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％。     

Wistar大鼠Pdia3cDNA的克隆及原核表达

哺乳动物的精卵融合是一个多分子参与的复杂过程,目前只鉴定出几种影响精卵融合的蛋白因子,对其分子机制还知之甚少.其中位于精子上的Pdja3具有二硫键异构酶活性,可能催化其他精卵融合相关蛋白的二硫键变化而参与精卵膜融合.克隆了Wistar大鼠的Pdia3 cDNA片段,长度为1 620 bp,其中编码区为1 518 bp.测序结果显示,编码区内有两个碱基与GenBank公布的大鼠Pdia3cDNA不同,分别为第147个密码子GAG(Glu)的第1位碱基和第410个密码子AAG(Lys)的第3位碱基,前者造成氨基酸替换,后者仅为多态性.此外,还在大肠杆菌中成功地表达并纯化了GST-Pdia3融合蛋白,为下一步研究大鼠Pdia3在体外影响精卵融合和与其它蛋白的相互作用奠定了基础.     

大鼠RVLG cDNA的克隆、原核表达和小鼠抗RVLG蛋白多克隆抗体的制备

为了识别大鼠卵巢中的生殖细胞,在原核系统中表达和纯化RVLG蛋白并制备了多克隆抗体.采用RT-PCR方法从大鼠睾丸组织中扩增获得RVLG cDNA片段,然后克隆到pMD19-T载体上进行测序,经双酶切回收目的基因片段后,将其插入到原核表达载体pGEX-4T-1上,转入大肠杆菌BL21(DE3)中诱导表达.纯化后的GST-RVLG融合蛋白免疫昆明(KM)小鼠,最后给小鼠腹腔注射S180细胞制备抗RVLG腹水多克隆抗体.用Western blotting及免疫组织化学法鉴定RVLG腹水多克隆抗体的特异性,间接ELISA法测定该抗体的效价.序列分析表明,所克隆的RVLG cDNA片段比GenBank中报道的大鼠RVLG cDNA(NM_001077647)多60 bp,原因是由于RVLG的可变剪切方式造成的.本研究成功构建了重组表达质粒pGEX-RVLG,且GST-RVLG融合蛋白在大肠杆菌BL21(DE3)中高效表达,表达的目的蛋白占菌体总蛋白的10%以上.制备的抗体可特异性识别RVLG蛋白,其效价达1:20 000.获得的高效价、高特异性的小鼠抗RVLG蛋白腹水多克隆抗体为下阶段研究RVLG的特异性表达奠定了基础.     

凋亡抑制因子Survivin的克隆、高效表达与纯化

Survivin蛋白抑制细胞的凋亡并参与调控细胞的分裂,在绝大多数肿瘤细胞中均有过量表达。本实验以人肝癌细胞系MHCC-97L总RNA为模板,应用RT-PCR的方法得到survivin cDNA,并构建了重组原核表达载体pET-21b(+)-survivin,导入BL21（DE3）菌株进行表达,表达产物以包涵体形式存在,表达量超过总蛋白的60%。Western blot结果表明表达产物与抗人survivin抗体发生特异性反应,经凝胶过滤层析后纯度达到95%以上,为进一步研究靶向Survivin的诊断试剂与抑制剂奠定了基础。     

Wistar大鼠CD9的cDNA克隆、原核表达及生殖腺中的检测

为了用Wistar大鼠研究CD9对精卵融合的影响和与其它精卵融合相关蛋白的作用,克隆了Wistar大鼠的CD9cDNA.测序结果显示,Wistar大鼠的CD9 cDNA编码区与GenBank中发布的SD( Sprague-Dawley)大鼠相同,但在3′非翻译区多一个T.用Western blotting方法检测Wistar大鼠睾丸和卵巢总蛋白发现睾丸和卵巢里均表达内源性CD9蛋白,分子量相同.此外,在大肠杆菌中表达了GST-CD9融合蛋白,并用GST标签纯化CD9蛋白,为体外研究CD9与其它精卵融合相关蛋白的作用提供参考.     

小鼠RMND5A的原核表达、纯化及多克隆抗体制备

RMNDSA通过MAPK信号途径参与了对细胞生命活动的调控过程.通过PCR扩增RMND5A基因,并将其克隆至表达载体pGEX-4T-1上,转化至大肠杆菌BL21（DE3）中,再通过IPTG进行诱导表达GST-RMND5A融合蛋白.通过尿素洗涤包涵体并切胶回收纯化融合蛋白,免疫新西兰大白兔制备多克隆抗体,Western blot检测抗体活性.结果说明,获得了RMND5A原核表达重组融合蛋白及高效价的特异性兔抗RMND5A多克隆抗体,为RMND5A进一步的功能研究奠定了基础.     

水牛CD14 cDNA的克隆、表达及鉴定

目的:克隆水牛白细胞分化抗原14(buffalo cluster of differentiation antigen14,bCD14)基因,表达bCD14蛋白,并进行Western Blot鉴定.方法:采用RT-PCR方法从水牛外周血白细胞中扩增bCD14基因,构建重组质粒pET28a-bCD14,转化入E coli BL21,IPTG诱导表达,对表达蛋白进行可溶性分析及Western blot鉴定.结果:bCD14基因含有一个1 122bp的开放阅读框,编码373个氨基酸;与印度水牛、挪威大鼠和人CD14的cDNA序列同源性分别为97.95%、68.78%、78.60%,氨基酸同源性分别为96.78%、61.27%、72.34%;主要以包涵体形式表达,表达蛋白经Western Blot鉴定,得到了一条约46 kD的特异性条带.结论:该文成功克隆了bCD14基因,表达了bCD14蛋白,为进一步揭示水牛抵抗革兰氏阴性菌感染的免疫机制奠定了基础.     

拟南芥MnSOD的原核表达、纯化及抗体制备

PCR扩增拟南芥MnSOD cDNA的保守区段,构建pET-SOD重组质粒,转化大肠杆菌JM109(DE3),IPTG诱导融合蛋白高效表达;经检测表达产物占菌体总蛋白的69％,且以不溶的包涵体形式存在;表达产物变性后经Ni—NTA Superflow亲和柱分离纯化,得到相对分子质量约为29000的PAGE纯蛋白。融合蛋白经还原复性处理后,比活达到200U／mg;免疫家兔制备MnSOD融合蛋白抗体,抗体效价为1：10000。以上结果为进一步大规模制备MnSOD及其功能研究奠定基础。     

海南坡鹿种群发展动态与保护建议

就地保护的海南坡鹿种群分布于海南大田国家级自然保护区,有野生和半野放两种类型,其中野生种群个体数量为65头,在1986～1992年间年均增长率为2.0%;半野放种群个体数量为1093头,同期年均增长率为22.4%.迁地保护的海南坡鹿的分布种群有野放、半野放和圈养等3种类型,种群个体数量分别为524、337和86头.野放种群个体已有产仔现象;海南邦溪自然保护区的半野放种群在1994～2004年间的年均增长率为18.9%;枫木鹿场的圈养种群同期年均增长率为12.4%.目前,半野放是就地保护的主要方式;而野放是迁地保护的主要方式.应该注重发展迁地保护的半野放种群及其栖息地.对坡鹿的就地保护和迁地保护提出了一些建议.     

CDC42 抑制剂ML141对喉癌Hep-2 细胞增殖的抑制作用

目的:探讨CDC42抑制剂ML141对喉癌细胞增殖的抑制作用,为喉癌的分子治疗提供新的靶点。方法:体外培养人喉癌Hep-2细胞。实时定量聚合酶链反应(Real-time PCR)检测CDC42在Hep-2细胞中的表达。利用GLISA法检测ML141对CDC42活性的抑制效果。利用CCK8法检测ML141对Hep-2细胞增殖能力的抑制效果。结果:1Real-time PCR结果显示在人喉癌Hep-2细胞中CDC42显著高表达(P0.001),证明全基因组的结果准确。2GLISA结果显示表皮生长因子作用的Hep-2细胞中CDC42的活性明显高表达,但加入ML141的Hep-2细胞中CDC42的活性受到明显抑制。(P0.001)。3CCK8结果显示24 h,48h和72 h时,ML141处理的Hep-2细胞的增殖能力与对照组相比均受到明显抑制。(P0.001)。结论:促癌基因CDC42抑制剂ML141能够抑制人喉癌Hep-2细胞增殖,具有成为抗喉癌新药的潜力,为喉癌的分子治疗提供新的切入点。     

海南坡鹿恢复生态学研究进展

海南坡鹿的个体总数已经超过1600头,分布地点已经扩展到10多个。海南坡鹿的恢复生态学研究主要包括种群恢复、栖息地管理和行为恢复等3个方面。就种群恢复而言,半野放是一种较稳妥的种群恢复管理方式,应加大投入;野生种群和野放种群的恢复现状缺乏了解,应加强监测与研究。栖息地管理是半野放种群恢复的基础和保障：而圈养、野放和野生种群的栖息地管理工作有待深入。海南坡鹿的恢复也面临行为恢复问题。这方面值得受到更多关注。     

CDC42 switches IRSp53 from inhibition of actin growth to elongation by clustering of VASP

Filopodia explore the environment, sensing soluble and mechanical cues during directional motility and tissue morphogenesis. How filopodia are initiated and spatially restricted to specific sites on the plasma membrane is still unclear. Here, we show that the membrane deforming and curvature sensing IRSp53 (Insulin Receptor Substrate of 53 kDa) protein slows down actin filament barbed end growth. This inhibition is relieved by CDC42 and counteracted by VASP, which also binds to IRSp53. The VASP:IRSp53 interaction is regulated by activated CDC42 and promotes high‐density clustering of VASP, which is required for processive actin filament elongation. The interaction also mediates VASP recruitment to liposomes. In cells, IRSp53 and VASP accumulate at discrete foci at the leading edge, where filopodia are initiated. Genetic removal of IRSp53 impairs the formation of VASP foci, filopodia and chemotactic motility, while IRSp53 null mice display defective wound healing. Thus, IRSp53 dampens barbed end growth. CDC42 activation inhibits this activity and promotes IRSp53‐dependent recruitment and clustering of VASP to drive actin assembly. These events result in spatial restriction of VASP filament elongation for initiation of filopodia during cell migration, invasion, and tissue repair.     

NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity

N-myc downstream regulated gene-1 (NDRG1) has been identified as a putative metastasis suppressor gene and proved to be a key player in cancer spreading and proliferation in our previous work. However, the effects of NDRG1 on tumor invasion and the mechanisms behind it are rarely understood. Here we provided in silico evidence that NDRG1 plays a crucial role in actin reorganization in colorectal cancer (CRC). Through in vitro experiments, we next observed filopodia formation was altered in NDRG1-modified cell lines, while cell division cycle-42 (CDC42) displayed excessive activation in NDRG1-silenced cells. Mechanistically, NDRG1 loss disrupts the binding between RhoGDIα and CDC42 and triggers the activation of CDC42 and the downstream cascades PAK1/Cofilin, thereby promotes the formation of filopodia and invasiveness of CRC. The knockdown of NDRG1 led to enhanced dissemination of CRC cells in vivo and correlates with active CDC42 expression. Using clinical sample analysis, we found an elevated level of active CDC42 in patients with advanced T stage, and it was negatively related to NDRG1 expression. In sum, these results uncover a mechanism utilized by NDRG1 to regulate CDC42 activity in coordinating cytoskeleton reorganization, which was crucial in cancer invasion.     

海南坡鹿栖息地植被覆盖变化研究

根据不同时期的Landsat TM卫星遥感图像,分别提取海南坡鹿Cervus eldi hainanus栖息地海南大田国家级自然保护区及周边地区的归一化植被指数(Normalized Difference Vegetation Index, NDVI).1991年保护区和周边地区的NDVI平均值分别为0.2485和0.291,而2001年的NDVI平均值分别为0.1272和0.0827,表明海南大田国家级自然保护区和周边地区的植被覆盖均下降,且周边地区植被覆盖下降幅度较大.保护区植被覆盖的下降是植被改造的结果,有利于坡鹿及栖息地的保护与发展;保护区周边地区植被覆盖下降是垦荒和采伐等人为干扰所致,是导致坡鹿栖息地丧失或破坏的重要原因.人类活动是植被覆盖变化的主要因素.建议自然保护区的规划应为濒危物种保护的未来留下空间.     

Biochemical analysis of the interactions of IQGAP1 C-terminal domain with CDC42

AIM:To understand the interaction of human IQGAP1 and CDC42,especially the effects of phosphorylation and a cancer-associated mutation. METHODS:Recombinant CDC42 and a novel C-termi- nal fragment of IQGAP1 were expressed in,and puri- fied from,Escherichia coli.Site directed mutagenesis was used to create coding sequences for three phos- phomimicking variants(S1441E,S1443D and S1441E/ S1443D)and to recapitulate a cancer-associated mu- tation(M1231I).These variant proteins were also ex- pressed and purified.Protein-protein crosslinking using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to investigate interactions between the C-terminal fragment and CDC42.These interactions were quanti- fied using surface plasmon resonance measurements.Molecular modelling was employed to make predictions about changes to the structure and flexibility of the protein which occur in the cancer-associated variant. RESULTS:The novel,C-terminal region of human IQGAP1 (residues 877-1558)is soluble following expression and purification.It is also capable of binding to CDC42,as judged by crosslinking experiments.Interaction appears to be strongest in the presence of added GTP.The three phosphomimicking mutants had different affini- ties for CDC42.S1441E had an approximately 200-fold reduction in affinity compared to wild type.This was caused largely by a dramatic reduction in the associa- tion rate constant.In contrast,both S1443D and the double variant S1441E/S1443D had similar affinities to the wild type.The cancer-associated variant,M1231I, also had a similar affinity to wild type.However,in the case of this variant,both the association and dis- sociation rate constants were reduced approximately 10-fold.Molecular modelling of the M1231I variant, based on the published crystal structure of part of the C-terminal region,revealed no gross structural changes compared to wild type(root mean square deviation of 0.564over 5556 equivalent atoms).However,pre- dictions of the flexibility of the polypeptide backbone suggested that some regions of the variant protein had greatly increased rigidity compared to wild type.One such region is a loop linking the proposed CDC42 bind- ing site with the helix containing the altered residue.It is suggested that this increase in rigidity is responsible for the observed changes in association and dissocia- tion rate constants. CONCLUSION:The consequences of introducing nega- tive charge at Ser-1441 or Ser-1443 in IQGAP1 are dif- ferent.The cancer-associated variant M1231I exerts its effects partly by rigidifying the protein.     

Advances in the study of CDC42 in the female reproductive system

CDC42 is a member of the Rho‐GTPase family and is involved in a variety of cellular functions including regulation of cell cycle progression, constitution of the actin backbone and membrane transport. In particular, CDC42 plays a key role in the establishment of polarity in female vertebrate oocytes, and essential to this major regulatory role is its local occupation of specific regions of the cell to ensure that the contractile ring is assembled at the right time and place to ensure proper gametogenesis. The multifactor controlled ‘inactivation‐activation’ process of CDC42 also allows it to play an important role in the multilevel signalling network, and the synergistic regulation of multiple genes ensures maximum precision during gametogenesis. The purpose of this paper is to review the role of CDC42 in the control of gametogenesis and to explore its related mechanisms, with the aim of further understanding the great research potential of CDC42 in female vertebrate germ cells and its future clinical translation.     

海南坡鹿对采食场地及食物的选择

2001年10月至2002年9月在海南大田国家级自然保护区,采用直接观察法和采食痕迹估计法对海南坡鹿(Cenrus eldi hainanus)的食性进行了研究.共记录取食地1273个,其中旱季748个,雨季525个.海南坡鹿取食植物234种,其中木本植物71种,草本植物161种,蕨类植物2种.其中取食频率超过0.01%的有76种,取食频率占总取食频率的81.85%.无论旱季和雨季坡鹿对采食场地都具有明显的选择性(旱季X2=21.476,P=0.000 7;雨季X2=19.882,P=0.001 3;df=5).尽管旱季坡鹿更多地选择在低平地热带草原生境中采食、雨季则喜欢季雨林,但是它们选择采食场地的季节性差异没有达到显著的水平(X2=9.447,P=0.092 5,df=5).坡鹿对植物种类的选择存在季节性差异,旱季喜欢采食的植物种类比雨季更多(30种vs.18种),采食的木本植物种类的比例也高于雨季(14种vs.6种).