Molecular cloning and characterization of Phoneutria nigriventer toxins active on calcium channels.

The aim of the present study was the molecular cloning of toxins active on calcium channels expressed by the spider Phoneutria nigriventer. Clones encoding the toxins Pn3-3A, Pn3-4A, Tx3-5, Pn3-5A, Tx3-6, Pn3-6A and Pn3-6B were identified from a cDNA library derived from the venom gland of this spider, revealing toxins of 49, 76, 45, 39, 55 and 58 amino acids residues, respectively, with polypeptide precursors being composed of three major portions: a signal peptide, a propeptide and finally, the mature toxin. A high degree of homology with the amino acid sequence was found between Pn3-3A and the neurotoxin Tx3-3 (identity of 79%), and between Pn3-4A and the neurotoxin Tx3-4 (identity of 95%). The deduced amino acid sequence for the mature polypeptides Tx3-5 and Tx3-6 confirms the polypeptide sequence previously published for these neurotoxins. In addition, the toxin Pn3-5A showed 58% identity to the Tx3-5 amino acid sequence, and the toxins Pn3-6A and Pn3-6B showed 85 and 33% identity, respectively, to the Tx3-6 amino acid sequence.     

Cloning of cDNAs encoding neurotoxic peptides from the spider Phoneutria nigriventer

A cDNA library made from venom glands of the spider Phoneutria nigriventer was constructed and used to clone neurotoxic peptides. A cDNA of about 360 nucleotides encoding the precursor for the toxin Tx2-1 active on mammals has been isolated. The deduced amino acid sequence for the mature polypeptide confirms the polypeptide sequence previously published. In addition, two new putative toxins called Pn2-1A and Pn2-5A have been characterized and their complete amino acid sequence show 92% similarity to Tx2-1 and 94% similarity to Tx2-5 respectively. The cDNAs revealed that the precursors contain signal peptides characterized by a very hydrophobic core and a propeptide interposed between the signal sequence and the peptide toxin.     

Cloning, cDNA sequence analysis and patch clamp studies of a toxin from the venom of the armed spider (Phoneutria nigriventer)

The cDNAs (Tx3-2 and Pn3A) encoding precursor of toxin Tx3-2 and an isoform called Pn3A have been isolated from a library constructed from stimulated venom glands of the spider Phoneutria nigriventer. The cDNA of Tx3-2 reveals the presence of a signal peptide of 21 amino acids and of an intervening propeptide (with 16 amino acids) preceding the toxin sequence, which was followed by additional amino acid residues at the C-terminus (C-terminal peptide), implying post-translational modifications of the synthesised peptide. The deduced amino acid sequence for the mature toxin confirms the previous sequence published. In addition, by using the whole-cell patch clamp technique, we have determined that purified Tx3-2 decreases L-type currents present in GH3 cells. Finally, the presence of the cDNA Pn3A, with high sequence identity with Tx3-2, reveals the existence of a putative new toxin showing, at the cDNA level, 85.4% identity in its whole segment.     

内蒙古白绒山羊Izumo蛋白的基因编码区cDNA序列扩增及结构初步分析

目的  对内蒙古白绒山羊精子膜蛋白Izumo的基因编码区cDNA序列进行扩增并对其结构作初步分析。方法  以已知的内蒙古白绒山羊Izumo部分基因序列为模板,采用cDNA末端快速扩增技术扩增3′端和5′端未知序列,钓取基因编码区,并分析编码区氨基酸序列。结果  经序列扩增和拼接,得到1035 bp的Izumo编码区cDNA序列,后者编码由344个氨基酸组成的蛋白。氨基酸序列中包含免疫球蛋白样结构域。Izumo编码区的疏水性平均值为-0.181。找到4个位于Izumo蛋白胞外区的潜在磷酸化位点。结论  进一步确定Izumo属于免疫球蛋白超家族的一员,初步得出Izumo是一种亲水性蛋白,根据潜在磷酸化位点的位置,推断磷酸化可能不是Izumo蛋白发挥作用的机制。     

广西菲牛蛭水蛭素基因cDNA的克隆与序列分析

目的克隆广西菲牛蛭水蛭素基因的cDNA,并对该基因以及氨基酸进行序列分析。方法根据已发表的马尼拉菲牛蛭水蛭素基因cDNA设计一对引物,从广西菲牛蛭的头部提取总RNA后,采用RT-PCR扩增cDNA,将产物克隆至载体PMD-19T,PCR鉴定后进行测序。结果广西菲牛蛭水蛭素基因cDNA序列长度为251 bp,编码框由83个氨基酸组成,其中包括由20个氨基酸的信号肽,以及63个氨基酸组成的成熟肽。广西菲牛蛭水蛭素基因的氨基酸序列和已报道的广东菲牛蛭、马尼拉菲牛蛭水蛭素基因HM1、HM2基因的氨基酸序列相比较,同源性分别为94%,91.8%,84.7%。结论广西菲牛蛭水蛭素基因cDNA序列长度为251 bp,由83个氨基酸组成。     

猪带绦虫成虫烯醇酶基因的生物信息学分析

目的 分析猪带绦虫成虫烯醇酶(enolase,Ts ENO)基因结构并预测其编码蛋白的结构和功能.方法 利用生物信息网站如美国国家生物技术信息中心(NCBI)和瑞士生物信息学研究所的蛋白分析专家系统(ExPASY)中生物信息学分析工具,并结合其它分析软件,从获得猪带绦虫成虫全长cDNA质粒文库的表达序列标签(expre...     

Purification and amino acid sequence of a highly insecticidal toxin from the venom of the brazilian spider Phoneutria nigriventer which inhibits NMDA-evoked currents in rat hippocampal neurones.

A new insecticidal toxin Tx4(5-5) was isolated from the fraction PhTx4 of the venom of the spider Phoneutria nigriventer by reverse phase high performance liquid chromatography (HPLC) and anion exchange HPLC. The complete amino acid sequence determined by automated Edman degradation showed that Tx4(5-5) is a single chain polypeptide composed of 47 amino acid residues, including 10 cysteines, with a calculated molecular mass of 5175 Da. Tx4(5-5) shows 64% of sequence identity with Tx4(6-1), another insecticidal toxin from the same venom. Tx4(5-5) was highly toxic to house fly (Musca domestica), cockroach (Periplaneta americana) and cricket (Acheta domesticus ), producing neurotoxic effects (knock-down, trembling with uncoordinated movements) at doses as low as 50 ng/g (house fly), 250 ng/g (cockroach) and 150 ng/g (cricket). In contrast, intracerebroventricular injections (30 microg) into mice induced no behavioural effects. Preliminary electrophysiological studies carried out on whole-cell voltage-clamped rat hippocampal neurones indicated that Tx4(5-5) (at 1 microM) reversibly inhibited the N-methyl-D-aspartate-subtype of ionotropic glutamate receptor, while having little or no effect on kainate-, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid- or gamma-aminobutyric acid-activated currents.     

cDNA cloning and sequence analysis of Xenopus laevis preproendothelin-1

Endothelin (ET)-like immunoreactivity has been observed not only in mammals, but also in amphibians. The biological actions of ET are similar in amphibians and mammals, and amphibian ET-related receptors have been cloned and characterized. The cDNA sequences of mature and precursor forms of ET-related peptides, however, have not been reported in any amphibian until now. To identify the ET-related peptides, we screened the Xenopus laevis intestine cDNA library using the rapid amplification of cDNA ends method and cloned cDNAs encoding preproendothelin-1. The deduced amino acid sequence of X. laevis preproendothelin-1 comprises 223 amino acids, including a putative signal sequence of 19 amino acids, a mature ET-1 of 21 amino acids, as well as big ET-1 and ET-1-like sequences. X. laevis ET-1 is identical to mammalian ET-1 as well as ET-1 peptide, recently purified from the stomach of the European green frog, Rana ridibunda. This is the first report describing the cDNA encoding preproendothelin-1 in an amphibian species.     

Isolation and characterization of human liver cytochrome b5 cDNA

Three cDNA clones encoding human cytochrome b5 have been isolated and sequenced from a lambda gt 11 cDNA library of a human liver. The largest clone (b5-C) contains 765 base pairs corresponding to a complete coding sequence of 134 amino acids and sequences for 5' non-coding (56 bases) and 3' non-coding regions (307 bases). In Northern blots, a band hybridizing to 32P-labelled b5-B cDNA clone was detected at around 12S in mRNA of human and rat livers. The deduced amino acid sequence was identical with the partial amino acid sequence (2-100th) previously reported, except that two amino acid replacements were observed between the 89th and 91st positions. The deduced amino acid sequence of human cytochrome b5 shares 88.0, 86.5, 86.5 and 74.4% similarity with those of rat, pig, horse and chicken, respectively. The sequences in the haem binding region were highly conserved among these species. In addition, high similarities in the hydrophobicity profiles were maintained throughout their entire regions, although some diversity was observed in the N-terminal sequences between human and chicken.     

小鼠脂肪组织脂联素基因编码区的克隆及序列分析

目的：克隆小鼠脂肪组织脂联素（ACRP30）编码区基因并进行序列分析。方法：用TRIzol试剂从小鼠脂肪组织提取总RNA，经RT-CR方法扩增全长的ACRP30 cDNA片段，将PCR产物克隆于pGEM-T载体，对重组质粒pGEM-ACRP30进行序列验证。结果：小鼠脂肪组织ACRP30基因编码序列不同于来自3T3-L1脂肪细胞的编码序列．IRM-2（Institute of Radimion Medicine-2）小鼠和C57BL／6J小鼠脂肪组织的ACRP30基因编码序列相同。337位点上的碱基A被G置换导致在113位的蛋氨酸被缬氨酸取代。来自脂肪组织的ACR30基因编码序列已被提交到GenBank^TM数据库。C57BL／6J小鼠的收录编号为AY749429。IRM-2小鼠为AY754346。结论：小鼠脂肪组织ACRP30编码序列是ACRP30序列的新成员，cDNA克隆的构建为进一步的蛋白表达和生物学活性研究奠定了基础。     

Molecular characterization of L-amino acid oxidase from king cobra venom.

An L-amino acid oxidase from Ophiophagus hannah snake venom (Oh-LAAO) was purified by successive gel filtration, ion-exchange and heparin chromatography. Oh-LAAO did not induce platelet aggregation; however, it had potent inhibitory activity on platelet aggregation induced by ADP and U46619, but showed no effect on platelet aggregation induced by thrombin, mucetin, ristocetin and stejnulxin. By RT-PCR and 5'-RACE methods, the complete Oh-LAAO cDNA was cloned from the venom gland total RNA preparations. The cDNA sequence contains an open-reading frame (ORF) of 1476-bp, which encodes a protein of 491 amino acids comprising a signal peptide of 25 amino acids and 466-residue mature protein. The predicted protein sequence of Oh-LAAO was confirmed by N-terminal and trypsin-digested internal peptides sequencing together with peptide mass fingerprinting. cDNAs encoding for ORF of LAAOs from Bungarus fasciatus and B. multicinctus were cloned and reported in this study. In addition, partial cDNA encoding for Naja atra LAAO was also reported. Oh-LAAO shared approximately 50% protein sequence identity with other known snake venom LAAOs. Phylogenetic analysis indicated that Oh-LAAO is evolutionary distant to other snake venom LAAOs.     

PnTx4-3, a new insect toxin from Phoneutria nigriventer venom elicits the glutamate uptake inhibition exhibited by PhTx4 toxic fraction.

Several pools of neurotoxic peptides obtained from fractionated Phoneutria nigriventer venom induce different toxicological effects. One of them, PhTx4, is highly toxic towards insects and displays only a slight toxicity when injected in mice. Also, this fraction contains a class of peptides that are able to inhibit glutamate uptake in preparations of mammalian central nervous systems (CNS). In this work a new toxin called PnTx4-3 was isolated from the PhTx4 fraction by reverse phase and anion exchange steps using high performance liquid chromatography (HPLC). Edman sequencing of PnTx4-3 revealed that it was a polypeptide of 48 amino acid residues, containing 10 cysteines cross-linked by five disulfide bridges. The molecular mass measured by ES-Q-TOF mass spectrometry was 5199.49+/-0.64 Da, which is very close to the calculated mass from amino acid sequence (5199.99 Da). This toxin induces immediate excitatory effects when injected intrathoracically in house flies and cockroaches. Intracerebroventricular injections of 30 microg of PnTx4-3 in mice resulted in no apparent signs of intoxication. In order to make an orthologous comparison, pharmacological characterisation were carried out in rat brain synaptosomes by using [3H]-L-glutamate, showed that the whole PhTx4 fraction as well as the pure toxins PnTx4-3, Tx4(6-1) and Tx4(5-5) obtained of this fraction, were able to inhibit the glutamate uptake in the micromolar concentration range. PnTx4-3 inhibits the glutamate uptake in a dose dependent manner, with an IC50 of approximately 1 microM. PnTx4-3 is highly homologous to the Tx4(6-1) and Tx4(5-5) toxins previously described from the same fraction.     

Isolation of a bovine full length cytochrome P450 (CYP3A) cDNA sequence and its functional expression in V79 cells

From a bovine liver cDNA library in λMaxl a 1870 bp cDNA was isolated using the human CYP3A4 cDNA as a probe. The cDNA-deduced amino acid sequence encoded a protein of 507 amino acids and exhibited homologies of 76, 72 and 64% with canine CYP3A12, human CYP3A4 and rat CYP3A1, respectively. Furthermore, a very high homology of 91.7% was observed with the deduced amino acid sequence of a partial CYP3A cDNA from dwarf goat. A striking observation was that both the bovine and the goat cDNA exhibit a 4 amino acid extension at the C-terminus, which is due to a frame-shifting insertion of 2 nt. The bovine CYP3A cDNA was cloned in a retroviral vector, transfected to V79 cells and cells were selected for cytochrome P450 expression. The expressed enzyme was shown to catalyze the 6β-hydroxylation of testosterone, which could also be observed in a V79 cell line expressing human CYP3A4. In the bovine CYP3A cell line, however, 6β-hydroxytestosterone was not found to be the major metabolite. This cell line additionally showed high levels of hydroxylase activity at the 2β and 12β position of testosterone. The cDNA-expressed testosterone hydroxylase activity could be inhibited with the specific CYP3A inhibitors, tiamulin and ketoconazole.     

Molecular cloning and sequence analysis of the porcine precursor of endothelin-2

The amino acid sequences of two of the three endothelin (ET) family peptides, ET-1 and ET-3, are identical among mammals, whereas for the other family member, ET-2 or vasoactive intestinal contractor (VIC), the mouse and rat sequences differ from the human counterpart ET-2 by one amino acid residue. To examine more deeply the structural diversity among ET-2/VIC orthologs (EDN2), we screened porcine ET-2/VIC-like cDNAs using the 5' rapid amplification of cDNA ends (RACE) method with degenerate primers based on ET-2/VIC mature peptides. Sequence analysis of the cDNAs showed that ET-2 is present in pig. The full-length cDNA sequence, produced by combining 5' RACE and 3' RACE products, revealed the porcine precursor protein of ET-2 (PPET-2). Porcine PPET-2, made up of 214 amino acids, includes a 26-residue putative signal sequence, big ET-2, mature ET-2, and ET-2-like peptide. The percent sequence identity of porcine PPET-2 with human PPET-2, and rat or mouse precursor protein of VIC runs between approximately 70% and 74%. ET-2, although expressed in intestine, has no anti-microbial activity.     

Molecular cloning of HR1a and HR1b, high molecular hemorrhagic factors, from Trimeresurus flavoviridis venom.

HR1a and HR1b are two high molecular weight (P-III class) hemorrhagic factors in the venom of Trimeresurus flavoviridis. In this study, we cloned cDNAs of the HR1a and HR1b precursors and analyzed their nucleotide sequences. The cDNA for HR1a was 2368 nucleotides in length and encoded an open reading frame (ORF) of 609 amino acids; that for HR1b was 2237 nucleotides and encoded an ORF of 614 amino acids. Both cDNAs belonged to the N-III class consisting of signal, pro, metalloproteinase, disintegrin-like and cysteine-rich regions, and shared strong amino acid sequence similarity (74.4%). The HR1b precursor was found to have an additional seven amino acid sequence at the carboxyl terminus compared with a mature form of HR1b.     

结肠黑变病相关基因金属泛激蛋白-1cDNA和氨基酸序列分析

目的对结肠黑变病相关基因金属泛激蛋白-1(MPS-1)cDNA序列和氨基酸序列进行综合分析,为其结构和功能的研究奠定理论基础。方法从Pubmed数据库中获得目标cDNA序列,利用基因和蛋白质分析处理软件DNAMAN、NCBI ORF finder、BLAST、Conserved Domains、GOR、SWISS-MODEL等软件对该目标的基因序列和氨基酸序列进行综合分析。结果 MPS-1蛋白cDNA序列长为361 bp,编码84个氨基酸残基,基因序列比对显示该蛋白与核糖体蛋白S27(RPS27)mRNA的编码序列同源度最高,两者核苷酸的相似度为361/361(100%),氨基酸序列比对显示该蛋白与RPS27的氨基酸序列同源度最高,两者氨基酸的相似度为84/84(100%),目标序列中存在1段Ribosomal_S27e家族保守结构域,二级结构和三级结构预测显示目标蛋白中主要存在α-螺旋、β-折叠和无规卷曲的结构。结论 MPS-1蛋白与RPS27同源度高,与肿瘤的发生密切相关。