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1.
用DNA合成仪合成了分别带有PsI位点和SaiI位点及张终止密码子的2个用于扩增hIDGF1cDNA的PCR引物,利用合成的引物,700bp长的hIGF-D1cDNA模板和Taq聚合酶进行PCR扩增。 相似文献
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3.
HSV1-tk基因的部分DNA序列分析 总被引:3,自引:0,他引:3
采用PCR技术从商品质粒pHSV106扩增出HSV1-tk基因(1128bp),PCR产物用BamHI和EcoRI双酶切后定向克隆至真核表达载体pcDNA3,然后以重组质粒pcTK为模板tk基因的5端引物为DNA测序引物进行部分DNA序列分析,部分DNA序列分析证明PCR产物为HSV1-tk基因正确无误,成功筛选到HSV1-tk基因的真核表达载体pcTK,并为利用tk基因在实验动物体内进行自杀性基 相似文献
4.
类志贺氏菌毒素Ⅱ型变异体基因的克隆与鉴定 总被引:3,自引:2,他引:1
用聚合酶链式反应(PCR)技术,从大肠杆菌TB1中扩增出类志贺氏菌毒素Ⅱ型变异体(SLTⅡe)的A亚单位基因(slt-ⅡeA)的960bp的编码序列和B亚单位基因(slt-ⅡeB)的107bp的编码序列,将这两个PCR扩增产物在EcoR1和BamH1位点分别克隆进PUC18质粒载体,并转化到大肠杆菌TG1中,再根据生内切酶酶切分析筛选到含有slt-ⅡeA的重组质粒P18slt-ⅡeA和含有slt- 相似文献
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通过PCR方法扩增了人胰岛素(HI)基因1.34kb的DNA片段并克隆于pUC18的SmaⅠ位点内。经DNA序列分析表明,该片段为HI基因的氨基酸编码区及3'端调控区序列。同时,应用PCR方法对牛α-乳白蛋白(BαLA)基因进行了扩增,得到了0.84kbDNA扩增片段。将其克隆于去除了EcoRI位点的pUC18SmaI位点内,经内切酶分析和DNA测序证明该片段是BαLA基因5'调控区序列。EcoR 相似文献
6.
报道BMP-3及BMP-5在不同组织细胞中的表达.将人的神经母细胞瘤SK细胞的总RNA及人的脑、肝、胸腺、脾、胎盘及睾丸的总RNA反转录成cDNA作为模板,利用设计的编码BMP-3和BMP-5成熟蛋白的专一性引物分别扩增出相应的片段.PCR产物的琼脂糖凝胶电泳结果表明,BMP-3及BMP-5在不同组织细胞中表达类型不同. 相似文献
7.
半套式PCR技术在植物基因克隆中的应用 总被引:1,自引:0,他引:1
杨明挚 《云南大学学报(自然科学版)》1998,20(5):377-379
常规PCR方法在分离克隆已知序列的基因中发挥着重要的作用,但要求目的基因片段与引物间的同源性几乎是100%,一旦引物序列与目的基因间存在有差异,便往往会导致扩增的特异性不强,扩增效率不高,给克隆带来很大的困难.在常规PCR基础上,若所分离的基因在不同植物间存在保守序列,依据这一序列再合成一对引物,进行半套式PCR则能大大提高扩增的特异性及扩增效率.对于较长的基因片段而言还能同时完成亚克隆.本文依据南瓜GPAT(甘油-3-磷酸酰基转移酶)基因cDNA序列及比较拟南芥菜、豌豆间在该基因内的保守区段序列合成相应引物来分离克隆黑子南瓜及西瓜GPAT基因的cDNA片段为例来说明半套式PCR技术在植物基因克隆中的应用 相似文献
8.
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
9.
根据人骨髓单核细胞表面分化抗原CD14(hCD14)基因的核苷酸序列,设计hCD14基因5'端和3'端的两个引物.以人血中提取的基因组总DNA为模板,利用聚合酶链式反应(PCR)技术特异性扩增该基因1245加的编码序列.扩增产物经Xbal、KpnⅠ双酶切后,克隆到pUC18质粒XbaI-KpnI位点间,随后转化大肠杆菌JM109.用PCR方法筛选出重组菌落.经酶切和序列分析方法检测后,证明获得了含hCD14基因的重组克隆pHCD14.巳测出的插入片段5'端部分中包含着人CD14基因488bp的序列,与巳报道的相应DNA序列相比具有99%的同源性. 相似文献
10.
新型抗菌肽基因设计、合成及其在酵母中的表达(Ⅰ)——杂合抗菌肽基因的设计与合成 总被引:1,自引:0,他引:1
以化学合成并证实抗菌活性和抗菌谱都高于天然抗菌肽的杂合肽CecropinA1-11D12-37的氨基酸序列为基础,选用酵母高频使用密码子设计了一种新型抗菌肽基因,基因合成采用二次PCR(聚合酶链式扩增)方法.设计和合成的基因全长140个碱基对,包括氨基酸编码序列、起始密码子、终止密码子和两端限制性内切酶BamHI、EcoRI、SalI识别顺序,合成的基因克隆于PCRTM2.1载体上.经DNA序列分析证实,合成基因碱基序列与设计序列完全一致. 相似文献
11.
YUXuping ZHUJunli YAOXunping HEShicheng HUANGHaining CHENWeiliang LIDebao 《科学通报(英文版)》2004,49(13):1370-1375
To understand the antagonistic mechanism of the broad spectrum antagonistic Enterobacter cloacae B8,Tn5 transposon-mediated mutagenesis is performed using suicide plasmid pZJ25. Two mutant strains that lost antagonistic character are isolated. Tagging with kanr gene on Tn5,an antagonistic related DNA fragment, the F fragment, right of the Tn5 insertion site is cloned in a plasmid named pTLF,from one of the mutant strains B8F. The 733 bp F fragment is then sequenced after subcloning. Genomic DNA of the original B8 strain is isolated, digested with Pst I and ligated to Pst I cassette. DNA fragments left and right of the F fragment are amplified from the Pst I cassette library using cassette primer and specific primers designed according to known sequence. 1106 bp sequence left of the F fragment and 664bp sequence right of the F fragment are finally obtained. Bioinformatics analysis shows that the contig assembled from the sequences of the cloned antagonistic related DNA fragments of B8 encodes three ORFs and is homogeneous to admM,admN and admO genes of Pantoea agglomerans andrimid biosynthetic gene cluster (AY192157). The ORF, named anrF gene which encodes a polyketide synthase, knocked out by Tn5 insertion, is a homology of admM and the insertion site of Tn5 is at 214 bp upstream of the stop codon. It is concluded that the anrF gene is a gene related to the antagonistic activity of E. cloacae B8, and speculated that the antagonistic substance produced by B8 is an andrimid. 相似文献
12.
A型口蹄疫病毒(FMDV)VP1蛋白的141~160和200~213位氨基酸序列是决定FMDV免疫原性的抗原决定簇片断.人工合成一个免疫活性肽基因的串连片段(20AA-14AA-20AA)、全部选用大肠杆菌偏爱密码子.在5’端带有EcoR1位点和一个起始密码子ATG,在3’端带有BamH1位点和一个终止密码子TGA.利用这两个酶切位点把该基因连接到pWR590质粒上.并筛选高表达的菌株.结果表明该融合蛋白在大肠杆菌JM101中能高表达.经斑点ELISA实验证明.大肠杆菌细胞中表达的融合蛋白有A型FMDV抗原活性. 相似文献
13.
A different genetic code in human mitochondria. 总被引:54,自引:0,他引:54
Comparison of the human mitochrondial DNA sequence of the cytochrome oxidase subunit II gene and the sequence of the corresponding beef heart protein shows that UGA is used as a tryptophan codon and not as a termination codon and suggests that AUA may be a methionine and not an isoleucine codon. The cytochrome oxidase II gene is contiguous at its 5' end with a tRNAAsp gene and there are only 25 bases at its 3' end before a tRNALys gene. These tRNA'S are different from all other known tRNA sequences. 相似文献
14.
根据已知的大麦黄矮病毒GPV株系的外壳蛋白(Coat Protein CP)和移动蛋白(Movement Protein MP)基因序列合成了CP,MP基因的上下游引物,通过PCR扩增获得目的片段,经过Sal I和Rst I酶切、连接、转化、重组质粒的酶切鉴定及基因测序,构建了酵母表达载体pGBKT7-GPV-CP和pGBKT7-GPV-MP,用于在酵母双杂交分析中表达诱饵融合蛋白,为进一步筛选小麦cDNA文库内与大麦黄矮病毒相互作用的寄主因子、克隆寄主因子,推测其种类和功能打下基础。 相似文献
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人血管生长素基因的化学合成与克隆 总被引:4,自引:0,他引:4
采用固相亚磷酸胺法化学合成并克隆人血管生长素基因,该基因全长423bp,其中369bp的结构基因全部选用大肠杆菌偏爱的密码子,在结构基因上游设置了适合原核非融合蛋白表达的核糖体结合位点的S-D序列。采用两级退火一步拼接法成功克隆全长基因,该基因适合在多种融合或非融合原核表达载体中表达。 相似文献
16.
叶绿体是植物细胞中重要的细胞器,大部分叶绿体蛋白质都是由核基因组编码,在细胞质中合成分子量较大的前体蛋白,转运至叶绿体实施其功能.TOC33/TOC34是叶绿体上发现的一个外膜蛋白转运器构件蛋白,它与TOC159、TOC75和TOC64相互作用,构成了叶绿体外被膜上的一个蛋白转运器.目前已从豌豆(Pisumsa tizrurn)、拟南芥(Arabidopsis thaliana)、玉米(Zea mays)、小立碗藓(Physcomitrella patens)、诸葛菜(Orychophragmus violaceus)和油菜(Brassica napus)克隆到TOC33或TOC34的cDNA或DNA的编码区.与其功能研究相比,Toc33基因的表达调控研究较少,该基因5’端调控区域的克隆及序列分析均未见报道.为此,在本实验室已经克隆到甘蓝型油菜Toc33基因编码区的基础上,采用单引物PCR方法进行染色体步移,克隆出Toc33基因的启动子,为进一步研究Toc33基因的转录调控机制奠定基础. 相似文献
17.
A 6.0 kb DNA fragment related to nikkomycin biosynthesis was cloned from nikkomycin-producingStreptomyces ansochromogenes 7100. Sequence analysis showed that the 1.9 kbTth111 I fragment, a part of the 6.0 kb DNA fragment, contains one complete ORF designatedsanB (GenBank accession No. AF224501), which is composed of 1740 bp encoding a protein consisting of 580 amino acid residues.
Its start codon is GTG at 100 bp position and stop codon is TGA at 1840-bp position. Database searching indicated that the
deduced protein ofsanB is homologous to the histidinol-phosphate aminotransferase inStreptomyces coelicolor with 31% identities and 47% positives. Gene disruption was performed to study the function ofsanB. It was found that disruptants ofsanB lost the ability to synthesize nikkomycin, which reveals thatsanB is a novel gene essential for nikkomycin biosynthesis. 相似文献
18.
短柄五加(Acanthopanax brachypus)rbcL基因的结构分析 总被引:2,自引:0,他引:2
克隆了含完整短柄五加rbcL基因的3.2kb EcoRI片段,测定了该基因的核苷酸序列.所测核苷酸序列总长度为1924bp,其中编码区1428bp,编码475个氨基酸的蛋白质.测定的基因5’上游区共278bp,包含原核性质-35区(TTGCGC),-10区(TACAAT)及类似真核的TATA box元件(TATATA).5’前导区长194bp,其中SD序列为GGAGG,紧邻起始密码子上游.测定的3’下游区共218bp,含2个相邻的转录后可形成茎环结构的反向重复序列.短柄五加rbcL基因编码区推导的氨基酸序列与烟草、菠菜、豌豆、苜蓿、玉米、水稻、松树、地钱、衣藻和Anacystis的同源性分别为93.5%、94.11%、94.53%、94.74%、89.68%、92.21%、92.21%、92.63%、87.58%和80.84%.本文还对不同植物rbcL基因的启动区及部分5’和3’非编码区进行了比较分析. 相似文献
19.
Most of the coding region of rat ACTH beta--LPH precursor gene lacks intervening sequences 总被引:14,自引:0,他引:14
The peptide hormones ACTH, beta-endorphin, alpha- and beta-melanotropin(MSH) and possibly gamma-MSH are synthesized in the pituitary gland by the processing of a 32,000-molecular weight (MW) polypeptide called proopiomelanocortin (POMC). The existence of a further precursor (pre form) to POMC containing an additional N-terminal 'leader' peptide has been suggested by analysis of the in vitro translation products of poly(A)-containing RNA from AtT-20 cells, a mouse ACTH-producing cell line of pituitary origin. Nakanishi et al. cloned and sequenced a cDNA copy of the bovine prePOMC mRNA. This sequence confirmed the known structure of the carboxyl half of POMC and revealed the presence of a new MSH-like moiety, gamma-MSH, within the 16,000-MW amino half of the precursor (16K fragment). Recent experiments have suggested that this peptide may act in synergy with ACTH to increase corticosterone and aldosterone production in vivo and in vitro. We have now isolated from a rat genomic DNA library a segment of a DNA encoding most of POMC, using as probe a mouse 144-base pair cloned cDNA fragment encoding beta-MSH and beta-endorphin. The cloned rat gene is one of two (or more) closely related POMC genes. The DNA sequence obtained shows that the cloned POMC gene is not interrupted by any intervening sequence (IVS) between the codon for amino acid 19 and the presumptive poly(A) addition site. This region of POMC encodes all the biologically active peptides mentioned above. The DNA sequence encoding the putative gamma-MSH and the coding sequence that precedes it are highly conserved between rat and cow. This may indicate an as yet unrecognized biological function(s) for the NH2-terminal portion of the 16K fragment. 相似文献
20.
《科学通报(英文版)》1999,44(14):1289-1289
Based on the cDNA fragment sequence of vernalization-related gene verc203 cloned by differential screening in our lab, the 5' primer has been designed. The cDNA 3' end of ver203 gene (1 197 bp) has been cloned by the RACE method. And it is identified by Northern blotting that its expression is special in vernalization treatment. After comparing the sequence in the nucleotide sequence databases of Genbank, EMBL and DDBJ, the gene has homology with Hordeum vulgare jesmonate-induced protein gene. It is suggested that this gene might be related to the signal transduction mediated by jamonate. 相似文献