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1.
hSOD基因在大白鼠肺上皮细胞中的表达 总被引:1,自引:1,他引:0
吴晓英 《华南理工大学学报(自然科学版)》1998,26(12):40-44
本文以哺乳动物细胞表达载体pRc/CMV为运载体,构建了受控于CMV启动子的hSOD基因的表达载体pRc/CMV_SOD,用脂质体转染法转染大白鼠肺上皮细胞L2细胞后,经抗生素G418选择性培养,获得与细胞染色体DNA稳定整合的hSODcDNA导入细胞,经SouthernBlots和WesternBlots分析,结果表明,hSOD在细胞中得到稳定表达,同时测定细胞SOD酶活性,hSODcDNA导入细胞的SOD酶活性是空载体导入细胞和父本细胞的约2倍,并且在选择细胞克隆之后的60天内是持续的. 相似文献
2.
用PCR扩增和克隆鸡贫血病毒衣壳蛋白VP2基因 总被引:1,自引:0,他引:1
接种鸡贫血病毒(CAV)Cux-1毒株于MDCC-RP1细胞中,并用经狄高辛标记的CAV衣壳蛋白VP1基因克隆DNA作为探针在斑点杂交中检测和比较了各代细胞中CAV DNA水平。通过聚合酶链式反应(PCR),从CAV DNA水平较高的MDCC-RP1细胞基因组DNA中扩增出CAV衣壳蛋白VP2基因片段约650bp的编码序列,将该PCR扩增的产物于EcoRI和KpnI位点克隆到pUC19质粒载体中, 相似文献
3.
斜纹夜蛾NPV多角体基因的克隆和部分测序 总被引:1,自引:1,他引:1
本文对SINPV基因组作了酶解分析,测得其基因组大小为145kb,并用双酶法确定了SINPV基因组的HindⅢ和PstⅠ物理图谱。以含AcNPV多角体基因的质粒pAC-Ⅰ的SalI-C片段为探针,对SINPVDNA酶切片段southern转印杂交结果,初步判断多角体基因定位于PstI-B/C/D片段、BglⅡ-C/D片段、BamHI-B/C片段和EcoRI-A/B片段上,且SINPV与AcNPV多角体蛋白基因有64%的同源性,而以大肠仟菌质粒pUC19为载体对SINPV的多角体基因试克隆,得到带有BglⅡ-PstⅠ双酶切片段的2个克隆子。对这两个杂交阳性克隆子之一的核苷酸序列测定,表明插入片段与BmNPV多角体基因上游序列亦有一定同源性。 相似文献
4.
在scu-PA32K的cDNA分子基础上经定点突变,在N端紧接Leu^1以前引入编码GHRP四肽的寡核苷酸序列(GGTCATAGGCCT),构建了GHRP-scu-PA-32K的突变体cDNA。将它克隆到表达载体pCM-β-dhfr共转染CHO/DHFR^-细胞。筛选到的稳定表达株在无 清培养基的表达量为580IR/(10^6细胞.24h)。经锌离子螯合亲和柱纯化的产物,SDS-PAGE显示为一蛋 相似文献
5.
乙型肝炎病毒(HBV)DNA免疫的初步研究 总被引:1,自引:1,他引:0
用聚合酶链反应(PCR)扩增了编码HBVsAg的基因序列,将其插入到pcDNA3载体中,位于人巨细胞病毒(CMV)早期启动子下游.重组质粒pcDNA3-sAg转染细胞后检测到HBsAg表达.用纯化后的重组质粒直接注射到BALB/C小鼠骨骼肌内,诱发实验小鼠产生了抗HBsAg特异性抗体.PCR扩增未检测到注射部位及肝脏细胞染色体中有外源HBVDNA整合 相似文献
6.
用聚合酶链反应(PCR)扩增了编码HBVsAg的基因序列,将其插入到pcDNA3载体中,位于人巨细胞病毒(CMV)早期启动子下游,重组质粒pcDNA3-sAg转染细胞后检测到HBsAg表达,用纯化后的重组质粒直接注射用BALB/C小鼠骨骼细内,诱发实验小鼠产生了抗HBsAg特异性抗体,PCR扩增未检测到注射部位及肝脏细胞染色体中有外源HBVDNA整合。 相似文献
7.
从蓝藻Calothrixsp.PCC7601染色体DNA中分离出含藻蓝蛋白基因cpcB2A2的略3.8KbDNA片段,与质粒pUC18重组,构建成一种重组质粒,pPC218-PCZ,转化E.codiJM109,获得了一系列克隆子,经斑点分子杂交和限制性内切核酸酶分析,筛选出了初步认为含有cpcB2A2基因的克隆子。 相似文献
8.
《南京大学学报(自然科学版)》1995,(3)
ONTHEEXPONENTSETOFPRIMITIVELOCALLYSEMICOMPLETEDIGRAPHS¥ZhangKeming1);BuYuehua2)(1)DepartmentofMathcmaties,NanjingUniversity,N... 相似文献
9.
新型数控遥测生产测井系统SGX—2的研制 总被引:2,自引:0,他引:2
鞠晓东 《石油大学学报(自然科学版)》2000,24(1):88-90,97
介绍了一种命名为SGX-2的生产测井专用地面系统,它可与大多数常规生产测井下井仪配接,完成类似AT^+系统的功能。该测井系统以高性质工业PC为控制中心,具有8个12位BGA模拟输入通道,其中有2个专门用CCL和电缆记号采集。3个可程控门槛电平的脉冲输入通道支持Manchester及单芯半双工电缆数据传输。数据采集、遥测及CBL-VDL均为内嵌MPU的接口模块。系统软件是以OOP方法设计的纯32位软 相似文献
10.
邹荣贤 《西华师范大学学报(哲学社会科学版)》1996,(1)
简易多用微型实验装置邹荣贤(四川师范学院化学系,南充637002)ASIMPLEBUTMULTI-PURPOSEMINI-EXPERIMENTDEVICE¥ZhouRongxian(Dept.ofChemistry,SichuanTeachersCi... 相似文献
11.
We had isolated and identified two Cucumber mosaic virus (CMV) isolates, the CMV red bean (CMV-RB) isolate and the CMV pea (CMV-P1) isolate. CMV-RB induces necrotic local lesions
on inoculated leaves of broad bean, pea, cowpea and bean, and could not infect these hosts systemically. However, CMV-P1 was
able to infect these legumes systemically. To study the difference of pathogenicity on the legumes induced by these two CMV
isolates, the full-length infectious cDNA clones of CMV-Fny, which induced similar symptoms as CMV-RB in the four legumes,
were used. The 243 nucleotides fragment, which encodes highly conserved GDD amino acid motif on 2a replicase gene of CMV-Fny
RNA2, was replaced with that of CMV-P1. The constructed chimeric virus FP could infect these legumes systemically. The exchange
of this region changes the virus symptoms on the legumes, indicating that this 243 nucleotides fragment has major effect on
pathogenicity of CMV on the legumes. 相似文献
12.
The pathogenicity on legumes of Cucumber mosaic virus was determined by 243 nucleotides on 2a polymerase gene of viral RNA2 总被引:1,自引:0,他引:1
We had isolated and identified two Cucumber mosaic virus (CMV) isolates, the CMV red bean (CMV-RB)isolate and the CMV pea (CMV-P1) isolate. CMV-RBinduces necrotic local lesions on inoculated leaves of broad bean, pea, cowpea and bean, and could not infect these hosts systemically. However, CMV-P1 was able to infect these legumes systemically. To study the difference of pathogenicity. on the legumes induced by these two CMV isolates, the full-length infectious cDNA clones of CMV-Fny, which induced similar symptoms as CMV-RB in the four legumes,were used. The 243 nucleotides fragment, which encodes highly conserved GDD amino acid motif on 2a replicase gene of CMV-Fny RNA2, was replaced with that of CMV-P1. The constructed chimeric virus FP could infect these legumes systemically. The exchange of this region changes the virus symptoms on the legumes, indicating that this 243 nucleotides fragment has major effect on pathogenicity of CMV on the legumes.`` 相似文献
13.
对侵染十字花科小青菜的黄瓜花叶病毒YN分离物(CMV-YN)RNA3进行全长克隆和序列分析.CMV-YNRNA3全长2220nt,分别编码279个氨基酸的3a蛋白和218个氨基酸的CP.序列同源性比较结果如下;CMV-YNRNA3核苷酸及其编码蛋白的氨基酸序列与亚组IA株系CMV-Fny、亚组IB株系CMV-Nt9、亚组Ⅱ株系CMV-Q的同源性,RNA3序列分别为92.7%、96.7%、74.2%,3a蛋白氨基酸序列分别为96.4%、98.6%、83.2%,CP氨基酸序列分别为97.7%、98.2%、83.1%.该结果表明CMV-YN与亚组IB株系CMV-Nt9的同源关系更密切.对CP核苷酸序列的系统进化树分析表明:CMV-YN归属于亚组IB,本研究为首次报道侵染我国十字花科植物的CMV基因组序列. 相似文献
14.
Min Zhang Long Yu Qiang Tu Peirong Hu Qi Zhang Anding Bi Chunling Jiang Shouyuan Zhao 《科学通报(英文版)》1999,44(9):799-799
An EST (gb/AA115239) with high identity to the mouse cytokine signal transduction inhibitor genemmSOCS-2 was selected in GenBank EST database by the homologous screening method. The cDNA with the same sequence of the EST was got
in human placenta cDNA library by PCR and a 1011 bp cDNA fragment was selected using above cDNA as probes to perform walking
hybridization in placenta cDNA library. The cDNA fragment contains one 594 bp open reading frame (ORF) which encodes 198 amino
acid residues. It was proved to be novel after NCBl database screening. Homology comparison showed that this gene has 93%
identity tommSOCS-2 at the amino acid level and it has high identities to other related genes in SH2 domain and SOCS box, so it was namedhumSOCS-2 and the accession number in GenBank is gb/AF020590. The expression analysis showed that the gene is expressed obviously higher
in prostate than in other 15 human tissues. 相似文献
15.
JIA Chunping YAN Jingbin XIAO Yanping FANG Yudan HUANG Shuzheng ZENG Yitao 《自然科学进展(英文版)》2003,13(9):672-677
The expression plasmids CMV/GFP, HS2ALL, HS3ALL and HS23ALL were selected to investigate the effect of HS2 and HS3 element on erythroid-specific expression in transgenic mice. These plasmids were digested with restriction enzymes and purified. And five DNA fragments, CMV/GFP, HS2/GFP, CMV/HS2/GFP, HS23/GFP and HS3/GFP were obtained. After purification, the above DNA fragments were microinjected into the pre-nuclei of the mice fertilized eggs and transgenic mice were generated, with an integration rate of 10.89%. The green fluorescence protein(GFP) expression in many transgenic mouse tissues was determined by FACS analysis. The results showed that the HS2 and 1.7 kb of β-globin gene promoter were sufficient for the erythroid-specific expression of β-globin gene. The GFP expression of different recombinant constructs was also analyzed in blood of all the transgenic mice with FACS. The results indicated that HS2 and HS3 had the same enhancement activity on the regulation of β-globin gene expression. Moreover, these two elements showed a significant synergistic effect on gene expression at the transgenic mouse level, although the GFP expression varied largely among different transgenic mouse litters. 相似文献
16.
为了得到长白猪蛋白激酶Akt1和Akt2基因序列并分析其表达模式,本研究使用RT-PCR方法,首先克隆了蛋白激酶Akt1和Akt2的cDNA.序列分析显示:长白猪Akt1基因的cDNA全长1461bp,编码具480个氨基酸残基的前体蛋白,其氨基酸序列与人,牛,大鼠,小鼠同源性达到97%以上.长白猪Akt2基因cDNA全长为1505bp,编码具481个氨基酸残基的前体蛋白,其氨基酸序列与人,牛,大鼠,小鼠的同源性高达97%以上.SMART分析表明,猪Akt1和Akt2蛋白均包含了与PI-3K结合的PH结构域及2个具有丝氨酸/苏氨酸激酶催化活性的S_TKc结构域.RT-PCR检测结果显示:Akt1mRNA在垂体、心脏、肝脏、脾脏、肌肉组织中高表达,在大脑、小脑、肾脏表达丰度较低.Akt2则在小脑、垂体、心脏、肝脏、脾脏和肌肉组织中高表达,而在大脑和肾脏中表达丰度较低. 相似文献
17.
Yuting Chen B. Bardoni Ming Yu Ning Zhu Guanyun Wu J. L. Mandel Yan Shen 《科学通报(英文版)》2000,45(6):516-520
Fragile X syndrome is the most common form of inherited mental retardation disease, resulting from absent of expression of
its disease geneFMR1. To study the function of the fragile X mental retardation protein (FMRP) through protein/protein interaction, a mouse embryo
cDNA library was screened by the yeast two-hybrid system. A clone was found to interact specifically with FMRP. The cDNA of
this clone (Genbank accession number af 102875) encoded a protein highly homologous to human G/T mismatch-specific DNA thymine
glycosylase (hTDG). Interactions between various alternatively spliced FMRP isoforms and a series of mTDG deletion proteins
were further studied in the yeast two-hybrid system and their interaction amino acid regions were determined. Interaction
between FMRP and TDG existed inside exon 13 of FMRP (amino acid residue 397–425) and around amino acid residue 122–346 of
TDG. These results will be helpful to the study of the biological role of FMRP. 相似文献
18.
构建携带p53和p21基因的重组腺病毒转移载体以研究p53和p21联合基因治疗效果.将p53cDNA克隆到转移载体pCMV5GFP替代gfp,得到pCMVp53,使其受到CMV启动子的调控;同时将p21基因克隆到pCMVp53,得到重组腺病毒转移载体pCMVp53/p21.得到携带p53和p21基因的重组腺病毒转移载体. 相似文献
19.
ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of
suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using
degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR
and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal
1–25 amino acid sequence is a predicted signal peptide and the other 26–216 amino acid sequence is a mature peptide. The 26–45
amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results
indicated that the expression protein is a Ca2+-dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation
for elucidating biological functions of ECBP21 by using molecular biological means. 相似文献
20.
钠离子依赖的中性氨基酸转运蛋白SNAT2在哺乳动物组织中广泛表达,具有转运中性氨基酸的功能,在谷氨酸-谷氨酰胺循环、肝脏糖质新生等生物通路中发挥重要作用.为了方便测定SNAT2在细胞膜上的表达和定位,本研究采用PCR扩增和酶切连接将HA标签蛋白与SNAT2的C末端连接,构建了真核生物表达载体pBK-CMVA(1098—1300)-SNAT2-HA表达载体.用脂质体转染法将该表达载体瞬时转染到HEK293T细胞中,通过Westernblot法检测到SNAT2-HA融合蛋白在细胞膜上的正确表达和定位.pBK-CMV△(1098-1300)-SNAT2-HA表达载体的成功构建,为今后对SNAT2的结构和功能的研究提供了有效方法. 相似文献