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胃癌相关cDNA片段的快速克隆和表达分析 总被引:3,自引:0,他引:3
利用差异显示PCR技术获得的一条在胃癌和正常组织有差异表达的表达序列标签(EST)-W123(GenBank登录号为AF150631),通过与GenBank的dbest库进行电子杂交,选取了与其同源度高的若干EST,在它们共有的保守序列设计了用于扩增的寡聚核苷酸引物,利用cDNA末端快速扩增PCR(RACE)技术得到了7条带有polyA尾的3′EST,进行序列分析后,发现它们均是代表新基因或不同剪接体的EST,且具有共同的保守序列,已登录GenBank.采用RNA印迹对目的序列进行初步鉴定,并进行了这些基因的组织分布分析.RACE技术和生物信息学相结合,具有快速、高效的特点,有助于疾病相关基因的克隆. 相似文献
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以戊型肝炎(Hepatitis)病人高热期血清为原材料,设计特定引物,经RT-PCR扩增获得开放阅读框2(ORF2)1.3kb基因片段,用EcoRI和BamHI插入克隆载体pUC18,测定了该克隆基因片段的核苷酸序列并进行了比较分析。其片段长度为1309bp,其A=249(19.2%),G=318(24.29%),T=339(25.9%),C=403(30.79%);与印度株和缅甸株的同源性在90 相似文献
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以戊型肝炎(Hepatitis E)病人高热期血清为原材料,设计特定引物,经RT-PCR扩增获得开放阅读框2(ORF2)1.3 kb基因片段,用EcoR I和BamH I插入克隆载体pUC18,测定了该克隆基因片段的核苷酸序列并进行了比较分析.其片段长度为1 309 bp,其A=249(19.2%),G=318(24.29%),T=339(25.9%),C=403(30.79%);与印度株和缅甸株的同源性在92%以上,而与乍得和墨西哥株的同源性分别为90.7%和77.4%. 相似文献
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猪瘟病毒兔化弱毒株cDNA片段的克隆及序列分析 总被引:2,自引:0,他引:2
猪瘟是猪最重要的传染病之一,往往给养猪业造成重大经济损失,猪瘟的病原为猪瘟病毒(HCV),属黄病毒科,瘟病毒属成员,其基因组为单股正链RNA,长度为123kb,仅含有一个大的开放阅读框架,编码一个含3898个氨基酸残基(AA)的多聚前体蛋白[1,2]。目前已经定位的蛋白有5种,即Npro、C、E0、E1和E2,它们均由HCVRNA5′端所编码,除Npro外,其它4种均为HCV的结构蛋白[3]。Npro为具有自我催化功能的蛋白水解酶,也是多聚蛋白N端的第一个蛋白水解酶,分子量为23kD,C为构成… 相似文献
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马哈利樱桃PGIP cDNA克隆序列分析 总被引:3,自引:1,他引:2
以马哈利樱桃(Prunus mahaleb L.)为材料,通过RT-PCR获得了1045bp的目的段,经克隆测序,证实该片段包含1个完整的开放阅读框架,该阅读框架由990碱基组成,编码330个氨基酸。该序列与杏、梨、苹果的PGIP cDNA序列同源性分别达97.2%、83.4%和83.6%,可能编码的氨基酸与杏、梨、苹果的PGIP cDNA所编码的氨基酸的同源性分别达到96.7%、85.2%和85.2%。与已经克隆的PGIP DNA序列的对比分析表明,PGIP DNA序列中包含2个外显子和1个内含子,内含子全长147 ,符合TG-AG规律,2个外显子长度分别为581bp、464bp。 相似文献
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抗肿瘤单抗3H11对应抗原cDNA片段的克隆 总被引:1,自引:0,他引:1
单克隆抗体3H11能与多种肿瘤细胞特异结合,克隆其对应抗原无疑具重要意义.用胃癌细胞MGC803构建cDNA表达文库,通过抗体3H11对其进行原核表达筛选,获得一株能与3H11特异反应的阳性克隆.其cDNA插入片段为554bp.GenBank不含其同源序列.将此cDNA片段与谷胱甘肽转移酶表达质粒pGEX-4T重组,Westernblot和竞争抑制实验表明,表达产物依然保持同3H11反应的特异性.可见它是3H11对应抗原的cDNA. 相似文献
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菠菜甜菜碱醛脱氢酶基因的克隆和序列分析 总被引:7,自引:0,他引:7
以耐盐的菠菜mRNA为模板,经反转录合成甜菜碱醛脱氢酶(BADH)基因第一链cDNA。在人工合成的两端引物引导下,通过多聚酶链式反应(PCR),扩增获得双链cDNA。把重组有BADH基因的pUC19转化至E.coli DH5α菌株,亚克隆后测定了基因的全序列。所得到的BADH基因全长序列为1491bp,编码497个氨基酸。与文献报道的相比较,核苷酸序列同源性99.8%,氨基酸序列同源性达99.6%。在此基础上,构建了BADH基因的高等植物表达载体。 相似文献
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A cDNA encoding for a sperm antigen, designated NZ-2, was cloned and sequenced from human testis cDNA-λgt11 expression library by using antibodies to human sperm surface antigens belonging to 14–18 kD molecular regions. These sperm antigens are involved in binding to zona pellucida of the human oocyte. Computer generated translation analysis of 963-bp cDNA yielded an open reading frame (ORF) of 163 amino acids (aa) with first ATG, Met start codon at nucleotide (nt) 335 and the stop codon TAA at nt 824. The NZ-2 cDNA has 335-bp 5′ and 139-bp 3′ noncoding regions. The translated protein has a calculated molecular weight of ∼19 kD, and has two casein kinase II (CK-2) sites at aa 94–97 and 149–152, respectively. Extensive computer search in the GenBank, National Biomedical Research Foundation (NBRF), and Swiss database indicates it to be a novel protein, having 99.5% nt sequence similarity, except for the first 40-bp, only with the human bacterial artificial chromosome (BAC) containing cloned human sperm DNA, at position 76935–76009. The in vitro translated product of T3 RNA polymerase by using NZ-2 cDNA digested with XhoI yielded a protein band of ∼20 kD, indicating it to be sense strand. The in vitro translated product of T7 RNA polymerase by using NZ-2 cDNA digested with NotI did not yield any protein band, indicating it to be antisense strand. The ∼20 kD protein was recognized specifically by the antisperm IgG, not by the control IgG in the Western blot procedure. Neither antisperm IgG nor control IgG recognized any protein band in the in vitro translation products of the antisense strand. The human genomic DNAs from three different cells/tissues namely, sperm, kidney, and testis when cut by HindIII, and then hybridized with the NZ-2 cDNA probe in the Southern blot procedure, showed restriction fragment length polymorphism (RFLP). The recombinant human sperm NZ-2 antigen may find applications in the development of a contraceptive vaccine, and diagnosis and treatment of infertility in humans. Mol. Reprod. Dev. 51:176–183, 1998. © 1998 Wiley-Liss, Inc. 相似文献
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使用RT-PCR方法克隆了Wistar大鼠脑α_1型甲状腺激素受体的cDNA,得到包含起始及终止密码子共1233bp、编码409个氨基酸的受体全长编码序列.酶切分析后,将此特异DNA片段重组入质粒pUC系统,得重组质粒pTRA.双脱氧末端终止法测定了全部核苷酸顺序,结果与文献报导的SD大鼠的结果一致,同时对长片段DNA的RT-PCR扩增进行了方法学的探讨。 相似文献
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A cDNA encoding cat growth hormone (Fc) GH) has been isolated and sequenced. This is the first report of a feline GH nucleotide and deduced amino acid (aa) sequences. This cat pituitary cDNA resembles a typical mammalian pre-GH cDNA with its encoded mature hormone differing from dog GH only by a single as residue. 相似文献
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Cloning, sequencing and restriction fragment length polymorphism analysis of a porcine cDNA for OCT2
A full-length pig cDNA for the POU-domain protein OCT2 has been isolated and sequenced. The 478 amino acid-long reading frame in pig OCT2 is 97% identical to human OCTZA, indicating strong conservation of function for this immunoglobulin regulatory protein. To investigate the potential use of this cDNA for mapping and identifying linkage of OCT2 to economic traits, restriction fragment length polymorphism (RFLP) analysis was used to identify a TaqI polymorphism. A population of 60 unrelated animals, as well as multigenerational families, were typed for this RFLP and showed variability in several American and Chinese breeds. The Taq I polymorphism was also detected by a non-POU-domain OCT2 probe, demonstrating that this RFLP is located in the OCT2 gene. This result suggests that OCT2 is likely to be a single-copy gene in swine as seen in other mammals. 相似文献
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A recombinant plasmid has been constructed containing a sequence of 186 nucleotides encoding a potent neurotoxin found in the venom of the sea-snake Laticauda semifasciata and designated as erabutoxin a. This sequence is flanked, in the upstream region, by a sequence of 60 nucleotides encoding a hydrophobic peptide fragment presumably involved in the secretion process of the neurotoxin. The sequence coding for the toxin ends with a termination codon which is followed by a 3'-untranslated sequence of approximately 240 nucleotides (excluding the poly(A) tract). 相似文献
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Liguo Feng Han Ding Jia Wang Meng Wang Wei Xia Shu Zang Lixia Sheng 《Saudi Journal of Biological Sciences》2015,22(4):417-423
Salt stress is one important factor influencing the growth and development of plants, and salt tolerance of plants is a result of combined action of multiple genes and mechanisms. Rosa rugosa is not only an important ornamental plant, but also the natural aromatic plant of high value. Wild R. rugosa which is naturally distributed on the coast and islands of China has a good salt tolerance due to the special living environment. Here, the vacuolar Na+/H+ reverse transporter gene (NHX1) and the vacuolar H+-ATPase subunit C gene (VHA-c) closely related to plant salt tolerance were isolated from wild R. rugosa, and the expression patterns in R. rugosa leaves of the two genes under NaCl stress were determined by real-time quantitative fluorescence PCR. The results showed that the RrNHX1 protein is a constitutive Na+/H+ reverse transporter, the expression of the RrNHX1 gene first increased and then decreased with the increasing salt concentration, and had a time-controlled effect. The RrVHA-c gene is suggestive of the housekeeping feature, its expression pattern showed a similar variation trend with the RrNHX1 gene under the stress of different concentrations of NaCl, and its temporal expression level under 200 mM NaCl stress presented bimodal change. These findings indicated that RrNHX1 and RrVHA-c genes are closely associated with the salt tolerance trait of wild R. rugosa. 相似文献
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Identification of gene expression patterns in mangroves grown under salinity will help to reveal the molecular mechanisms of salt tolerance. Here, 10 cDNAs of genes were isolated from Kandelia candel and identified by representational difference analysis of cDNA (cDNA RDA) under different NaCl concentrations. Of five genes expressed preferentially under salt condition, two were unknown, three were two kinds of low molecular mass heat-shock proteins (sHSPs) and ADP-ribosylation factor, respectively. The expressions of other five genes were repressed under NaCl stress, two encoded cyclophilins, three were tonoplast intrinsic protein, early light-induced protein and 60S ribosomal protein, respectively. 相似文献