重组小鼠canstatin N端片段对鸡胚新生血管生成的抑制作用(英)

为了研究重组小鼠canstatin N端片段的体内抗血管生成活性, 通过PCR扩增小鼠canstatin N端片段cDNA,定向克隆于原核表达载体pET30a(+)中,构建小鼠canstatin N端片段重组表达载体pET-mCanN, 转化E.coli BL21(DE3), IPTG诱导表达,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹检测小鼠canstatin N端片段的表达. 结果表明,IPTG诱导原核表达载体pET-mCanN在大肠杆菌E.coli BL21(DE3)中高效表达, 小鼠canstatin N端片段表达量约占菌体总蛋白量的18%, 小鼠canstatin N端片段主要以包涵体形式存在,包涵体经过洗涤、裂解、Ni-spin column亲合柱层析以及蛋白质复性等步骤纯化后,获得了纯度约为92%的重组小鼠canstatin N端片段. 鸡胚绒毛尿囊膜(chicken embryo choriollantoic membrane,CAM)实验表明,原核表达的小鼠canstatin N端片段能有效地按剂量依赖的方式抑制鸡胚新生血管的形成.     

小鼠canstatin C端片段基因的克隆及其在大肠杆菌中的表达

为了构建小鼠canstatinC端片段的原核表达载体并在大肠杆菌中表达。以小鼠肝脏组织总RNA为模板,通过RT-PCR扩增小鼠canstatinC端片段(mCan-C)基因,克隆到pMD18-T载体中并进行序列分析。将mCan-C基因定向克隆于原核表达载体pET30a(+)中,构建表达载体pET／mCan-C,转化大肠杆菌BL21(DE3),IPTG诱导表达。结果表明,小鼠canstatinC端片段的cDNA长度为399bp,含有1个终止密码,编码132个氨基酸,与已知的人canstatinC端片段氨基酸的同源性为61％。IPTG诱导mCan-C在大肠杆菌E．coliBL21中表达,表达量约占菌体总蛋白量的28％,重组蛋白主要以包涵体形式存在。首次克隆了小鼠canstatinC端片段的cDNA,IPTG诱导mCan-C在大肠杆菌E．coliBL21中高效表达。小鼠canstatinC端片段的cDNA序列已收入GenBank,接受号为:AY502947。     

Purification of full-length human Pregnane and Xenobiotic Receptor： polyclonal antibody preparation for immunological characterization

Pregnane and Xenobiotic Receptor （PXR; or Steroid and Xenobiotic Receptor, SXR）, a new member of the nuclear receptor superfamily, is thought to modulate a network of genes that are involved in xenobiotic metabolism and elimination. To further explore the role of PXR in body＇s homeostatic mechanisms, we for the first time, report successful prokaryotic expression and purification of full-length PXR and preparation of polyclonal antibody against the whole protein. The full-length cDNA encoding a 434 amino acids protein was sub-cloned into prokaryotic expression vector, pET-30b and transformed into E. coli BL21（DE3） cells for efficient over expression. The inclusion body fraction, containing the expressed recombinant protein, was purified first by solubilizing in sarcosine extraction buffer and then by affinity column chromatography using Ni-NTA His-Bind matrix. The efficacy of anti-PXR antibody was confirmed by immunocytology, Western blot analysis, EMSA and immunohistochemistry. The antibody obtained was capable of detecting human and mouse PXR with high specificity and sensitivity. Immunofluorescence staining of COS-1 cells transfected with human or mouse PXR showed a clear nuclear localization. Results from immunohistochemistry showed that level of PXR in liver sections is immunologically detectable in the nuclei. Similar to exogenously transfected PXR, Western blot analysis of cell extract from HepG2 and COLO320DM cells revealed a major protein band for endogenous PXR having the expected molecular weight of 50 kDa. Relevance of other immunodetectable bands with reference to PXR isoforms and current testimony are evaluated. Advantages of antibody raised against full-length PXR protein for functional characterization of receptor is discussed and its application for clinical purposes is envisaged.     

Identification of a Differentially Expressed Gene PPP1CB between Porcine Longissimus dorsi of Meishan and Large White×Meishan Hybrids

To study the molecular basis of heterosis,suppression subtractive hybridization was used toinvestigate the differences in gene expression between porcine Longissimus dorsi of F1 hybrids LargeWhite×Meishan and their female parents Meishan.From two specific subtractive cDNA libraries,the clonesselected by reverse Northern high-density blot screening were chosen to clone full-length cDNA by rapidamplification of cDNA ends.An expression-upregulated gene for Meishan skeletal muscle,designated proteinphosphatase 1,catalytic subunit,beta isoform(PPPICB),was identified.Porcine PPPICB contains an openreading frame encoding 327 amino acid residues with 13 and 1763 nucleotides in the 5′ and 3′ untranslatedregions,respectively.A DNA fragment of 721 nucleotides was amplified and a mutation that creates/disruptsa restriction site for endonuclease RsaI was found.The derived amino acid sequence of PPPICB has highhomology with the PPPICB of three species,Mus musculus(99%),human(99%)and mouse(100%).Thetissue expression analysis indicated that the swine PPP1CB gene is generally expressed in most tissues.Thepossible role of PPP1CB and its relation to porcine heterosis are discussed.     

Human augmenter of liver regeneration: molecular cloning, biological activity and roles in liver regeneration

The complete amino acid sequence of human augmenter of liver regeneration (hALR) was reported by deduction from nucleotide sequence of its complementary DNA . The cDNA for hALR was isolated by screening a human fetal liver cDNA library and the sequencing of this insert revealed an open reading frame encoding a protein with 125aa and highly homologous (87% ) with rat ALR encoding sequence. The recombinant hALR expressed from its cDNA in transient expression experiments in cos-7 cells could stimulate DNA synthesis of HTC hepatoma cell in the dose-dependent and heat-resistant way. Northern blot analysis with rat ALR cDNA as probe confirmed that ALR mRNA was expressed in the normal rat liver at low level and that dramatically increased in the regenerating liver after partial hepatectomied rat. This size of hALR mRNA is 1.4 kb long and expressed in human fetal liver, kidney and testis. These findings indicated that liver itself may be the resource of ALR and suggested that ALR seems to be an im-portant parac     

cDNA cloning and recombinant expression of the general odorant binding protein II from Spodoptera litura

A cDNA encoding the general odorant binding protein II (GOBP II) was isolated from the antennae of Spodoptera litura (SlGOBP II, GenBank Accession No. EU086371) by homologous cloning and rapid amplification of cDNA ends (RACE). Sequencing and structural analyses revealed that the open reading frame (ORF) of SlGOBP II was 489 bp, encoding 162 amino acids with a predicted MW of 18.2 kD and pI of 5.72. SlGOPB II shared typical structural features of odorant binding proteins with other insects, including the six conservative cysteine residues. The deduced amino acid sequence of SlGOPB II shared significant identity with the GOBP II from S. frugiperda and S. exigua. RT-PCR and Northern blot analyses showed that SlGOBP II was specifically expressed in the antennae. cDNA encoding SlGOBP II was constructed into the pET-32a vector and the recombinant protein was highly expressed in Es-cherichia coli BL21 (DE3) after induction with IPTG. SDS electrophoresis and Western blot analysis confirmed the molecular weight of the recombinant SIGOBPII i.e, 32 kD, which has a 6×His tag at the N-terminus. The recombinant SlGOBP II was purified by single-step Ni-NTA affinity chromatography and used to raise antiserum in rabbits. ELISA showed that the titer of antiserum was 1︰12800, while Western blot analysis showed that the recombinant SlGOBP II was recognized as anti-SlGOBP II an-tiserum.     

Two Potent α3/5 Conotoxins from Piscivorous Conus achatinus

Every cone snail produces a mixture of different conotoxins and secretes them to immobilize their prey and predators. α3/5 Conotoxins, isolated from fish-hunting cone snails, target muscle nicotinic acetylcholine receptors. The structure and function of α3/5 conotoxin from the piscivorous Conus achatinus have not been studied. We synthesized two pentadecamer peptides, Ac 1.1 a and Ac 1.1 b, with appropriate disulfide bonding, based on cDNA sequences of α3/5 conotoxins from C. achatinus. Ac 1.1 a and Ac 1.1 b differ by only one amino acid residue. They have similar potency on blocking recombinant mouse muscle acetylcholine receptor expressed in Xenopus laevis oocytes, with IC_(50) values of 36 nM and 26 nM, respectively. For Ac 1.1b, deletion of the first three N-terminal amino acids did not change its activity, indicating that the Nterminus is not involved in the interaction with its receptor. Furthermore, our experiments indicate that both toxins strongly prefer the α1-δ subunit interface instead of the α1-γ binding site on the mouse muscle nicotinic acetylcholine receptor. These peptides provide additional tools for the study of the structure and function of nicotinic receptor.     

非洲菊查尔酮合酶基因的克隆、序列分析及在大肠杆菌中的表达

利用RT-PCR方法,从非洲菊(Gerbera hybrida)花瓣的CDNA中克隆到了查尔酮合酶(Chalcone Synthase,CHS)基因CHS,进行了序列分析。结果表明,克隆到的CHS基因全长为1197bps,编码一个由398个氨基酸残基组成的多肽,与Helariutta等发表的非洲菊查尔酮合酶CHSI基因的CDNA序列的CHS基因同源性高达99％。进一步将该基因克隆到表达载体pET32a上,经IPTG诱导表达,得到高效表达的融合蛋白。     

人血管能抑素基因N端片段的表达、纯化及其生物活性测定

以中国人胎盘脐带组织为材料 ,提取组织总RNA ,用RT PCR方法合成人血管能抑素cDNA ,将该cD NA克隆进 pSP72载体获得重组质粒 pSP72C。以pSP72C为模板 ,PCR方法合成编码血管能抑素N端 189aa的基因片段 ,将其克隆进pET 3c载体获得重组表达质粒 pET CN ,转化E .coliBL2 1(DE3) ,SDS PAGE分析显示 ,在IPTG诱导下 ,人血管能抑素N端基因片段获得了有效表达 ,表达量约占菌体总蛋白质的 35 .3% ,主要以包涵体形式存在。包涵体经过洗涤、裂解、蛋白质复性以及SephadexG 10 0凝胶过滤层析等步骤纯化后 ,获得了纯度约92 .6 %的人血管能抑素N端片段 ,CAM实验证明具有显著抑制鸡胚新生血管生成活性。     

人血管能抑素基因的克隆、表达及其表达产物的纯化和生物活性测定

以人胎盘脐带组织为材料,提取组织总RNA,用netRTPCR方法合成人血管能抑素cDNA基因,将该cDNA克隆进pSP72载体获得重组质粒pSP72C, DNA序列分析结果与预期序列一致。用BamHⅠ和NdeⅠ双酶切,切下pSP72C上的血管能抑素cDNA,插入pET3c载体的相应位点获得重组表达质粒pETC, 转化E. coli BL21(DE3), SDSPAGE分析显示：在IPTG诱导下,血管能抑素基因获得了高效表达,表达量约占菌体总蛋白的 27.9 %,主要以包涵体形式存在。包涵体经过洗涤、裂解、蛋白复性以及Sephadex G75凝胶过滤层析等步骤后,获得了纯度达91.4 %的人血管能抑素。CAM实验证明10 μg纯化蛋白就能显著抑制鸡胚新生血管生成。     

球毛壳菌（Chaetomium globosum）过氧化物膜蛋白过敏原基因克隆、序列分析及原核表达

以球毛壳菌cDNA文库中获得过氧化物膜蛋白（pero）基因片段（GenBank Accn:BP099709）为基础,用RACE 技术获得该基因的全长cDNA序列。序列长747bp,由412bp的3′RACE产物和508bp的5′RACE产物拼接而成。开放阅读框501bp,编码166个氨基酸,蛋白分子量为17.5kD,理论等电点为5.75。利用cDNA两侧非编码区序列作引物克隆出该基因的DNA序列,序列分析表明该基因由2个内含子和3个外显子组成。ClustalX多序列比对表明：该基因与粗糙脉孢菌（Neurospora crassa）的过氧化物膜蛋白过敏原同源性最高（83%）。将pero基因编码区克隆到原核表达载体pET28a中,构建成表达质粒pET28a-pero并转化大肠杆菌BL21,IPTG诱导后SDS-PAGE检测表达情况,结果发现在21kD处有一特异性融合蛋白带,大小与预期相符,说明该基因已经在大肠杆菌中表达。克隆的cDNA序列、DNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY555771,AY584753,AAS66898)。     

拟南芥中柯浩体蛋白Atcoilin的克隆、表达及纯化

旨在制备柯浩体的标志蛋白——Atcoilin蛋白,利用pET-28a与目的基因构建重组表达质粒,经DNA测序证实插入序列与设计完全一致后,将重组质粒转化大肠杆菌BL21(DE3),用IPTG进行诱导表达,产物用SDS-PAGE及Western blotting分析鉴定。通过分别改变IPTG的浓度、培养时间、培养温度等来优化Atcoilin蛋白的表达条件。表达出的重组蛋白经过镍柱、分子筛进行纯化。结果显示,原核表达载体pET28a-At1g13030成功构建,可在大肠杆菌BL21(DE3)中诱导表达,得到相应的重组蛋白经Western blotting鉴定正确。在IPTG浓度为0.7 mmol/L,18℃培养20 h的条件下,目的蛋白表达量最高。经过SDS-PAGE分析鉴定,过镍柱、分子筛后得到的重组蛋白纯度较高。     

人内皮生长抑制素基因克隆表达及其抑瘤作用

内皮生长抑制素（ｅｎｄｏｓｔａｔｉｎ是最近报道具有抑制肿瘤生长和铁蛋白质,为此,从正常人肝细胞株Ｌ０２中抽提总ＲＮＡ,以ＲＴ－ＰＣＲ法获得了人内皮生长抑制素编码序列,序列分析表明,内皮生长抑制素ＣＤＮＡ开放阅读框架全长５５５ｂｐ,此基因（ＧｅｎＢａｎｋ登录号为Ａ地８４０６０）,共编码１、８４个氨基酸残序列为５个碱基与文献报道不同,蛋白质序列中有３个氨基酸残基与文献报道不同,说明存在人种间差异。将人     

人铜锌超氧化物歧化酶cDNA的克隆,测序及表达

用逆转录聚合酶链反应（ＲＴＰＣＲ）,以人胎肝组织总ＲＮＡ为模板,扩增了人铜锌超氧化物歧化酶（ｈＣｕ,ＺｎＳＯＤ）的ｃＤＮＡ,并进行序列分析,将该ｈＣｕ,ＺｎＳＯＤｃＤＮＡ重组到Ｔ７启动子控制下的分泌型表达载体ｐＥＴ２２ｂ（＋）中,构建表达质粒ｐＥＴＳＯＤ,并转化大肠杆菌ＢＬ２１（ＤＥ３）。ＳＤＳＰＡＧＥ及蛋白质印迹分析表明,经１ｍｍｏｌ／Ｌ异丙基硫代βＤ半乳糖苷（ＩＰＴＧ）诱导后,可高效表达一分子量为１９ｋＤ的蛋白质,与抗人ＳＯＤ多抗有特异的免疫反应,表达量约为菌体总蛋白质的３０％,具有特异性ＳＯＤ酶活性,酶活力可达１７９７ｕ／ｍｌ培基。     

人生长激素基因的cDNA克隆,原核高效表达及纯化

用ＲＴ－ＰＣＲ方法,从含有全长人生长激素（ＨＧＨ）基因的ＣＨＯ细胞中获得ｈｇｈ的ｃＤＮＡ,将其克隆入ｐＥＴ－１１ｂ载体中,在大肠杆菌中获得高效表达,表达量占菌体蛋白总量的２０％。经色谱纯化后,纯度大于９５％。