抗菌肽P113多拷贝表达载体的构建

目的:构建唾液抗菌肽P113基因多拷贝串联重组体,并将其克隆到表达载体pET-28a.方法:根据大肠杆菌偏爱的密码子设计并合成人唾液抗菌肽P113基因,采用PCR技术构建P113基因的自融合多拷贝串联重组体polyP113,BAMH I和HindⅢ双酶切表达载体pET-28a和polyP113基因,将两者连接后转化大肠杆菌DH5α,氨苄青霉素筛选阳性菌落,PCR、酶切、测序鉴定重组质粒.结果:所构建的含有十拷贝P113基因的重组体正确克隆到表达载体pET-28a上,无突变.结论:成功构建含十拷贝唾液抗菌肽P113基因表达框的大肠杆菌表达载体.     

猕猴桃抗菌肽截短体原核表达载体的构建

抗菌肽是生物体先天防御系统中的多肽,协助机体抵御外源微生物的侵害.天然抗菌肽提取难度大,不利于其开发利用,其旨在探究猕猴桃抗菌肽体外高效制备方法.本研究通过生物信息学方法进行序列分析与基因克隆合成猕猴桃截短体抗菌肽(Truncated Antimicrobial Peptide,TrAMP)序列,并将其连接到原核表达载体pET-47b(+)+Fa-AMP上,转化获得其BL21(DE3) pLysS工程菌,经PCR和测序鉴定,表明重组表达载体构建成功,并经生物信息学模型预测显示抗菌肽具有抑菌活性,为进一步研究猕猴桃抗菌肽体外诱导表达及生物学功能验证奠定基础.     

例析未知序列的PCR扩增

本文例析并介绍了未知序列的PCR扩增方法.要对已知序列两侧的未知序列进行测序分析,可将其连成环状,利用已知序列设计引物,进行反向PCR.为减少随机片段的含量,可再进行巢式PCR.未知序列也可用热不对称交错PCR来扩增.     

基于可调子块迭代的加速SAGE算法在PET图像重建中的应用

提出了一种可调子块迭代(RBI)方法加速空间交替广义期望最大(SAGE)算法的收敛性.新的可调子块迭代的空间交替广义期望最大算法(RBI-SAGE)组合了RBI算法和SAGE算法的优点用于加速正电子发射断层(PET)图像重建.RBI-SAGE将投影数据分成不连续的子块,每一次迭代仅包含一个这样的子块.在每一个子块中用SAGE算法序列更新参数.实验中,运用RBI-SAGE算法与SAGE算法对PET图像进行重建.结果表明,RBI-SAGE收敛性能比SAGE算法优越,且重建图像质量较高.     

人铁蛋白基因的克隆与原核表达

通过PCR技术扩增出人铁蛋白基因,经酶切后与表达载体质粒pGEX-4T-2连接,重组质粒转化感受态大肠杆菌,利用菌落PCR、质粒双酶切、测序,证实成功地构建了人铁蛋白基因表达载体,利用IPTG对重组菌进行诱导表达,通过尿素洗涤纯化目的蛋白用于制备抗体．     

FTA滤膜用于PCR检测肉中志贺氏菌的研究

建立直接从肉中检测志贺氏菌的快速、敏感、特异的PCR检测方法．根据GenBank公布的志贺氏菌属的侵袭性质粒抗原基因IpaH的保守序列设计特异性引物．经过PCR扩增得到393bp的产物．经过DNA测序证实该产物为目的扩增产物．使用FTA滤膜处理样品．再通过PCR方法检测志贺氏菌．猪肉、牛肉、羊肉检测的检出限均为10CFU／mL匀浆,可在6h内完成对肉中志贺氏菌的检测．FTA滤膜用于PCR检测肉中志贺氏菌灵敏度高,耗时短,为肉中志贺氏菌的快速检测提供了新的方法．     

猪圆环病毒Ⅱ型河南地方株ORF4的克隆及原核表达

根据GenBank数据库中猪圆环病毒Ⅱ型的基因组序列设计引物,采用PCR技术从病料基因组DNA中扩增出PCVⅡ河南地方株ORF4基因,全长180 bp,编码59个氨基酸.将该基因克隆至载体pGEX-4T-3中形成pGEX-4T-3-ORF4表达载体.经PCR、酶切和测序鉴定后,转化表达菌株BL21（DE3）诱导表达.SDS-PAGE结果显示：ORF4能够在大肠杆菌中表达,产物的分子量约为32 kD,且以包涵体形式存在.Western Blot检测结果显示,纯化后的ORF4蛋白能够与鼠抗6＆#215;His标签单克隆抗体发生特异性反应,为进一步研究PCVⅡORF4蛋白的特性与功能奠定了基础.     

Txt5腺病毒载体构建及其在心肌细胞中的表达

设计小鼠Txt5基因的特异性引物,将小鼠c DNA作为模板,用PCR扩增出m Txt5编码区,并加入HA标签序列.将这一片段插入p MD-18T载体,再亚克隆至p Adtrack-cmv穿梭载体上.线性化后,转化BJ5183感受态细胞,在BJ5183中发生同源重组获得p Ad-Txt5质粒.p Ad-Txt5线性化后转染293A细胞,包装得到含Txt5基因的病毒.将病毒溶液感染大鼠原代心肌细胞,一段时间后观察绿色荧光,然后通过RT-PCR和免疫印迹法检测HA标签蛋白的表达.结果表明成功构建小鼠Txt5腺病毒表达载体并实现在大鼠原代心肌细胞中的表达.     

幼儿社会技能——进入儿童群体的重要社会标签

社会标签是社会、他人或社会组织给有关人员加上的一个身份证明,在儿童群体中以人缘的好坏来衡量。社会技能是个体经过学习获得的,从“同伴接纳”的角度分析,主要用于评定同伴接纳或者人际关系(如同伴社会测量地位)。幼儿社会技能是进入儿童群体的重要社会标签,社会技能的获得将促使幼儿良好的心理行为的发生。根据社会技能干预的理论,本文提出社会技能干预训练的方法,以帮助幼儿成功获得进入儿童群体的社会标签。     

Study of Resistin gene expression in peripheral blood mononuclear cell and its gene polymorphism in a small range population

INTRODUCTION Recently discovered as an endocrine organ, adi-pose tissue can secrete many cytokines such as Re-sistin, adiponectin and free fatty acids, and receives much attention in the study of insulin resistance and type 2 diabetes mellitus (T2DM). As a peptide hor-mone secreted by adipocyte, Resistin is considered to be the linkage between obesity and insulin resistance (Steppan et al., 2001). Most studies on Resistin in-vestigate adipose tissue. In this experiment we de-tected t…     

Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B

INTRODUCTION Chronic hepatitis B virus (HBV) infection is aserious clinical problem because of its wide distribu-tion and possible adverse consequences, such as he-patic decompensation, cirrhosis and/or primary livercancer (PLC). The natural course of chronic HBVinfection is characterized by a series of hepatitic flaresor exacerbations and remissions (Ganem and Prince,2004). The severity, extent, duration and frequency ofhepatic histopathological changes in hepatitic flaresare d…     

A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1×106ml-1, the percentages of conidium germination and appressorium formation were (97.98±0.67)% and (97.88±0.45)%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 pg total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA (complementary DNA) library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.     

基因克隆常见方法简介

本文对目前常用的一些基因克隆方法包括:抑制性差减杂交、差异显示PCR、DNA代表性差 异分析、cDNA微量列阵法(microarray)、外显子捕获法(exon capturation)、序列基因表达测(serial analysis of gene expression,SAGE)和图位克隆(mapbased cloning)等等作了简要的介绍;同时还介绍了从基因片段获得 其全长的3'RACE或5'RACE技术。可供相关研究人员参考。     

SSR分子标记技术在遗传学实验教学中的应用

该实验是科研成果转化为实验教学的一个案例。选择位于水稻不同连锁群上的6对SSR引物构建了2个杂交稻及其亲本的SSR指纹图谱,建立了一套适合于本科生实验的杂交水稻亲本鉴定的稳定的SSR技术体系。筛选的6对引物进行PCR扩增得到的杂交稻均有2条带。由SSR指纹图谱分析可得:杂交稻P5的亲本是P2和P4,P6的亲本是P1和P3。通过本实验巩固了学生关于分离定律的学习,并有所延伸。     

SARS肺组织毒的分离及其全基因组cDNA文库的构建

利用常规病毒分离方法，自死亡病人肺组织病料剖检样品中成功分离到了强致病性SARS-Coy肺组织毒。根据已发表的SARS-Coy Tor2株序列，设计并合成了38条覆盖全长基因组的PCR引物。应用RT-PCR技术从实验感染的Vero E6细胞上清中成功地扩增出了各相应的cDNA重叠片段，分别克隆至pGEM-T载体后，构建了SARS-Coy肺组织毒全基因组cDNA文库。SARS-Coy肺组织毒全基因组cDNA文库的构建为进一步研究SARS-Coy的分子生物学特性奠定了基础。     

粉渣替代部分粮食喂猪试验

用粉渣代替部分粮食喂肥育猪。试验用 30千克左右的长白杂交猪 2 0头 ,分成试验和对照组 ,每组 1 0头 ,公母各半 ,试验组饲喂用粉渣代替部分粮食的自配饲料 ,而对照组饲喂常规配合饲料。结果表明试验组与对照组净增重、日增重和日耗料无显著差异 ,而试验组的日支出低于对照组的 ,日增收高于对照组的。暗示粉渣可代替部分粮食喂猪     

Characterization of enhancer trap and gene trap harboring Ac/Ds transposon in transgenic rice

INTRODUCTIONAfterthewholericegenomewassequencedsuccessfully,moreandmoregenescanbepredictedwithdifferentsoftwaresandannotationsystem.However,thefunctionsofpredictedgeneshavetobefurtheridentifiedbybiologicalanalysis.Oneofthemostpowerfulmethodsassigningfunctiontogeneisthroughinsertionmutagenesiswithtrans-posableelementasDNAsequencetag.Ac/Dstrans-posonsystemhasbecomeaverypopulartoolforgenetaggingandfunctionalgenomicsinvariousplantspecies(Altmannetal.,1995;Chinetal.,1999;Enokietal.,1999;Gre…     

Characterization of enhancer trap and gene trap harboring Ac/Ds transposon in transgenic rice

Insertion mutagenesis has become one of the most popular methods for gene functions analysis. Here we report a two-element Ac/Ds transposon system containing enhancer trap and gene trap for gene tagging in rice. The excision of Ds element was examined by PCR amplification. The excision frequency of Ds element varied from 0% to 40% among 20 F2 populations derived from 11 different Ds parents. Southern blot analysis revealed that more than 70% of excised Ds elements reinserted into rice genome and above 70% of the reinserted Ds elements were located at different positions of the chromosome in rice. The result of histochemical GUS analysis indicated that 28% of enhancer trap and 22% of gene trap tagging plants displayed GUS activity in leaves, roots, flowers or seeds. The GUS positive lines will be useful for identifying gene function in rice.