双齿围沙蚕诱导型热休克蛋白70基因的克隆及Cu胁迫下的表达分析

本研究主要评估了双齿围沙蚕热休克蛋白70(HSP70)基因的分子特征,记录了其对于液态Cu²⁺胁迫的基因表达情况,并通过测序获得的HSP70 cDNA序列与其他沙蚕及无脊椎动物HSP70同源性比对来判定蛋白特性.结果表明: 该HSP70基因全长cDNA序列共2161 bp,包括5′非翻译区48 bp,3′非翻译区142 bp,一个多聚腺苷酸信号序列(AATAAA)和Poly A尾巴以及开放阅读框1971 bp.阅读框共编码656个氨基酸,总分子量为71.43 kD,理论等电点为5.15.该氨基酸序列中含有HSP70家族的3个签名序列——IDLGTTYS、IFDLGGGTFDVSIL和IVLVGGSTRIPKIQK,以及细胞质特异性调控基序EEVD,C端重复序列GGMP.同源性分析表明,本研究所获双齿围沙蚕HSP70氨基酸序列与已报道的序列相似性高达94%,与其他无脊椎生物的HSP70相似性也高达79%以上.荧光实时定量PCR分析表明,Cu²⁺(0.2～5.0 mg·L^-1)胁迫能够显著诱导沙蚕HSP70 mRNA表达,并于1 d后达到峰值.本研究系统描述了双齿围沙蚕HSP70的分子特性,其可被液态Cu²⁺诱导表达,具备作为环境污染分子生物标记物的潜力.     

粘虫hsc70克隆及其密度胁迫下的表达模式

【目的】昆虫种群密度的升高往往成为一种胁迫因子。粘虫在低密度下形成散居型,而在高密度下则形成群居型。本研通过比较hsc70在两型粘虫中的表达模式,究旨在探索hsc70在粘虫Mythimna separata响应密度胁迫中的作用。【方法】采用RT-PCR和RACE技术克隆粘虫hsc70cDNA全长序列,利用生物信息学软件分析该基因及其编码蛋白质的序列特性;运用实时荧光定量PCR技术(qRT-PCR)分析该基因在两型粘虫不同发育阶段(卵、1-6龄幼虫、蛹和成虫)、5龄幼虫不同组织(头、表皮、前肠、中肠、后肠和马氏管)以及群居和分离处理后mRNA表达量的变化。【结果】克隆得到的HSC70基因命名为Mshsc70(GenBank登录号:MH669276),全长cDNA长2 184 bp,开放阅读框为1 965 bp,编码653个氨基酸。序列分析发现MsHSC70具有HSP70家族典型的结构域,即IDLGTTYS(11-18aa),IFDLGGGTFDVSLL (198-211 aa)和IVLVGGSTRIPKVQQ (341-355 aa)。序列比对结果显示,MsHSC70与斜纹夜蛾Spodopteralitura,棉铃虫Helicoverpaarmigera,大螟Sesamiainferens、甘蓝夜蛾Mamestra brassicae HSC70的相似性高达99%;基于HSC70的系统发育分析显示,粘虫与夜蛾科(Noctuidae)昆虫的亲缘关系最近。Mshsc70在两型粘虫的各个发育阶段均表达,hsc70的表达量在1龄和5龄中的表达量较散居型显著上调,而在2-3龄中的表达量显著下调。除表皮和前肠外,群居型粘虫的其他组织中hsc70的表达量显著高于散居型。散居型粘虫经群聚饲养36h后,hsc70显著上调表达,表达量是对照的3.17倍,而群居型粘虫经分离饲养36 h后,hsc70的表达量与对照无显著差异。【结论】粘虫的种群密度是决定粘虫hsc70表达的一个重要因子。     

黄颡鱼HSC70基因及其组织表达分析

热休克蛋白70（HSP70）与生物体的抗胁迫能力密切相关。本文采用RACE (Rapid amplification of cDNA ends) 技术,从黄颡鱼Pelteobagrus fulvidraco克隆到一种组成型热休克蛋白（HSC70）基因及其cDNA。该cDNA全长2245bp,包括5′非编码区82bp,3′非编码区225bp,开放阅读框(ORF) 1938bp,编码645个氨基酸组成的蛋白质。黄颡鱼HSC70基因含有8个内含子,与人、鼠、虹鳟和花斑溪鳉的HSC70基因内含子数目相同,位置相似。其中,最长内含子（873bp）位于5′端非编码区,其余内含子（长度在80－251bp之间不等）均在编码区以内。黄颡鱼HSC70基因编码的氨基酸序列与南方鲶的相似度最高,达96.13%,与欧洲银鲫和团头鲂的相似度分别为94.45%和94.14%。RT-PCR检测显示,正常情况下黄颡鱼HSC70在血细胞、心脏、肝、头肾、脾、鳃、肌肉和脑中均有表达,但表达量在鳃中最高,肌肉中最低;统计结果显示,热激后HSC70在血细胞、肝、头肾和脑中的表达量显著上升(p<0.05),而在其余组织中热激前后的表达差异不显著(p>0.05)。     

异色瓢虫热休克蛋白70基因的克隆与分析

本研究通过RT-PCR和RACE方法,首次克隆了异色瓢虫Harmonia axyridis(Pallas)热休克蛋白70(HSP70)cDNA全序列(GenBank登录号:EF668009).获得的cDNA全长2200 bp,其中开放阅读框1956 bp,编码一个651氨基酸的蛋白,计算分子量为70 kDa,等电点(PI)为5.32.同源序列比对结果表明,异色瓢虫HSP70与其它真核生物的HSP/HSC70有着较高的序列同源性(85%～93%).其中与赤拟谷盗Tribolium castaneum热休克蛋白70(HSP70)同源性最高,达到93%.与其它真核生物HSP70一样,该序列包含真核生物HSP70高度保守的全部三个家族标签以及ATP/GTP结合位点、一个Bipartive nuclear localization signal和细胞核定位信号.     

哈维氏弧菌铁超氧化物歧化酶基因表达及免疫作用研究

哈维氏弧菌(Vibrio harveyi)是鱼虾等海水动物的重要病原菌。超氧化物歧化酶(Superoxide dismutase)通过催化超氧阴离子自由基(O2-)形成O2 和H2O2, 以保持细胞自由基产生和清除之间的平衡, 在病原菌适应环境及细菌致病性方面发挥重要作用。用PCR 从哈维氏弧菌基因组扩增得到600 bp 的目的片段, 序列分析表明与弧菌Fe-SOD 的序列相似性为91%—99%。将目的基因片段克隆到原核表达载体进行表达。SDS-PAGE电泳分析显示纯化的蛋白为单一条带, 分子量为27 kD。具有典型的Fe-SOD 吸收光谱, 对氯仿-乙醇和H2O2敏感, 表明纯化的蛋白属于Fe-SOD。用邻苯三酚自氧化法测得酶的最适pH 7, 最适温度20℃。该酶在pH6—8 的范围内稳定, 当温度超过40℃时酶的活力迅速丧失。纯化的重组蛋白免疫大菱鲆, 4 周后用致病性哈维氏弧菌进行人工感染试验, 对大菱鲆的免疫保护率为80.00%。Western blot 可以检测到免疫大菱鲆的血清中的特异抗体。       

鲢鱼解偶联蛋白2全长cDNA序列的克隆及其组织表达

从淡水食毒藻鱼类鲢鱼(Hypophthalmichthysmolitrix)肝脏,通过简并引物克隆解偶联蛋白2(un-couplingprotein2,UCP2)cDNA核心序列,应用5′RACE和3′RACE技术分别扩增该序列的5′末端和3′末端序列,最后通过序列拼接获得鲢鱼肝脏UCP2cDNA全序列。序列分析结果表明,鲢鱼肝脏UCP2cDNA全长1452bp,其中5′-UTR长337bp,3′-UTR长182bp,编码区933bp,编码310个氨基酸,推测的氨基酸序列包含线粒体内膜载体蛋白3个特征结构及解偶联蛋白(UCPs)的特征序列。对鲢鱼不同组织UCP2的表达调控研究发现,鲢鱼组织UCP2基因在肠道、肝脏、肌肉、脂肪组织均大量表达,而在脑组织表达量较低,这与鲢鱼体内微囊藻毒素在这几个组织的分布完全一致,表明UCP2的功能可能与抑制微囊藻毒素引发过量活性氧(ROS)生成有关。     

人热休克蛋白70和热休克固有蛋白70的基因克隆和表达

目的:克隆人热休克蛋白70(HSP70)和热休克固有蛋白70(HSC70)基因,并在大肠杆茵中表达,获得重组蛋白.方法:用RT-PCR法从HepG2细胞中扩增HSP70及HSC70cDNA序列.测序后,将相应的cDNA插入pRSET-A表达载体,在大肠杆菌中表达,重组蛋白纯化后用SDS-PAGE及Western Blotting分析.结果:DNA序列结果显示.本研究所获得的HSP70及HSC70 cDNA序列与参考序列一致.将全长cDNA分别插入表达质粒后,转化BL21(DE3)细菌,在IPTG的诱导下,表达产物SDS-PAGE显示相应的分子量(70kDa)位置有明显的蛋白条带.Western Blotting结果证实了其为目的蛋白,经镍树脂柱纯化,获得了相应的重组多肽.结论:成功构建了原核表达重组质粒HSP70-pRSET-A和HSC70-pRSET-A,并获得了纯化的重组人HSP70和HSC70蛋白,为进一步研究这两种蛋白的结构、功能及临床应用奠定了基础.     

鲢鱼解偶联蛋白2全长cDNA序列的克隆及其组织表达

从淡水食毒藻鱼类鲢鱼(Hypophthalmichthys molitrix)肝脏，通过简并引物克隆解偶联蛋白2(uncoupling protein 2，UCP2) cDNA核心序列，应用5′RACE和3′RACE技术分别扩增该序列的5′末端和3′末端序列，最后通过序列拼接获得鲢鱼肝脏UCP2 cDNA全序列。序列分析结果表明，鲢鱼肝脏UCP2 cDNA全长1 452 bp，其中5′-UTR长337 bp，3′-UTR长182 bp，编码区933 bp，编码310个氨基酸，推测的氨基酸序列包含线粒体内膜载体蛋白3个特征结构及解偶联蛋白(UCPs)的特征序列。对鲢鱼不同组织UCP2的表达调控研究发现，鲢鱼组织UCP2基因在肠道、肝脏、肌肉、脂肪组织均大量表达，而在脑组织表达量较低，这与鲢鱼体内微囊藻毒素在这几个组织的分布完全一致，表明UCP2的功能可能与抑制微囊藻毒素引发过量活性氧(ROS)生成有关。     

二化螟热休克蛋白70基因的克隆及热胁迫下的表达分析

热休克蛋白70是已知热休克蛋白家族中最重要的一种, 它在细胞内的大量表达可以明显改善细胞的生存能力, 提高对环境胁迫的耐受性。为探讨热胁迫对二化螟Chilo suppressalis幼虫热休克蛋白70表达的影响, 采用RT-PCR及RACE技术从二化螟血淋巴细胞中克隆了热休克蛋白70基因全长cDNA序列。该基因全长2 102 bp, 开放阅读框 (open reading frame, ORF)为1 959 bp, 编码652个氨基酸; 5′非编码区（untranslated region, UTR)为81 bp, 3′UTR为62 bp。从该基因推导的氨基酸序列与其他昆虫的同源序列比较有很高的相似性（73%~97%)。实时定量PCR显示二化螟HSP70基因能被热胁迫诱导表达, 幼虫血淋巴细胞的HSP70基因在36℃时表达量最高。流式细胞术研究发现HSP70在蛋白质水平上的表达变化与在mRNA水平上高度一致, 说明二化螟HSP70基因在转录及翻译水平上受到热应激的调节。     

Molecular cloning, characterization and expression analysis of cathepsin D gene from turbot Scophthalmus maximus

Cathepsin D is a lysosomal endoproteolytic aspartic proteinase which also has been found in endosomes of macrophage. It is thought to play key roles in the developmental and physiological process of animals. The EST sequence of turbot (Scophthalmus maximus L.) cathepsin D was obtained from a subtractive cDNA library. In the present study, 5'-RACE and 3'-RACE were carried out to obtain the complete cDNA sequence of turbot cathepsin D, which contained a 91 bp 5'-UTR, a 1191 bp open reading frame encoding 396 amino acids, and a 329 bp 3'-UTR. The deduced amino acid sequence of the cathepsin D consisted of a signal peptide of 18 aa, a leader peptide extending 43 aa, and a mature peptide of 335 aa. BLAST analysis revealed that turbot cathepsin D shared high similarity with other known cathepsin D, and it showed significant homology with that of Barramundi (Lates calcarifer B., 89% aa similarity). Quantitative real-time PCR (q PCR) demonstrated that the highest expression level of the turbot cathepsin D was in liver. After turbot were challenged with Vibrio harveyi, the lowest expression levels of cathepsin D in liver, spleen and head kidney were detected at 8 h. This result was different from the expression of MHCII of which the expression lever was increased upon challenge. The expression levels of cathepsin D in liver and head kidney increased gradually after 8 h and exceeded the background level after 24 h. In spleen, the expression level was reinforced after 8 h and kept at level that was higher than the original level after 12 h. The results suggested that cathepsin D might process antigens for presentation to the immune system and have synergetic effect with apoptosis pathway until 12 h after injection.     

Secretion of the heat shock proteins HSP70 and HSC70 by baby hamster kidney (BHK‐21) cells

The HSPs (heat‐shock proteins) of the 70‐kDa family, the constitutively expressed HSC70 (cognate 70‐kDa heat‐shock protein) and the stress‐inducible HSP70 (stress‐inducible 70‐kDa heat‐shock protein), have been reported to be actively secreted by various cell types. The mechanisms of the release of these HSPs are obscure, since they possess no consensus secretory signal sequence. We showed that baby hamster kidney (BHK‐21) cells released HSP70 and HSC70 in a serum‐free medium and that this process was the result of an active secretion of HSPs rather than the non‐specific release of the proteins due to cell death. It was found that the secretion of HSP70 and HSC70 is independent of de novo protein synthesis. BFA (Brefeldin A) did not inhibit the basal secretion of HSPs, indicating that the secretion of HSP70 and HSC70 from cells occurs by a non‐classical pathway. Exosomes did not contribute to the secretion of HSP70 and HSC70 by cells. MBC (methyl‐β‐cyclodextrin), a substance that disrupts the lipid raft organization, considerably reduced the secretion of both HSPs, indicating that lipid rafts are involved in the secretion of HSP70 and HSC70 by BHK‐21 cells. The results suggest that HSP70 and HSC70 are actively secreted by BHK‐21 cells in a serum‐free medium through a non‐classical pathway in which lipid rafts play an important role.     

HSP70 interacting protein prevents the accumulation of inclusions in polyglutamine disease1

Heat shock proteins (HSPs) are associated with the proteinaceous inclusions that characterise many neurodegenerative diseases. This suggests they may be associated with disease aetiology and/or represents an attempt to remove abnormal protein aggregates. In this study the adenoviral mediated over‐expression of HSP70 interacting protein (HIP) alone was shown to significantly reduce inclusion formation in both an in vitro model of Spinal Bulbar Muscular Atrophy and a primary neuronal model of polyglutamine disease. Experiments to determine the mechanism of action showed that: denatured luciferase activity (a measure of protein refolding) was not increased in the presence of HIP alone but was increased when HIP was co‐expressed with HSP70 or Heat Shock cognate protein 70 (HSC70); the expression of polyglutamine inclusions in cortical neurons mediated an increase in the levels of HSC70 but not HSP70. Our data suggest that HIP may prevent inclusion formation by facilitating the constitutive HSC70 refolding cycle and possibly by preventing aggregation. HIP expression is not increased following stress and its over‐expression may therefore reduce toxic polyglutamine aggregation events and contribute to an effective therapeutic strategy.     

Cloning and nucleotide sequencing of three heat shock protein genes (hsp90, hsc70, and hsp19.5) from the diamondback moth, Plutella xylostella (L.) and their expression in relation to developmental stage and temperature

Heat shock protein genes, hsp90, hsc70, and hsp19.5, were cloned and sequenced from the diamondback moth, Plutella xylostella (L.) by RT-PCR and RACE method. The cDNA sequence analysis of hsp90 and hsp19.5 revealed open reading frames (ORFs) of 2,151 and 522 bp in length, which encode proteins with calculated molecular weights of 82.4 and 19.5 kDa, respectively. Analysis of cDNA from hsc70 revealed an ORF of 1,878 bp coding a protein with a calculated molecular weight of 69.3 kDa. Furthermore, the analysis of genomic DNA from hsc70 confirmed the presence of introns while no introns were apparent in hsp90 and hsp19.5. Southern blot analysis suggested the presence of multiple copies of each gene family in the DBM genome. Detectable expression of hsp19.5 was observed at the pupal stage while expression of hsp90 and hsc70 was detected at both pupal and adult stages. At adult stage, females showed a higher expression of hsp90 and hsc70 than males. An increased expression was observed in all three genes after exposure to a high temperature in both sexes. These results suggest that in addition to a heat shock response, these HSP genes might be involved in other functions during the course of development in DBM.     

大螟HSC70基因克隆及表达模式分析

【目的】近年来,大螟Sesamia inferens (Walker)对水稻的为害逐渐加重并成为水稻的重要害虫之一。随着全球气候变暖,大螟的分布区域也在逐渐向北延伸。HSP70家族作为分子伴侣参与生物生长发育并对外界刺激产生响应,对生物功能蛋白质的正确折叠及其转运有着重要意义。本研究旨在明确HSP70家族HSC70基因在大螟不同组织、不同发育阶段以及低温胁迫下的表达差异,初步探讨大螟对环境适应的分子机理。【方法】应用RT-PCR及RACE技术从大螟5龄幼虫中克隆得到HSC70基因;进行基因组验证,得到其基因组序列,分析内含子的位置及大小;应用实时定量PCR技术分析大螟HSC70基因的表达模式。【结果】大螟HSC70基因长2 160 bp,命名为Sihsc70（GenBank登录号：KJ639908）,开放阅读框长1 962 bp,编码653个氨基酸,推测分子量为71.6 kDa。其氨基酸序列中含有3个HSP70家族保守序列,在C-末端存在细胞质定位信号,说明大螟HSC70是细胞质热激蛋白家族成员。大螟HSC70基因组序列长度为3 522 bp（GenBank登录号：KJ639909）,含有2个内含子,长度分别为685 bp（位于编码区上游）和803 bp（位于编码区内）。在大螟5龄幼虫的不同组织中Sihsc70表达量差异不显著（P＞0.05）,其中在中肠、后肠和体壁中的表达量较高,在唾腺中的表达量最低;在大螟不同发育阶段中,Sihsc70的表达量在雌成虫最低,较高的3个阶段依次为卵、2龄幼虫和5龄幼虫,分别为雌成虫表达量的6.33,3.21和1.86倍;相对于对照组（27℃）,低温胁迫对大螟5龄幼虫HSC70基因表达的影响差异不显著（P＞0.05）。【结论】结果说明,大螟HSC70基因在不同发育阶段和幼虫不同组织中具有不同的表达水平,而低温胁迫不能诱导该基因大量表达。