly, plant expression vector pBTWG2/AttlsfAla overexpressing Arabidopsis heat shock factor AtHsfAla was constructed based on attL and attR site-specific recombination method （LR reaction）, to screen E. coli transformants harboring target plasmid. [ Result] Plant expression vector of Arabidopsis heat shock factor gene AtHsfAla was constructed successfully. [ Conclusion] This study not only provided experimental materials for acquiring transgenic plants overexpressing heat shock transcription factor AtHsfAla, but also laid the foundation for further investigation of the diversity of adversity stress tolerance functions reanlated by HSFs.

Review: Plant Binary Vectors of Ti Plasmid in <i>Agrobacterium tumefaciens</i>with a Broad Host-Range Replicon of pRK2, pRi, pSa or pVS1

This review chronicles the development of the plant binary vectors of Ti plasmid in Agrobacterium tumefaciens during the last 30 years. A binary vector strategy was designed in 1983 to separate the T-DNA region in a small plasmid from the virulence genes in avirulent T-DNA-less Ti plasmid. The small plant vectors with the T-DNA region have been simply now called binary Ti vectors. A binary Ti vector consist of a broad host-range replicon for propagation in A. tumeraciens, an antibiotic resistance gene for bacterial selection and the T-DNA region that would be transferred to the plant genome via the bacterial virulence machinery. The T-DNA region delimited by the right and left border sequences contains an antibiotic resistance gene for plant selection, reporter gene, and/or any genes of interest. The ColEI replicon was also added to the plasmid backbone to enhance the propagation in Escherichia coli. A general trend in the binary vector development has been to increase the plasmid stability during a long co-cultivation period of A. tumefaciens with the target host plant tissues. A second trend is to understand the molecular mechanism of broad host-range replication, and to use it to reduce the size of plasmid for ease in cloning and for higher plasmid yield in E. coli. The broad host-range replicon of VS1 was shown to be a choice of replicon over those of pRK2, pRi and pSA because of the superior stability and of small well-defined replicon. Newly developed plant binary vectors pLSU has the small size of plasmid backbone (4566 bp) consisting of VS1 replicon (2654 bp), ColE1 replicon (715 bp), a bacterial kanamycin (999 bp) or tetracycline resistance gene, and the T-DNA region (152 bp).