Molecular cloning and expression analysis of interferon-gamma-inducible-lysosomal thiol reductase gene in orange-spotted grouper, Epinephelus coioides

In mammals, interferon-gamma-inducible-lysosomal thiol reductase (GILT) has been demonstrated to play a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction, thus unfolding native protein Ag and facilitating subsequent cleavage by proteases. In this study a cDNA containing the orange-spotted grouper GILT (OsgGILT) coding sequence has been cloned and its complete sequence determined. The full-length cDNA of OsgGILT gene is 1066 bp nucleotides (nt) encoding a protein of 260 amino acids (aa), with a putative molecular weight of 28.7 kDa. The deduced OsgGILT possesses the typical structural feature of mammalian GILT, including an active-site CXXC motif, a GILT signature sequence CQHGX(2)ECX(2)NX(4)C, and 10 conserved cysteines. The result of real-time PCR showed that OsgGILT mRNA was expressed in heart, liver, brain, gill, kidney and muscle and more highly expressed in spleen. The OsgGILT expression is obviously up-regulated in spleen and kidney after induction with LPS, these results suggest that OsgGILT may be involved in the immune response to LPS challenge in orange-spotted grouper.     

Molecular cloning,characterization and expression analysis of a C-type lectin (Ec-CTL) in orange-spotted grouper,Epinephelus coioides

C-type lectins play crucial roles in pathogen recognition, innate immunity, and cell–cell interactions. In this study, a new C-type lectin (Ec-CTL) gene was cloned from grouper, Epinephelus coioides by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of Ec-CTL was composed of 840 bp with a 651 bp open reading frame (ORF) that encodes a 216-residue protein. The deduced amino acid sequence of Ec-CTL possessed all conserved features crucial for the fundamental structure, such as the four cysteine residues (Cys⁷¹, Cys¹⁵², Cys¹⁶⁷, Cys¹⁷⁵) involved in the formation of disulphide bridges and the potential Ca²⁺/carbohydrate-binding sites. Ec-CTL contains a signal peptide and a single carbohydrate recognition domain (CRD). The genomic DNA of the gene consists of three exons and two introns. Ec-CTL showed high similarity of 54% to the C-type lectin of killifish Fundulus heteroclitus. Ec-CTL mRNA is predominately expressed in liver and skin, and lower expressed in kidney, intestine, heart, brain and spleen. The expression of Ec-CTL was differentially up-regulated in orange-spotted grouper challenged with Saccharomyces cerevisiae, Vibrio vulnificus, Staphyloccocus aureus and Singapore grouper iridovirus (SGIV). Recombinant mature Ec-CTL (rEc-CTL) was expressed in E. coli BL21, purified and characterized as a typical Ca²⁺-dependent carbohydrate-binding protein possessing hemagglutinating activity. It bound to all examined bacterial and yeast strains, and aggregated with S. cerevisiae, V. vulnificus and S. aureus in a Ca²⁺-dependent manner.     

The therapeutic implications of clinically applied modifiers of heat shock protein 70 (Hsp70) expression by tumor cells

Evidence that membrane-bound and extracellular heat shock proteins (HSPs) with molecular weights of 70 and 90 kDa are potent stimulators of the immune responses has accumulated over the last decade. In this review, we discuss the modulation of Hsp70 expression, a major stress-inducible member of the HSP70 family, in the cytoplasm and on the plasma membrane of tumor cells by clinically applied interventions such as radio- and chemotherapy.     

烟蚜热激蛋白基因MpHsp70的克隆及在UV-B胁迫下的表达分析

【目的】为探讨烟蚜Myzus persicae适应UV-B胁迫的分子机制。【方法】采用RT-PCR和RACE技术克隆了烟蚜热激蛋白基因Hsp70的全长,利用生物信息学方法分析了其特征;采用实时荧光定量PCR检测了不同时长(0,15,30,60,90和120 min) UV-B胁迫下烟蚜成虫中该基因的相对表达量。【结果】克隆获得烟蚜Hsp70基因并命名为MpHsp70(GenBank登录号:MF509827),该基因全长为2 221 bp,开放阅读框(ORF) 1 965 bp,编码654个氨基酸,蛋白相对分子量为71. 41kD,等电点(p I)为5. 34,末端高度保守序列EEVD显示该蛋白属于胞质热激蛋白。系统进化关系分析表明,MpHsp70与多种昆虫的Hsp70的同源性较高,表现出Hsp70基因的高度保守性。实时荧光定量PCR分析表明,随着UV-B照射时间的延长,烟蚜成虫体内MpHsp70基因的表达量先上升后下降,当照射时间为30 min时表达量最大。【结论】烟蚜MpHsp70基因可以响应UV-B的胁迫,它可能在烟蚜适应UV-B胁迫过程中起到重要作用。     

cDNA cloning and expression characterization of serum transferrin gene from oriental weatherfish Misgurnus anguillicaudatus

In this study, the cDNA coding serum transferrin (stf) of Misgurnus anguillicaudatus (mastf) was cloned. mastf cDNA is composed of 2326 bp with a 2007 bp open reading frame encoding 668 amino acids. The deduced Mastf protein consists of a signal peptide, two lobes (N and C‐lobes) and signature motifs of transferrin (Tf) family. The results of tissue distribution indicated that mastf mRNA was predominantly expressed in the liver. The results indicate that the mastf expression increased significantly in liver, blood, spleen and head kidney after the challenge with Aeromonas sobria, acting as a positive acute protein, suggesting that mastf is related to the immune response. The cloning and expression analysis of mastf further demonstrates the evolutionary conservation of Stf and immune function in vertebrates.     

Identification and expression analysis of a heat-shock protein 70 gene in Polycelis sp.

Heat-shock protein 70 (HSP70) is ubiquitously found in a variety of organisms and plays an important role in cytoprotection, environmental monitoring, and disease resistance. In this study, the full-length complementary DNA (cDNA) of hsp70 from planarian Polycelis sp. was first cloned using rapid amplification of cDNA ends (RACE). The expression levels of Pyhsp70 were analyzed in the presence of various stressors by real-time PCR, and its temporal-spatial expression patterns were also examined in both intact and regenerative animals by whole-mount in situ hybridization. The results show that (1) the deduced amino acid sequence of Pyhsp70 includes three typical HSP70 family signature motifs and is highly conserved during evolution; (2) Pyhsp70 expression is induced by prolonged starvation, tissue damage, and ionic liquid but inhibited by high or low temperatures; and (3) Pyhsp70 mRNA is mainly expressed in the head peripheral region and in the regenerating blastema during regeneration. These results suggest that the highly expressed Pyhsp70 gene may contribute to enhance cytoprotection and tolerance against stress-induced molecular damage, and the migration of neoblasts to the wound, which might also be involved in the proliferation and differentiation of neoblasts. Our work provides basic data for the study of stress responses and regenerative mechanism in freshwater planarians.     

人热休克蛋白70的原核高效表达

将人热休克蛋白基因hsp70片段克隆到高效原核表达载体pMAL-c2X中,酶切鉴定并进行DNA测序。将该重组表达载体转化大肠杆菌DH50α,用IPTG在不同温度及时间下进行诱导表达。收集细菌,菌体裂解后进行SDS-PAGE及Western blot检测,并以凝胶薄层扫描分析表达水平。结果表明,成功地构建了含人hsp70基因的表达载体pMAL-c2X／hsp70,该载体能在大肠杆菌中表达相对分子质量为110000并具有抗原活性的融合蛋白;改变诱导温度和时间,目的蛋白表达总量及可溶性部分所占比例不同。对人hsp70基因的克隆、表达,并对其进行表达条件的优化,为研究HSP70的结构、功能与临床应用提供了必要条件。     

Cloning of an orange-spotted grouper Epinephelus coioides heat shock protein 90AB (HSP90AB) and characterization of its expression in response to nodavirus

The heat shock proteins (HSPs) family which consists of HSP90, HSP70, and low molecular mass HSPs are involved in chaperone activity. Here, we report the cloning and characterization of HSP90AB gene from orange-spotted grouper, Epinephelus coioides. The full-length of grouper HSP90AB was 727 amino acids and possessed an ATPase domain as well as an evolutionarily conserved molecular chaperone. The HSP90AB-green fluorescent protein fusion protein was evenly distributed in the cytoplasm. Immunohistochemistry (IHC) and real-time polymerase chain reaction (PCR) analyses indicated that the expression of grouper HSP90AB was marginally increased following nodavirus infection. Grouper E. coioides that received HSP90 inhibitor geldanamycin (GA) showed an increase in HSP90AB expression and growth of nodavirus supporting nodavirus replication.     

两种入侵粉虱热激基因Hsp70的克隆及温度胁迫下的种间差异表达

为了检测B型烟粉虱和温室白粉虱在受到温度胁迫时热激基因Hsp70的差异表达谱,克隆了2种粉虱Hsp70基因的全长cDNA序列,并用实时定量PCR的方法分析了在不同温度条件下该基因的表达谱。结果表明:B型烟粉虱和温室白粉虱的Hsp70基因BTH-sp70和TVHsp70(GenBank登录号分别为DQ093385和EU861391),在5’-非编码区(UTR)中有类似TATA-box样元件;在3’-UTR中有poly(A)信号AATAA和富含A-T区;根据基因开放阅读框序列(ORF)推导的氨基酸序列均含有全部3个Hsp70蛋白家族高度保守的基序。在整个检测的温度范围内(-19℃～46℃),诱导温室白粉虱Hsp70表达的起始温度(Ton)或最大温度(Tmax)要比B型烟粉虱低2.5℃～7.5℃。这些结果表明,所克隆的基因属于有功能的温度诱导型Hsp70基因;在基因表达水平上,温室白粉虱比B型烟粉虱更耐冷,而后者更耐热;Hsp70的Ton(或者Tmax)能代表这2种粉虱的温度耐受能力。本研究结果在一定程度上解释了自然界中这2种粉虱种群地理分布和季节发生差异的原因。     

Inducible heat shock protein 70 and its role in preconditioning and exercise

Heat shock proteins (Hsp) are well known to be expressed in response to a range of cellular stresses. They are known to convey protection against protein denaturation and a subsequent immediate stress. Inducible heat shock protein 70 (Hsp70) is among the most studied of these stress proteins and its role and function are discussed here in terms of thermal and in particular exercise preconditioning. Preconditioning has been shown to confer cellular protection via expression Hsp, which may be of benefit in preventing protein damage following subsequent periods of exercise. Many studies have used animal models to gather data on Hsp70 and these and the most recent human studies are discussed.     

朱砂叶螨热激蛋白HSP70基因TCHSP70-4的克隆与表达

为了研究朱砂叶螨Tetranychus cinnabarinus(Boisduval)热激蛋白HSP70的表达与其适应高温和低温胁迫的关系,我们利用物种的同源性及RACE 技术,获得朱砂叶螨热激蛋白HSP70基因1个,命名为TCHSP70-4（GenBank 登录号为EU977182）。该基因全长2 182 bp,包含1 959 bp的开放阅读框,编码653个氨基酸,理论分子量为70.9 kDa,等电点为5.4,含有HSP70家族高度保守的基序。运用real-time PCR分析冷激（4℃）和热激（40℃）1 h后TCHSP70-4在朱砂叶螨体内的表达量。结果显示冷激后TCHSP70-4表达量明显下降,而热激后TCHSP70-4表达量却明显上升。这些结果一方面表明该基因属于诱导型HSP70基因,另一方面揭示了朱砂叶螨分别受到冷和热胁迫后体内TCHSP70-4的表达及所起的保护作用是不同的。