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.

Construction and expression of the bicistronic expression vector with RANTES and SDF-1 genes

Objective: To construct bicistronic expression vector with RANTES and SDF-1 genes, the ligands of HIV-1 principal coreceptors, and identify its expression. Methods: RANTES-KDEL was amplified from plasmid pCMV-R-K by PCR and cloned into eukaryotic expression vector pCMV-S/K. Gene transfection into HeLa cells was carried out by lipofectin. Indirect immumofluorescence and radioimmunoprecipitation were used to confirm the expression of RANTES and SDF-1. Results: The construction of pCMV-R-K-S-K was confirmed by enzymatic digestion and sequencing. RANTES and SDF-1 were shown expressed in HeLa cells by indirect immumofluorescence and radioimmunoprecipitation. Conclusion: pCMV-R-K-S-K was constructed and expressed in cell line Hela successfully, which will contribute to further study of gene therapy of AIDS by HIV-1 coreceptors knockout.

Cloning and construction of sense and antisense eukaryotic expression vector of human Pin1

Objective： To clone and construct eukaryotic expressing vectors of sense and antisense human Pin1 （hPinl） genes. Methods： Total RNA was extracted from MG-63 cells, then the hPinl cDNA was amplified by RT-PCR. The same time the sense and antisense hPinl genes were formed by binding BamH Ⅰ and Hind Ⅲ in cis and trans-directions. At the end they were cloned into the eukaryotic expressing vector pIRES2-EGFP in cis and trans directions using DNA recombinant technology. The recombinant vectors were further identified by digestion of BamHⅠ and Hind Ⅲ. Results： The results of sequencing showed that the orientation of the ligations and the reading frame were correct. After digested by BamH Ⅰ and Hind Ⅲ, two fragments exhibiting 5.3 kb and 0.99 kb were formed in sense and antisense eukaryotic expressing vectors. Electrophoretic results were completely coincident with theoretical calculation. Conclusion： Human Pin1 sense and antisense genes were successfully cloned and eukaryotic expressing vectors were successfully constructed.

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.

Construction of Plant Antisense Expression Vector with Defective in Anther Dehiscence1 Gene Fragment of Chinese Kale

A pair of primers was designed according to the reported conserved sequence of the defective in anther dehiscencel （DAD1） gene ofArabidopsis thaliana and Brassica rapa. A 558 bp long fragment was amplified from genomic DNA of Chinese kale, showing more than 88% identity with the known DAD1 nucleotide sequence and no intron. The reverse of the amplified fragment was ligated to the downstream of the CaMV35S promoter in the plant expression vector pBIl21. Antisense expression vector pBII21-DAD1F was constructed with DAD1 fragment of Chinese kale, and was transferred into Agrobacterium tumefaciens, which will be used in the transformation to create male sterile materials of Chinese kale.

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.

Establishment of the Agrobacterium-mediated Genetic Transformation System of Ginkgo biloba and the Construction of the Expression Vector of Gb-DXR

[Objective] The research aimed to provide reference for increasing the genetic transformation efficiency of Ginkgo biloba mediated by Agrobacterium.[Method] Taking the mature embryos of Ginkgo biloba seeds as explants,after 48 hours＇ pre-cultivation on MS medium in the absence of phytohormone,GUS gene was transmitted into embryos of Ginkgo biloba mediated by three kinds of Agrobacterium.Transient expression of GUS gene activity was observed through histochemical staining,and the influencing factors of the expression of GUS gene were analyzed.And the expression vector of 1-deoxy-D-xylulose-5-phosphate reductoisomerase in the biosynthesis approach of biobalide precursor of Ginkgo biloba was constructed.[Result] A more suitable genetic transformation scheme was obtained as follows：taking embryos of Ginkgo biloba as explants,using EHA105 Agrobacterium with pCAMBIA1304＋ for infection,co-culture for 3 days and GUS staining.The results showed that transient expression rate of GUS after transformation was higher.[Conclusion] The research provide a more effective method for further study on the transgene of Ginkgo biloba.

Construction of PR domain eukaryotic expression vector and its inhibitory effect on esophageal cancer cells

Objective：PR domain is responsible for the tumor suppressing activity of RIZ1.The study aimed to construct human PR domain eukaryotic expression vectors,transfect human esophageal cancer cells （TE13）,and evaluate the anticancer activity of PR domain on human esophageal cancer TE13 cells.Methods：First,mRNA was extracted from human esophageal cancer tissue by RT-PCR,then reversetranscribed to cDNA.After amplifying from the DNA template,PR domain was linked to T vector.Second,after extraction,PR domain was cut using enzyme and linked to pcDNA3.1（＋）.Then,the plasmid was transfered to Trans1-T1 phage resistant competent cells,following by extracting the ultrapure plasmid,and transfecting into TE13 cells.In the end,the protein expression of pcDNA3.1（＋）/PR domain in TE13 was detected by Western blot,and the apoptosis of TE 13 by technique of flow cytometry.Results：More than 5,000 bp purposed band of pcDNA3.1（＋）/PR domain plasmid was found by agarose gel electrophoresis.After transfection,the PR domain （molecular weight of about 28 Da） was found only in 3,4 and 5 groups by Western blot.Flow cytometry assay showed apoptosis in experimental group was significantly more than that in the control group （P＜0.05）.Conclusions：The PR domain eukaryotic expression vector was constructed successfully.The protein of the PR domain could be expressed in esophageal cancer TE13 cells firmly after transfection,and a single PR domain could promote apoptosis of TE13 cells.

Expression of Aminopeptidase N1(APN1),the Main Receptor Protein for Bacillus thuringiensis CrylA Toxin from Helicoverpa armigera Larval Midgut in Trichoplusia ni cells

The aim of this article is to successfully express the Bt （Bacillus thuringiensis） toxin receptor protein located on the internal membrane of larval midgut of cotton bollworm （Helicoverpa armigera Hübner） within eukaryotic expression system, which is one of the key links for clarifying the relationship between receptor and Bt resistance. The fragments of aminopeptidase N1 （APN1） gene without signal peptide in the susceptible and the resistant H. armigera were cloned separately using PCR method, and were separately cloned into pUC 19 vector. After sequencing the gene, the fragments encoding for APN1 without signal peptide were cloned into the Bac-to-Bac baculovirus expression system with transfer vector pFastBacHTB under the polyhedron gene promoter. The recombinant transposing plasmid pFastBacHTB/APN1 was screened and then transformed into Escherichia coli DH10Bac. It was cultured in LB medium, which contained Te, Kan, Ge, X-gal, and IPTG. The resulting recombinant bacmid was transfected into cells of the insect Trichoplusia ni and recombinant baculoviruse was obtained. The lysate of cells infected with recombinant baculoviruse was analyzed by SDS-PAGE and blot analysis. The results showed that the recombinant baculoviruse was fully capable of expressing APN1. The APN1 gene successfully expressed in T. ni cell established the base for continuing the research on its function and relationshio of resistance with Bt.

人泛素启动子UbB介导Fat-1基因在绵羊成纤维细胞内的表达

旨在构建不饱和脂肪酸脱氢酶基因Fat-1表达载体,实现人泛素启动子UbB调控外源基因Fat-1在绵羊细胞内表达。PCR扩增得到UbB序列,秀丽隐杆线虫Fat-1基因密码子根据绵羊对同义密码子使用偏好性优化后,由公司合成获得oFat-1基因序列,以及pcDNA3.1中的GBHpolyA序列共同插入pCMV-DsRed2-1载体的多克隆位点(MCS),得到重组真核表达载体pUbB-oFat-polyA-CMV-DsRed,载体序列经酶切测序鉴定。线性化的表达载体采用脂质体包裹后转染绵羊成纤维细胞,经G418抗性和红色荧光标记筛选得到阳性细胞,并对阳性细胞进行PCR和反转录PCR鉴定,HPLC检测细胞中不饱和脂肪酸的变化。构建的真核表达载体序列正确,Fat-1基因能够在绵羊成纤维细胞基因组中正常表达,促进细胞ω-6PUFAs向ω-3PUFAs转化,ω-3PUFAs占总量的百分比从11.48%增加到24.41%,ω-6PUFAs的百分比从88.52%下降到75.59%,ω-6PUFAs/ω-3PUFAs比值从7.82±0.18下降到3.10±0.03,差异极显著(P<0.01)。成功构建了UbB启动子介导的Fat-1真核表达载体,筛选得到表达不饱和脂肪酸脱氢酶的绵羊细胞,为进一步制备转基因克隆绵羊奠定了基础。

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。

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。

PD-1胞外段cDNA在真核细胞的表达与其功能鉴定

PD L PD 1是参与肿瘤免疫逃避的一条抑制性信号途径。为了用可溶性的PD 1受体阻断PD L PD 1的相互作用 ,将小鼠PD 1胞外段 (aa1 aa16 7)作为独立可溶性分子进行了真核表达 ,并对其功能进行鉴定。构建了编码小鼠PD 1胞外段cDNA(sPD 1)的真核质粒表达载体pPD 1A和编码sPD 1 GFP重组蛋白的真核质粒表达载体pPD 1B ;细胞转染实验表明其表达产物主要是分泌到细胞外的可溶性产物 (sPD 1) ,流式细胞仪检测表明sPD 1可有效结合PD 1配体 ;肿瘤细胞杀伤实验表明 ,sPD 1作用于肿瘤细胞或在脾淋巴细胞激活过程中作用于淋巴细胞 ,均可增强Hsp70 H2 2抗原肽复合物激活的脾淋巴细胞杀伤肿瘤细胞的作用。sPD 1真核表达载体的构建为在肿瘤局部表达抑制性共刺激分子的可溶性受体 ,拮抗肿瘤微环境中负调节因素对T细胞的抑制作用 ,增强机体的抗肿瘤能力 。

编码1,3-丙二醇氧化还原酶同工酶基因yqhD的克隆与高效表达

利用PCR技术从大肠杆菌(Escherichia coli)中扩增出1.16 kb的编码1,3-丙二醇氧化还原酶同工酶的基因yqhD,将其连接到温控表达载体pHsh,得到重组载体pHsh-yqhD,重组载体在大肠杆菌JM109中得到高效表达。SDS-PAGE分析显示:融合表达产物的相对分子质量均为43 000,同核酸序列测定所推导的值相符。对含有yqhD的基因工程菌进行表达研究表明:42℃诱导4 h,1,3-丙二醇氧化还原酶同工酶的酶活力达到100 IU/mg,而对照菌株的酶活力仅为0.5IU/mg。

PLK1基因沉默抑制HeLa细胞凋亡

以高表达Polo样激酶1(Polo-likekinase1,PLK1)的宫颈癌细胞系HeLa细胞为模型,观察针对PLK1基因的短发夹状RNA(shorthairpinRNA,shRNA)对其凋亡和增殖的影响.设计并合成了针对PLK1的shRNA,将其导入构建的携带增强型绿色荧光蛋白(EGFP)的RNAi(RNAinterference)表达载体中.通过RT-PCR和Western印迹分别检测HeLa细胞PLK1基因和蛋白水平的表达,以流式细胞仪和PI-Hochest双染法测细胞凋亡,MTT法检测细胞的增殖水平.成功构建了携带EGFP的RNAi表达载体pEGFP-H1.转染shRNA后,HeLa细胞PLK1的表达降低至30%.与对照组和空载体转染组相比,shRNA转染组的HeLa细胞凋亡率明显增加,其增殖活性则明显降低.本课题构建的RNAi表达载体便于观察靶基因的转染情况,且不影响H1启动子的体内转录.PLK1的基因沉默能明显增加HeLa细胞的凋亡,抑制该细胞的增殖,有可能为未来肿瘤的治疗找到新的靶点和有效途径.

pVAX-WIF-1真核表达载体的构建及其抗肺癌作用

背景与目的 WIF-1是肺癌中重要的抑癌基因之一,其编码蛋白WIF-1能够通过抑制Wnt/β-catenin信号通路达到抑制肿瘤生长增殖及促进凋亡等抗肺癌作用。本研究旨在利用美国食品和药品管理委员会认可用于临床治疗的p VAX载体,构建p VAX-WIF-1真核表达载体,初步探究p VAX-WIF-1在体内外对A549肺癌细胞的抗肿瘤作用。方法应用PCR方法扩增人WIF-1编码序列片段,构建p VAX-WIF-1载体后转染肺癌A549细胞,Western blot检测WIF-1基因的表达;采用MTT法、Hoechst3235染色和流式细胞术分别检测p VAX-WIF-1在体外对肺癌A549细胞增殖、凋亡作用的影响;构建A549小鼠皮下瘤模型,探究p VAX-WIF-1在体内的抗肺癌作用。结果真核表达质粒载体p VAX-WIF-1构建成功,p VAX-WIF-1转染A549细胞后,WIF-1蛋白表达升高。p VAX-WIF-1转染A549后能抑制细胞增殖和诱导凋亡。动物体内结果表明,p VAX-WIF-1能有效抑制肺癌肿瘤生长。结论本研究成功构建了p VAX-WIF-1真核表达质粒,p VAX-WIF-1能明显抑制A549肺癌细胞增殖和促进凋亡,有效抑制A549皮下瘤生长。本研究将为WIF-1基因的肺癌临床治疗奠定了基础。