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.

ENO1蛋白及其相关活性位点缺失突变蛋白在昆虫杆状病毒表达系统中的表达与鉴定

目的:利用昆虫杆状病毒表达系统(昆虫BEVS)表达糖酵解酶α-烯醇化酶(ENO1)及其3种酶活性位点缺失突变的ENO1蛋白ENO1-M1、ENO1-M2和ENO1-M3,为后续宫颈癌的代谢治疗研究奠定基础。方法:利用分子克隆技术将优化后ENO1序列插入pFastBacTM1载体,获得含有目的基因的重组质粒pFastBac-ENO1。分别缺失ENO1发挥糖酵解酶功能的3个活性位点,进行优化后将其插入pFastBacTM1载体,获得3个活性位点缺失的重组质粒pFastBac-M1、pFastBac-M2和pFastBac-M3。通过转座、转染后获得重组杆状病毒rBV-ENO1、rBV-M1、rBV-M2和rBV-M3,利用WB法对目的蛋白的表达及特异性进行检测。结果:成功扩增重组杆粒rBacmid-ENO1、rBacmid-M1、rBacmid-M2和rBacmid-M3,获得大小约2000 bp的基因片段,与预期大小相符。昆虫BEVS可表达ENO1蛋白及其3个酶活位点缺失的重组蛋白ENO1-M1、ENO1-M2和ENO1-M3,其分子量约为52000,与预期相符。WB法鉴定这些蛋白能与特异性标签His-tag发生反应。结论:通过昆虫BEVS成功表达目的蛋白ENO1及其酶活性位点缺失蛋白ENO1-M1、ENO1-M2和ENO1-M3,这些蛋白具有反应原性,为后续测定这些蛋白与ENO1单抗亲和力创造了条件。

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。

2015–2022年漓江流域12天时间分辨率地表水体面积数据集

喀斯特地貌在广西地区分布广泛,具有汛期降水强度大、水文过程迅速且时空异质性高等特点。水是重要的生态基础和自然资源,及时、准确地获取地表水体的时空变化特征,对于水资源的保护与管理、旱涝灾害的快速评估以及实现各个经济产业的可持续发展等具有现实意义。本数据集以广西桂林漓江流域为研究区域,针对当前地表水体监测频次低、水体提取方法受限于样本信息以及在复杂地形区域内提取精度较低等问题,结合Sentinel-1雷达和Sentinel-2光学等多源遥感数据,基于雷达和光学传感器成像原理的不同,提出了一种基于经验阈值进行矢量分类的方法,剔除了绝大部分地物阴影的干扰,构建了2015–2022年漓江流域时间分辨率为12天、空间分辨率为10米的地表水体面积数据集,共包括196期地表水体矢量数据。通过对数据集的空间分布和定量精度验证,结果表明:本数据集对河流、湖泊和水库的入、出水口以及复杂地形区域内小型水体等的提取精度较高,总体精度达到92.73%,Kappa系数为0.85。本数据集可用于支撑漓江流域水资源保护和生态环境的可持续管理,能够为应急管理、灾害防治、水利开发和经济发展等领域的决策提供可靠数据。

双特异性磷酸酶-1对小胶质细胞极化及颞叶癫痫发作的影响

目的探讨双特异性磷酸酶-1(DUSP1)对小胶质细胞极化及颞叶癫痫(EP)发作的影响。方法采用慢病毒转染技术构建DUSP1正常表达组(DUSP1^(normal)组)、DUSP1高表达组(DUSP1^(over)组)、DUSP1低表达组(DUSP1^(low)组)小鼠,每组12只,将这些小鼠又分为DUSP1^(normal)+对照(Con)组、DUSP1^(normal)+EP组、DUSP1^(over)+Con组、DUSP1^(over)+EP组、DUSP1^(low)+Con组和DUSP1^(low)+EP组,每组6只。取DUSP1^(normal)+EP组、DUSP1^(over)+EP组及DUSP1^(low)+EP组小鼠分别腹腔注射氯化锂溶液(125 mg/kg)、硫酸阿托品溶液(1 mg/mL)及盐酸匹罗卡品溶液(50 mg/kg),构建颞叶EP模型。评估EP小鼠注射后2 h内EP发作等级,记录达到Ⅳ级的时间为潜伏期。将EP小鼠处死,取完整脑组织用于免疫荧光检测,取颞叶组织用于炎症细胞因子、DUSP1及小胶质细胞调控蛋白检测。结果与DUSP1^(normal)组比较,DUSP1^(over)组DUSP1/β-actin及DUSP1 mRNA表达水平均升高,DUSP1^(low)组DUSP1/β-actin及DUSP1 mRNA表达水平均降低,差异均有统计学意义(P<0.05)。与DUSP1^(normal)+EP组比较,DUSP1^(over)+EP组小鼠EP发作达Ⅳ级潜伏期明显延长,DUSP1^(low)+EP组小鼠EP发作达Ⅳ级潜伏期明显缩短,差异均有统计学意义(P<0.05)。与DUSP1^(normal)+Con组比较,DUSP1^(normal)+EP组小鼠颞叶组织中M1型小胶质细胞极化标志物CD16、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β及诱导型一氧化氮合酶(iNOS)mRNA水平均升高,炎症细胞因子TNF-α、IL-6水平均升高,IL-10水平降低,DUSP1诱导小胶质细胞极化相关蛋白p-胞外调节蛋白激酶(ERK)1/2、p-c-Jun氨基末端激酶(JNK)和p-p38蛋白表达水平均升高,差异均有统计学意义(P<0.05)。与DUSP1^(normal)+EP组比较,DUSP1^(over)+EP组小鼠颞叶组织中M1型小胶质细胞极化标志物CD16、TNF-α、IL-1β和iNOS mRNA水平均降低,M2型小胶质细胞极化标志物CD206、转化生长因子-β、精氨酸酶1与几丁质酶样蛋白mRNA水平均升高,炎症细胞因子TNF-α、IL-6水平均降低,IL-10水平升高,DUSP1诱导小胶质细胞极化相关蛋白p-ERK1/2、p-JNK和p-p38蛋白表达水平均降低,差异均有统计学意义(P<0.05);与DUSP1^(normal)+EP组比较,DUSP1^(low)+EP组小鼠颞叶组织中M1型小胶质细胞极化标志物TNF-α和iNOS mRNA水平均升高,炎症细胞因子TNF-α、IL-6水平均升高,IL-10水平降低,DUSP1诱导小胶质细胞极化相关蛋白p-ERK1/2、p-JNK和p-p38蛋白表达水平均升高,差异均有统计学意义(P<0.05)。结论氯化锂溶液-盐酸匹罗卡品溶液点燃EP小鼠颞叶组织中DUSP1蛋白表达水平降低,高表达DUSP1基因能够延长小鼠EP发作的潜伏期,其机制可能涉及抑制p-ERK1/2、p-JNK和p-p38蛋白表达来调节小胶质细胞向M2型极化。

过表达溶质载体家族1成员5和敲低慢病毒载体构建及稳定转染RAW264.7细胞株

背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供有力的实验工具。目的:构建小鼠SLC1A5过表达和敲低的慢病毒载体,以建立稳定转染的RAW264.7细胞株,为深入探讨SLC1A5在炎症中的作用提供实验基础。方法:根据SLC1A5基因序列设计合成引物并使用聚合酶链反应扩增该基因片段。将目的基因定向接入经Age I/Nhe I酶切的载体质粒GV492中构建重组慢病毒质粒,对阳性克隆进一步筛选后测序比对结果;pHelper1.0质粒载体、pHelper2.0质粒载体、目的质粒载体与293T细胞共同培养并转染,获得慢病毒原液进行包装和滴度测定;在此基础上,通过体外培养RAW264.7细胞,确定嘌呤霉素工作质量浓度;不同滴度的慢病毒分别与RAW264.7细胞共同培养,根据荧光强度确定转染效率;用嘌呤霉素挑选出稳定转染细胞,实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测稳定转染细胞株的SLC1A5基因和蛋白表达水平。结果与结论:(1)测序序列与目的序列一致提示重组慢病毒载体构建成功;(2)过表达SLC1A5慢病毒的滴度为1×10~9 TU/mL,敲低SLC1A5慢病毒的滴度为3×10~9 TU/mL;(3)确定RAW264.7细胞嘌呤霉素工作质量浓度为3μg/mL;(4)过表达/敲低SLC1A5慢病毒转染RAW264.7细胞的最佳条件皆为HiTransG P转染增强液且感染复数值等于50;(5)过表达SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量明显上调,而敲低SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量显著下调。结果表明,成功构建了小鼠SLC1A5过表达和敲低的慢病毒载体并获得稳定转染的RAW264.7细胞株。

Construction of Recombinant Retroviral Vector Containing HIV-1 Tat Gene and Functional Detection of Expressed Tat in Target Cells

Objective: To construct recombinant retroviral vector containing HIV-1 Tatgene and evaluate the junction of the expressed Tat in target cells. Methods: HIV-1 Tat_(101) genewas recovered from pEV plasmid by Hind Ⅲ digestion and cloned into expression plasmid LZESpBMN-Z toconstruct recombinant retroviral expression plasmid named LZRS-Tat_(101). Using the method ofcalcium phosphate, the construct of LZRS-Tat_(101) was then transfected into packaging cell linesPhoenix (ΦNX) which contained env and gal genes encoding structural proteins and pol gene codingfor 3 enzymes ( reverse transcriptase, protease and integrate) essential for retroviral integrationand replication . The stable transfected cell lines was obtained using puromycin to screen for morethan 3 days. Then, immunohistochemical (IHC ) staining was carried out to detect the expressionlevel of Tat_(101) protein in both transiently and stably trancfected ΦNX, respectively. Thesupematants containing recombinant virus collected from transient and stable transfected cells wereemployed to infect 293 cells, respectively, and the expressed Tat in 293 cells was tested by Westernblot. Meantime, the supematants of infected 293 cells was further added to HL3T1 cells which wereHela cell lines containing an HIV-1-LTR/CAT reporter construct to establish a co-culture system.After co-culture for 72 hours, the protein was extracted from HL3T1 cells and used for CAT activityassay. Results: After LZRS- Tat_(101) was transfected into ΦNX, the amount of expressed Tat intransient transfection cells was significantly higher than that in stable transfection cells; Tatcould be detected not only in 293 cells but also in the supematants from 293 cells culture, and Tatin the supematants could activate HIV-1 LTR promoter in HL3T1, resulting in high 'expression of CATlocated at the downstream of LTR. Conclusion: The construct of recombinant retrovirus LZRS-Tat_(101) could express Tat protein in target cells and the expressed Tat was functionally activeand can really exhibit the ability to activate transcription.

Cloning of Cotton Delta-12 Oleate Desaturase Gene FAD2-1 and Construction of Its ihpRNA and amiRNA Interference Vectors

Delta-12 oleate desaturase gene （FAD2-1） which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of cottonseed oil. By employing RT-PCR method, full length cDNA of cotton delta-12 oleate desat- urase gene GhFAD2-1 containing an open reading frame of 1 158 bp was cloned for constructing RNAi vector. A 515 bp long specific fragment of this gene was se- lected for constructing ihpRNA vector under the control of a seed-specific promoter NAPIN, named pFGC1008-NAPIN-FAD2-1; meanwhile miRNA gene-silencing vector pCAMBIA1302-amiRNA-FAD2-1 targeting GhFAD2-1 was also constructed.

Construction of Prokaryotic Expression Vectors of EBP1 Gene from Nervilia Fordii (Hance) Schltr.

[Objective] To construct prokaryotic expression vectors encoding gene Erb3binding protein （EBP1）, which plays important roles in regulating plant organ size from Nervilia fordii （Hance） Schltr. [Methods] PCR products of NfEBP1 with particular restriction sites and expression vectors, pET-28 and pET-16b were digested. Ligation, transformation and selection were performed to construct the recombinant plasmids pET-28-NfEBP1 and pET-16-NfEBP1. The recombinant plasmids were transformed into E. coli BL21 using heat -shock transformation. [Results] Recombinant plasmids pET-28-NfEBP1-1188 and pET-16-NfEBP1-1188 were constructed and transformed into expressional host cells, E. coli BL21, and validated by colony PCR, sequencing and double digestion. [Conclusion] Prokaryotic expression vectors of EBP1 gene from N. fordii were successfully constructed, which laid the foundation for characterization of the gene function.

Cloning of Broad-spectrum Anti-disease NPR1 Gene with RT-PCR and Construction of Its Protein Expression Vector

[Objective] It is to clone broad-spectrum anti-disease gene NPR1 and to construct its protein expression vector.[Method] First to extract total RNA of Arabidopsis thaliana and design relevant primers,and then the method of reverse transcription PCR was adopted to clone.With the method of enzyme digestion and ligation,this gene will be directed into protein expression vector.[Result] After relevant testing,NPR1 was inserted into vector pMXB10 to obtain pMXB10-NPR1 protein expression vector.[Conclusion] Protein expression vector including NPR1 was successfully constructed.

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.