抑癌蛋白PTEN的原核表达、抑癌活性研究和抗体制备

PTEN是具有蛋白质和酯类双重特异性磷酸酶活性的抑癌蛋白,在肿瘤治疗中具有广阔的应用前景。鉴于原核表达PTEN蛋白并用于抑癌实验的研究尚未见报道,因此尝试利用大肠杆菌表达有活性的PTEN蛋白,检测其抑癌效果。利用本室克隆的PTEN基因cDNA和原核表达载体pET44a( )分别构建带6×His和Nus标签的两种诱导型原核融合表达载体pETPTEN和pETNusPTEN,在不同的大肠杆菌表达宿主BL21(DE3)(简写为BL)和Rosettagami(DE3)pLysS(简写为RG)中诱导表达。SDSPAGE和Westernblot检测表明:在可溶性组分和包涵体中均含有目的蛋白,在BL中目的蛋白的表达量较高(18.7%)而在RG中可溶性蛋白的比例较高(6.6%)。经纯化和包涵体蛋白复性处理后,重组融合蛋白经Chariot转运入小鼠实体瘤及人前列腺癌DU145细胞。抑癌实验表明:与对照组相比,重组PTEN蛋白对小鼠实体瘤的生长抑制率为58.76%;对癌细胞DU145的生长抑制率可达46.16%;并可导致明显的G0G1期阻滞,其中在宿主RG中表达的重组蛋白抑癌效果明显高于BL宿主中表达的目的蛋白。证实在原核系统中表达的重组PTEN蛋白具有抑癌活性,同时制备了PTEN的高效价腹水多抗,为深入研究PTEN蛋白在癌症治疗中的应用打下了良好的基础。     

精子发生相关基因片段CG14的克隆及其蛋白的表达

为研究与精子发生相关的基因并探讨其功能 ,用差异显示法发现了 1个与精子发生相关的基因片段CG14 .将该基因片段克隆到表达载体pGEX 3X上 ,在大肠杆菌中表达了融合蛋白 .通过谷胱甘肽 Sepharose 4B亲和柱纯化该融合蛋白 .经Xa因子酶切后Western印迹方法证明 ,靶蛋白分子量约为 8kD ,与预期分子量相符 .用融合蛋白免疫家兔获得抗血清 .免疫印迹实验表明 ,血清中含有CG14蛋白的特异性抗体 ,为进一步研究CG14基因及其表达蛋白的功能打下基础     

水母雪莲通气组织形成的相关基因SmLSD1克隆及表达

该研究以水母雪莲为实验材料,通过RT-PCR结合RACE技术克隆了通气组织形成相关基因SmLSD1(GenBank登录号为OL690334),并对该基因在不同胁迫下的表达量及编码蛋白结构进行测定分析。结果表明：(1)水母雪莲SmLSD1基因全长965 bp,包含537 bp的开放阅读框,编码178个氨基酸。(2)同源序列比对发现,水母雪莲SmLSD1蛋白与菊科植物牛蒡LSD1的氨基酸序列相似性最高,达到98.31%。(3)亚细胞定位显示SmLSD1基因主要在细胞核和细胞膜上表达;原核表达显示,SmLSD1基因编码氨基酸的分子量约为18 kD。(4)荧光定量分析显示,SmLSD1基因在根、茎、叶中均有表达,且在叶片中表达量最高;在低温、低氧及紫外胁迫下,SmLSD1基因的表达量下调。研究推测,SmLSD1基因在水母雪莲通气组织的形成以及对逆境胁迫的响应中发挥着重要作用。     

人抑癌基因PTEN的原核表达载体的构建及融合表达

为研究抑癌因子PTEN蛋白的抑癌机理,掏建了PTEN cDNA的原核表达载体并进行融合表达。将含有PTEN cDNA的质粒pMD-PTEN经EcoR Ⅰ和Sal Ⅰ双酶切,回收PTEN基因片段与经相同酶切的高效原核表达载体pET-44a连接,经序列测定,证实融合型表达载体pET-Nus-PTEN构建成功。转化表达宿主BL21(DE3)后,IPTG诱导表达。经12％SDS-PAGE凝胶电泳,获得118kD的特异蛋白条带。目的蛋白占细菌总蛋白的17％。结果表明：PTEN基因和Nus基因融合表达成功,获得可溶性Nus-PTEN蛋白。该研究为PTEN蛋白的抑癌机理和基因工程药物的研究打下了基础,这是国内PTEN蛋白在原核细胞中成功表达的首次报道。     

肺癌中SOX4基因的调控网络分析验证和蛋白质预测

目的:分析SOX4基因的调控网络和蛋白产物特征,并进行验证。方法:利用生物信息学方法预测人SOX4基因的调控网络及其蛋白产物与其他蛋白的相互作用;在肺癌细胞系和肺癌临床样本中通过实时荧光定量PCR对mi RNA-30s进行定量分析。结果:人类SOX4蛋白可能与TP53、DICER1和SDCBP2等3种蛋白相互作用;SOX4基因可能受多种lnc RNA和mi RNA的调控;hsa-mi R30s在临床样本中表达模式不完全相同。与癌旁组织相比,hsami R30c-5p和hsa-mi R30d-5p在癌组织中显示低表达,hsa-mi R30d-5p则相反;与正常人胚肺成纤维细胞KMB17相比,肺癌细胞系A549和H1299中3个mi RNA-30成员的表达模式均为高表达。结论:SOX4基因及其蛋白产物的生物信息学分析为相关研究提供了重要信息基础,但尚须相关实验验证,以确定相关性。     

梭梭中HaDREB2A的克隆与表达分析

脱水响应元件结合蛋白(DREB)转录因子在应激反应相关基因的调控中起重要作用。本研究利用RT-PCR方法从梭梭组织中获得Ha DREB2A基因开放阅读框(ORF)全长(Gen Bank登录号为:KP765243)。生物信息学分析表明,该基因ORF长1 083 bp,编码360个氨基酸,蛋白的分子量为40.5 k D,理论等电点(p I)为5.07,该蛋白在90(K)~143(R)区域含有一个典型的AP2保守结构域。通过物种间系统发育树可以看出Ha DREB2A与Sb DREB2A进化关系最接近,二者属于同一进化分支。半定量RT-PCR表达模式分析显示,该基因在梭梭同化枝、根、茎、花和种子中均有表达,且在同化枝中的表达量最高,并且受干旱、高盐和外源ABA的诱导。     

4.1B蛋白的抑制肿瘤作用

4.1 R和Merlin是4.1蛋白超家族中两个功能比较清楚的成员,前者通过结合肌动蛋白和血影蛋白维持红细胞骨架结构的完整性:后者为抑癌蛋白,其缺失与脑膜瘤发生有关.4.1B蛋白是4.1R和Merlin的同源蛋白,与二者的结构和功能具有相似性.4.1B蛋白由三个保守的结构域构成,即FERM、SABD和CTD,通过这三个结构域,能与一系列蛋白质相互作用.4.1 B蛋白表达缺失与脑膜瘤、乳腺癌和非小细胞肺癌的发生相关,而过量表达则可激活JNK信号途径,促进细胞凋亡;此外,4.1 B蛋白还具有抑制肿瘤转移的功能.因此,目前多认为4.1B基因可能是一个抑癌基因.     

稳定表达外源性p16基因肺癌A549细胞株的建立及鉴定

为构建稳定表达外源性抑癌基因p16的肺癌A549细胞株,用脂质体介导的基因转染方法,借助真核质粒表达载体（pcDNA3)。将抑癌基因p16转移入此基因缺失的人肺癌细胞株A549细胞中,经G418筛选,获得稳定表达的细胞克隆,用逆转录聚合酶链反应（RT-PCR）及免疫组织化学鉴定p16基因的表达,同时对克隆细胞分泌蛋白进行活性检测。结果显示转染p16基因的A549细胞中可以检测到p16mRNA及蛋白的表达,说明建立的p16真核表达载体能在肺肿瘤细胞中分泌表达蛋白,表达P16抑癌蛋白的A549细胞株的建立有助于研究抑癌基因p16在肺癌发生中的作用。     

中华绒螯蟹IAG基因的克隆及生物信息学分析

采用RACE-PCR技术,获得中华绒螯蟹胰岛素样促雄激素腺基因(Es-IAG)全长cDNA序列,并利用相关生物信息学软件对该基因及其蛋白质的结构、理化特性进行生物信息学分析。结果发现,Es-IAG基因序列全长1 392 bp,编码151个氨基酸,Es-IAG多肽的相对分子量为16.61 kD,理论等电点pI为5.08,前体多肽中存在分泌型信号肽,存在两个糖基化位点和16个磷酸化位点,存在两个典型的R**R蛋白酶酶切位点,未发现跨膜结构域。Es-IAG为分泌性蛋白,蛋白需要通过结合细胞膜上的受体而发挥性别调控等作用。对22个近缘物种的IAG基因序列系统进化分析显示,中华绒螯蟹与蓝蟹、拟穴青蟹的亲缘关系较近。为深入研究Es-IAG基因及其蛋白的结构和功能提供了相关依据。     

垂体瘤中PTEN及CyclinD1抑癌基因的表达及临床意义

本研究以垂体瘤患者垂体组织为研究对象,探讨了抑癌基因PTEN及CyclinD1表达的临床意义,分析了其与垂体瘤侵袭性的相关性。结果发现,垂体瘤患者垂体组织抑癌基因PTEN的表达低于正常垂体组织,同时垂体瘤患者垂体组织CyclinD1表达高于正常垂体组织(p0.05)。垂体瘤组织PTEN的mRNA表达低于正常垂体组织,而CyclinD1的mRNA表达高于正常垂体组织(p0.05)。垂体瘤患者垂体组织抑癌基因PTEN低表达的患者CyclinD1呈现高表达,相关性分析发现两者呈负相关(p0.05)。抑癌基因PTEN及CyclinD1表达与垂体瘤侵袭性相关,可作为垂体瘤患者的诊断和预后指标。     

Overexpression of DOC-1R Inhibits Cell Cycle G1/S Transition by Repressing CDK2 Expression and Activation

DOC-1R (deleted in oral cancer-1 related) is a novel putative tumor suppressor. This study investigated DOC-1R antitumor activity and the underlying molecular mechanisms. Cell phenotypes were assessed using flow cytometry, BrdU incorporation and CDK2 kinase assays in DOC-1R overexpressing HeLa cells. In addition, RT-PCR and Western blot assays were used to detect underlying molecular changes in these cells. The interaction between DOC-1R and CDK2 proteins was assayed by GST pull-down and immunoprecipitation-Western blot assays. The data showed that DOC-1R overexpression inhibited G1/S phase transition, DNA replication and suppressed CDK2 activity. Molecularly, DOC-1R inhibited CDK2 expression at the mRNA and protein levels, and there were decreased levels of G1-phase cyclins (cyclin D1 and E) and elevated levels of p21, p27, and p53 proteins. Meanwhile, DOC-1R associated with CDK2 and inhibited CDK2 activation by obstructing its association with cyclin E and A. In conclusion, the antitumor effects of DOC-1R may be mediated by negatively regulating G1 phase progression and G1/S transition through inhibiting CDK2 expression and activation.     

p12(DOC-1), a growth suppressor, associates with DNA polymerase alpha/primase.

p12(DOC-1) is a growth suppressor identified and isolated from normal keratinocytes. Ectopic expression of p12(DOC-1) in squamous carcinoma cells led to the reversion of in vitro transformation phenotypes including anchorage independence, doubling time, and morphology. Here we report that p12(DOC-1) associates with DNA polymerase alpha/primase (pol-alpha:primase) in vitro and in cells. The pol-alpha:primase binding domain in p12(DOC-1) is mapped to the amino-terminal six amino acid (MSYKPN). The biological effect of p12(DOC-1) on pol-alpha:primase was examined using in vitro DNA replication assays. Using the SV40 DNA replication assay, p12(DOC-1) suppresses DNA replication, leveling at approximately 50%. Similar results were obtained using the M13 single-stranded DNA synthesis assay. Analysis of the DNA replication products revealed that p12(DOC-1) affects the initiation step, not the elongation phase. The p12(DOC-1) suppression of DNA replication is likely to be mediated either by a direct inhibitory effect on pol-alpha:primase or by its effect on cyclin-dependent kinase 2 (CDK2), a recently identified p12(DOC-1)-associated protein known to stimulate DNA replication by phosphorylating pol-alpha:primase. p12(DOC-1) suppresses CDK2-mediated phosphorylation of pol-alpha:primase. These data support a role of p12(DOC-1) as a regulator of DNA replication by direct inhibition of pol-alpha:primase or by negatively regulating the CDK2-mediated phosphorylation of pol-alpha:primase.     

The inhibitory role of DOC-2/DAB2 in growth factor receptor-mediated signal cascade. DOC-2/DAB2-mediated inhibition of ERK phosphorylation via binding to Grb2

DOC-2/DAB2 (differentially expressed in ovarian carcinoma-2/disabled 2) appears to be a potential tumor suppressor gene with a growth inhibitory effect on several cancer types. Previously, we have shown that DOC-2/DAB2 suppresses protein kinase C-induced AP-1 activation, which is modulated by serine 24 phosphorylation in the N terminus of DOC-2/DAB2. However, the functional impact of the C terminus of DOC-2/DAB2, containing three proline-rich domains, has not been explored. In this study, we examined this functional role in modulating signaling mediated by peptide growth factor receptor tyrosine kinase, particularly because it involves the interaction with Grb2. Using sequence-specific peptides, we found that the second proline-rich domain of DOC-2/DAB2 is the key binding site to Grb2 in the presence of growth factors. Such elevated binding interrupts the binding between SOS and Grb2, which consequently suppresses downstream ERK phosphorylation. Reduced ERK phosphorylation was restored when the binding between DOC-2/DAB2 and Grb2 was interrupted by a specific peptide or by increasing the expression of Grb2. Furthermore, the C terminus of the DOC-2/DAB2 construct can inhibit the AP-1 activity elicited by growth factors. We conclude that DOC-2/DAB2, a potent negative regulator, can suppress ERK activation by interrupting the binding between Grb2 and SOS that is elicited by peptide growth factors. This study further illustrates that DOC-2/DAB2 has multiple effects on the RAS-mediated signal cascades active in cancer cells.     

The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase.

Yeast vacuolar acidification-defective (vph) mutants were identified using the pH-sensitive fluorescence of 6-carboxyfluorescein diacetate (Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7027-7031). Vacuoles purified from yeast bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. The peripherally bound nucleotide-binding subunits of the vacuolar H(+)-ATPase (60 and 69 kDa) were no longer associated with vacuolar membranes yet were present in wild type levels in yeast whole cell extracts. The VPH1 gene was cloned by complementation of the vph1-1 mutation and independently cloned by screening a lambda gt11 expression library with antibodies directed against a 95-kDa vacuolar integral membrane protein. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is not essential for viability but is required for vacuolar H(+)-ATPase assembly and vacuolar acidification. VPH1 encodes a predicted polypeptide of 840 amino acid residues (molecular mass 95.6 kDa) and contains six putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity. Multiple sequence alignments show extensive homology over the entire lengths of the following four polypeptides: Vph1p, the 116-kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene), and the TJ6 mouse immune suppressor factor.     

p12(DOC-1) is a novel cyclin-dependent kinase 2-associated protein

Regulated cyclin-dependent kinase (CDK) levels and activities are critical for the proper progression of the cell division cycle. p12(DOC-1) is a growth suppressor isolated from normal keratinocytes. We report that p12(DOC-1) associates with CDK2. More specifically, p12(DOC-1) associates with the monomeric nonphosphorylated form of CDK2 (p33CDK2). Ectopic expression of p12(DOC-1) resulted in decreased cellular CDK2 and reduced CDK2-associated kinase activities and was accompanied by a shift in the cell cycle positions of p12(DOC-1) transfectants ( upward arrow G(1) and downward arrow S). The p12(DOC-1)-mediated decrease of CDK2 was prevented if the p12(DOC-1) transfectants were grown in the presence of the proteosome inhibitor clasto-lactacystin beta-lactone, suggesting that p12(DOC-1) may target CDK2 for proteolysis. A CDK2 binding mutant was created and was found to revert p12(DOC-1)-mediated, CDK2-associated cell cycle phenotypes. These data support p12(DOC-1) as a specific CDK2-associated protein that negatively regulates CDK2 activities by sequestering the monomeric pool of CDK2 and/or targets CDK2 for proteolysis, reducing the active pool of CDK2.     

新的端粒酶活性抑制蛋白LPTS-L的制备

LPTS基因是利用定位候选克隆策略克隆的一个新的肝相关候选肿瘤抑制基因。LPTS基因编码一个全长为328氨基酸的蛋白质(LPTS-L),该蛋白具有抑制细胞端粒酶活性的功能。为了进一步研究LPTS-L蛋白的结构与功能,利用DNA重组技术,将LPTS-L的cDNA克隆到表达载体pET-24a中构建重组克隆pET-24-LPTS,并在大肠杆菌BL-21中进行融合表达,获得可溶形式的LPTS-L融合蛋白。采用Ni Sepharose4B柱亲和层析,可以获得纯度较高的蛋白,但不适合大量制备。通过设计引物去掉了pET-24a载体上的6×His tag将LPTS-L基因进行了非融合表达,然后采用磷酸纤维素P11阳离子交换层析纯化LPTS-L蛋白,纯度可达到55%。再经Sephadex G-100凝胶过滤,LPTS-L蛋白的纯度可达到80%。Western blot实验显示经纯化后的LPTS-L蛋白可与兔抗GST-LPTS-L的多抗发生特异性结合。采用TRAP法测定蛋白质活性,结果显示纯化得到的LPTS-L蛋白可抑制端粒酶的活性,与采用Ni Sepharose4B纯化获得的LPTS-L融合蛋白比较,其抑制效率基本一致。因此,所建立的技术可以有效地制备LPTS-L蛋白。     

A recombinant trans-membrane protein hMnSOD–R9 inhibits the proliferation of cervical cancer cells in vitro

Human manganese superoxide dismutase (hMnSOD) is a new type of cancer suppressor. Nonamer of arginine (R9) is an efficient protein transduction domain (PTD). The aim of the study was to improve the transduction efficiency of hMnSOD and investigate its activity in vitro. In this study, we designed, constructed, expressed, and purified a novel fusion protein containing the hMnSOD domain and R9 PTD (hMnSOD–R9). The DNA damaged by Fenton’s reagent was found to be significantly reduced when treated with hMnSOD–R9. hMnSOD–R9 fusion protein was successfully delivered into HeLa cells. The MTT assay showed that proliferation of various cancer cell lines were inhibited by hMnSOD–R9 in a dose-dependent manner. In addition, the cell cycle of HeLa cells was arrested at the sub-G0 phase by hMnSOD–R9. hMnSOD–R9 induced apoptosis of HeLa cells in a dose-dependent manner. With hMnSOD–R9 treatment, Bax, JNK, TBK1 gene expression was increased and STAT3 gene expression was gradually down-regulated in HeLa cells. We also found that apoptosis was induced by hMnSOD–R9 in HeLa cells via up-regulation of cleaved caspase-3 and down-regulation phospho-STAT3 pathway. These results indicated that hMnSOD–R9 may provide benefits to cervical cancer treatment.     

斜纹夜蛾核多角体病毒p49基因在大肠杆菌中的表达及其在昆虫细胞中的表达时相分析

利用PCR方法从斜纹夜蛾核多角体病毒（SpltMNPV）基因组中扩增获得了细胞凋亡抑制基因〖STBX〗p49〖STBZ〗的完整ORF并将其克隆于pMD18T载体,其序列分析结果与文献报道一致。将基因重组于硫氧还蛋白融合表达载体pThioHis C,在大肠杆菌BL21(DE3)中获得了稳定表达,表达的P49融合蛋白占菌体总蛋白30%左右,主要以包涵体形式存在。分离纯化重组表达的SpltMNPV P49蛋白作为抗原,免疫家兔制备得到效价高于1∶10000的抗重组P49蛋白多克隆抗体。应用制备的抗体对受SpltMNPV感染的Sl细胞中P49蛋白的表达时相进行分析,结果显示P49蛋白在细胞感染后3h内便可检测到,并在整个感染期间维持着低水平表达。     

Cloning and sequencing of complement component C9 and its linkage to DOC-2 in the pufferfish Fugu rubripes

The Japanese pufferfish Fugu rubripes has a 400 Mb genome with high gene density and minimal non-coding complexity, and is therefore an ideal vertebrate model for sequence comparison. The identification of regions of conserved synteny between Fugu and humans would greatly accelerate the mapping and ordering of genes. Fugu C9 was cloned and sequenced as a first step in an attempt to characterize the region in Fugu homologous to human chromosome 5p13. The 11 exons of the Fugu C9 gene share 33% identity with human C9 and span 2.9 kb of genomic DNA. By comparison, human C9 spans 90 kb, representing a 30-fold difference in size. We have also determined by cosmid sequence scanning that DOC-2, a tumour suppresser gene which also maps to human 5p13, lies 6–7 kb from C9 in a head-to-head or 5′ to 5′ orientation. These results demonstrate that the Fugu C9/DOC-2 locus is a region of conserved synteny. Sequence scanning of overlapping cosmids has identified two other genes, GAS-1 and FBP, both of which map to human chromosome 9q22, and lie adjacent to the Fugu C9/DOC-2 locus, indicating the boundary between two syntenic regions.