AAV mediated carboxyl terminus of Hsp70 interacting protein overexpression mitigates the cognitive and pathological phenotypes of APP/PS1 mice

The E3 ubiquitin ligase,carboxyl terminus of heat shock protein 70(Hsp70)interacting protein(CHIP),also functions as a co-chaperone and plays a crucial role in the protein quality control system.In this study,we aimed to investigate the neuroprotective effect of overexpressed CHIP on Alzheimer’s disease.We used an adeno-associated virus vector that can cross the blood-brain barrier to mediate CHIP overexpression in APP/PS1 mouse brain.CHIP overexpression significantly ameliorated the performance of APP/PS1 mice in the Morris water maze and nest building tests,reduced amyloid-βplaques,and decreased the expression of both amyloid-βand phosphorylated tau.CHIP also alleviated the concentration of microglia and astrocytes around plaques.In APP/PS1 mice of a younger age,CHIP overexpression promoted an increase in ADAM10 expression and inhibitedβ-site APP cleaving enzyme 1,insulin degrading enzyme,and neprilysin expression.Levels of HSP70 and HSP40,which have functional relevance to CHIP,were also increased.Single nuclei transcriptome sequencing in the hippocampus of CHIP overexpressed mice showed that the lysosomal pathway and oligodendrocyte-related biological processes were up-regulated,which may also reflect a potential mechanism for the neuroprotective effect of CHIP.Our research shows that CHIP effectively reduces the behavior and pathological manifestations of APP/PS1 mice.Indeed,overexpression of CHIP could be a beneficial approach for the treatment of Alzheimer’s disease.

Effect of Ultraviolet Radiation on Hsp70 Protein Expression in HaCaT Cells

Ultraviolet radiation by its wavelength is divided into: UVA, UVB and UVC. Only UVA and UVB manage to penetrate the ozone layer, but due to anthropological activities, all of them are capable of interacting with humans to a greater or lesser extent, and can generate adverse effects such as cellular stress when interacting with intra-and extracellular biomolecules. The skin is the first organ in contact with UV radiation, and the stress it generates can be analyzed by the expression of a bioindicator of cellular damage such as Hsp70. Therefore, the objective of the project was: to determine the effect of UVA, UVB and UVC radiation on HaCaT epithelial cells, by analyzing the expression of Hsp70. Materials and methods: HaCaT cells were cultured in vitro, which were irradiated with UVA, UVB and UVC light at different doses, to subsequently determine the degree of Hsp70 expression by Immunodetection by PAGE-SDS and Western Blot. Results: Basal expression of Hsp70 was observed in no irradiated HaCaT cells. When HaCaT cells were irradiated with UVA, UVB, UVC, an increase in this Hsp70 protein was observed. With UVA, a higher degree of expression was observed at a time of 30 minutes of irradiation. With UVB the highest expression shifted to a time of 20 minutes. With UVC, overexpression was observed after 10 minutes. Conclusion: UV radiation generates cellular stress on HaCaT cells, evaluated by the stress bioindicator Hsp70. According to the wavelength of UV radiation, those that have a shorter wavelength have a greater potential for cellular damage, such as UVC.

Ischemic preconditioning induces chaperone hsp70 expression and inhibits protein aggregation in the CA1 neurons of rats

Objective To investigate the effect of ischemic preconditioning on chaperone hsp70 expression and protein aggregation in the CA1 neurons of rats, and to further explore its potential neuroprotective mechanism. Methods Two-vesseloccluded transient global ischemia rat model was used. The rats were divided into sublethal 3-min ischemia group, lethal 10- min ischemia group and ischemic preconditioning group. Neuronal death in the CA1 region was observed by hematoxylineosin staining, and number of live neurons was assessed by cell counting under a light microscope. Immunochemistry and laser scanning confocal microscopy were used to observe the distribution of chaperone hsp70 in the CA1 neurons. Differential centrifuge was used to isolate cytosol, nucleus and protein aggregates fractions. Western blot was used to analyze the quantitative alterations of protein aggregates and inducible chaperone hsp70 in cellular fractions and in protein aggregates under different ischemic conditions. Results Histological examination showed that ischemic preconditioning significantly reduced delayed neuronal death in the hippocampus CA1 region （P 〈 0.01 vs 10-min ischemia group）. Sublethal ischemic preconditioning induced chaperone hsp70 expression in the CA1 neurons after 24 h reperfusion following 10-min ischemia. Induced-hsp70 combined with the abnormal proteins produced during the secondary lethal 10-min ischemia and inhibited the formation of cytotoxic protein aggregates（P〈0.01 vs 10-min ischemia group）.Conelusion Ischemic preconditioning induced chaperone hsp70 expression and inhibited protein aggregates formation in the CA1 neurons when suffered secondary lethal ischemia, which may protect neurons from death.

Effects of moxibustion on heat-shock protein 70 expression in the spinal cord and colonic mucosa in a rat model of ulcerative colitis

Pathological changes in the colon are closely associated with the spinal cord, and innervation of spinal cord can regulate cellular functions. Our previous studies verified that moxibustion protects and restores the colonic mucosa, but the mechanisms of action remain unknown. The present study observed the effects of moxibustion and salicylazosulfapyridine on expression of heat-shock protein 70 （HSP70） and its mRNA in the spinal cord and colonic mucosa of ulcerative colitis rats. Results demonstrated that moxibustion and salicylazosulfapyridine increased HSP70 mRNA expression in the spinal cord and colonic mucosa of ulcerative colitis rats. The decreased transcriptional activity of HSP70 in the spinal cord and colonic mucosa might participate in damage to the colonic mucosa in ulcerative colitis rats. Moxibustion exerted protective effects on colonic mucosa by up-regulating HSP70 transcriptional activity in the spinal cord and colonic mucosa.

Novel heat shock protein Hsp70L1 activates dendritic cells and acts as a Th1 polarizing adjuvant

Heat shock proteins (HSPs) are reported to act as effective adjuvants to elicit anti-tumor and anti-infection immunity. Here, we report that Hsp70-like protein 1 (Hsp70L1), a novel HSP derived from human dendritic cells (DCs), has potent adjuvant effects that polarize responses toward Th1. With a calculated molecular weight of 54.8 kDa, Hsp70L1 is smaller in size than Hsp70 but resembles it both structurally and functionally. Hsp70L1 shares common receptors on DCs with Hsp70 and can interact with DCs, promoting DC maturation and stimulating secretion of the proinflammatory cytokines interleukin 12p70 (IL-12p70), IL-1beta, tumor necrosis factor-alpha (TNF-alpha), and the chemokines IP-10, macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and normal T cell expressed and secreted (RANTES). The induction of interferon-gamma-inducible protein 10 (IP-10) secretion by Hsp70L1 is not shared by Hsp70, and other functional differences include more potent stimulation of DC IL-12p70, CC-chemokine, and CCR7 and CXCR4 expression by Hsp70L1. Immunization of mice with the hybrid peptide Hsp70L1-ovalbumin(OVA)(257-264) induces an OVA(257-264)-specific Th1 response and cytotoxic T lymphocyte (CTL) that results in significant inhibition of E.G7-OVA tumor growth. The ability of Hsp70L1 to activate DCs indicates its potential as a novel adjuvant for use with peptide immunizations; the Hsp70L1 antigen peptide hybrid may serve as a more effective vaccine for the control of cancer and infectious diseases.

Effects of Batroxobin on Spatial Learning and Memory Disorder of Rats with Temporal Ischemia and the Expression of HSP32 and HSP70

The effect of Batroxobin on spatial memory disorder of left temporal ischemic rats and the expression of HSP32 and HSP70 were investigated with Morri`s water maze and immunohistochemistry methods. The results showed that the mean reaction time and distance of temporal ischemic rats in searching a goal were significantly longer than those of the sham-operated rats and at the same time HSP32 and HSP70 expression of left temporal ischemic region in rats was significantly increased as compared with the sham-operated rats. However, the mean reaction time and distance of the Batroxobin-treated rats were shorter and they used normal strategies more often and earlier than those of ischemic rats. The number of HSP32 and HSP70 immune reactive cells of Batroxobin-treated rats was also less than that of the ischemic group. In conclusion, Batroxobin can improve spatial memory disorder of temporal ischemic rats; and the down-regulation of the expression of HSP32 and HSP70 is probably related to the attenuation of ischemic injury.

Effect of chaperone–client interaction strength on Hsp70-mediated protein folding

Protein folding in crowding cellular environment often relies on the assistance of various chaperones. Hsp70 is one of the most ubiquitous chaperones in cells. Previous studies showed that the chaperone–client interactions at the open state tend to remodel the protein folding energy landscape and direct the protein folding as a foldase. In this work, we further investigate how the chaperone–client interaction strength modulates the foldase function of Hsp70 by using molecular simulations. The results showed that the time of substrate folding(including the whole folding step and substrate release step) has a non-monotonic dependence on the interaction strength. With the increasing of the chaperone–client interaction strength, the folding time decreases first, and then increases. More detailed analysis showed that when the chaperone–client interaction is too strong, even small number of chaperones–client contacts can maintain the substrate bound with the chaperone. The sampling of the transient chaperones–client complex with sparse inter-molecule contacts makes the client protein have chance to access the misfolded state even it is bound with chaperone. The current results suggest that the interaction strength is an important factor controlling the Hsp70 chaperoning function.

Creation of Male Sterile Line in Tobacco With HSP70 Anti-sense Fragment

In order to create the Male Sterile Line in tobacco, the anti-sense fragment of HSP70 gene was linked to anther specific expression promoter TA29 and the reconstructed vector was transformed into tobacco by Agrobacterium mediated transformation, and the transformants were then screened. Gus and spot blotting hybridization analysis of the transformants indicated that anti-sense fragment of HSF70 gene had been integrated into tobacco genome and expressed, thus the male sterile tobacco line was obtained. Microscope observation of anther and pollen showed that pistils of transgenic tobacco were normal, whereas anthers and pollens were fairly abortive in the same transgenic tobacco flower, comparing with pistils and stamens in control plants. The ratio of HSI：＇70 protein before and after heat shock in mitochondrial was found to be 1.39 in control tobacco plants and 1.01 in transgenic tobacco sterile lines. This is suggested that the anti-sense gene fragment of HSP70 can effectively inhibit the expression of HSP70 protein and lead to transgenic male sterility in tobacco flowers. The assay provided a new genetic engineering method for male sterility creation in plants.

Batroxobin Against Anoxic Damage of Rat Hippocampal Neurons in Culture:Morphological Changes and Hsp70 Expression

Batroxobin,the thrombin-like enzyme,is used for therapeutic defibrination. We have found that batroxobin has good therapeutic effect in ischemic reperfusion rats and clinical practices in vivo. But we have not studied the neuroprotective effect of batroxobin on anoxic hippocampal neurons in vitro. The purpose of this study was to obtain further information on the mechanism of the batroxobin-induced neuroprotection and examine the neuroprotective effect on neurons exposed to anoxia. The effect of batroxobin on anoxic damages in cultured hippocampal neurons of neonatal rats was investigated by using morphological changes and heat shock protein 70Kd (Hsp70) immunoreactive expression as indicators. The results indicate that batroxobin, besides its defibrination, may have a direct neuroprotective effect on anoxic damage of hippocampal neurons.

Frequency sensitivity of nanosecond pulse EMF on regrowth and hsp70 levels in transected planaria

Purpose: To study the effect of time varying/ pulsing electromagnetic fields (PEMF) on bio-logical systems by measuring regrowth and the induction of elevated levels of the stress protein hsp70 in the regenerative model Planaria Duge-sia dorotocethala. Objective: The outcomes of studies using electromagnetic fields (EMF) are dependent on pulse design, field strength (mG), frequency (Hz), duration and magnetic field/rise time (dB/ dt). Standardization of effective pulse design is necessary to avoid continuing confu-sion in the investigation of pulsing electro-magnetic field (PEMF) technology. Information from studies on hsp70 protein induction and regrowth in transected Planaria provides in-formation on EMF efficacy for potential clinical application in the treatment of ischemia reper-fusion injury and the eventual inclusion of EMF prophylaxis prior to surgery. Materials and methods: Planaria were transected equidistant between the tip of the head and the tip of the tail. Individual head and tail portions from the same worm were placed in pond water and exposed to 8, 16 or 72 Hertz PEMF for one hour daily post transection under carefully controlled exposure conditions. Regrowth of heads and tails was measured in PEMF-exposed and sham control. Protein lysates from PEMF-exposed and sham control transected heads and tails were ana-lyzed for hsp70 levels by Wes￥tern blot analy-ses. Conclusion: The degree of regrowth and hsp70 levels in transected heads and tails ex-posed to nanosecond PEMF exposures at 8, 16 or 72 Hz was frequency dependent. There are currently several views on the interaction mechanism involved in regrowth. Here we dis-cuss two: in one [7,8] we propose a direct effect on the DNA of the PEMF consensus sequence, nCTCTn, referred to as electromagnetic field response elements (EMRE) in the promoter re-gion of the stress response gene HSP70. In the second mechanism [28] it is proposed that EMF induce vibrations of proteins through a series of quantized low frequency phonon signals.

Effect of moxibustion on expressions of HSP70 mRNA and protein in gastric cancer-bearing rats

Objective:To observe the effect of moxibustion on the mRNA and protein expressions of heat-shock protein 70(HSP70)in gastric cancer-bearing rats.Methods:A total of 40 healthy Sprague-Dawley(SD)rats were adaptively fed for one week.The gastric cancer model was prepared by Walker-256 cancer tissue transplantation.After 7 d,10 rats were randomly selected to verify the successful modeling,and the remaining 30 rats were divided into a model group,a moxibustion group and an infrared group by the random number table method,with 10 rats in each group.After enrollment,the moxibustion group received suspended moxibustion at Zhongwan(CV 12),Guan yuan(CV 4)and bilateral Zusanli(ST 36),(the first group of acupoints)on the 1st day,and suspended moxibustion at bilateral Pishu(BL 20)and Weishu(BL 21),(the second group of acupoints)on the 2nd day,20 min each time,once a day.Moxibustion was alternately performed every other day at the two groups of acupoints for 21 d.From the day of en roll me nt,rats in the infrared group were irradiated with the infrared radiatio n at the stomach area on the 1st day,and at the T12-Ti3 interspinous region on the 2nd day,20 min each time,once a day,and the two locations were alternately irradiated every other day for 21 d.During the treatment,rats in the model group were intervened by grasping and fixation without treatment.At the end of the treatment,blood was collected from the inner eye orbit,and the HSP70 expression in peripheral blood was determined by enzyme linked immunosorbent assay(ELISA).Rats were sacrificed,the tumor volume and growth inhibition rate were measured.The position and changes of HSP70 in gastric cancer were observed by streptavidin-perosidase(SP);HSP70 protein expression was determined by ELISA;HSP70 mRNA expression in cancer tissues was determined by reverse transcription-polymerase chain reaction(R「PCR)assay.Results:In comparison of the model group,the volume growth of the gastric cancer in the moxibustion group was significantly restricted(P<0.01);the volume growth inhibition rate in the moxibustion group was 37.93%;the HSP70 expressi on in peripheral blood and the cancer tissues was sign ifica ntly in creased(both P<0.01);the expressio n of HSP70 mRNA and HSP70 content in gastric tumor were both obviously in creased in the moxibustion group(P<0.01);and a large amount of HSP70 was released to the outside of cancer cells in the moxibustion group.In comparison of the model group,the volume growth of the gastric cancer in the infrared group was slightly restricted(P<0.05)with a volume growth inhibition rate of 15.89%;the HSP70 expression in the infrared group was increased significantly in peripheral blood(P<0.01)and in the gastric cancer tissues(P<0.05);more HSP70 was released outside of the cancer cells in the infrared group.In comparison of the infrared group,the volume growth of gastric cancer was more restricted in the moxibustion group(P<0.05)z and the HSP70 expression in the gastric cancer tissues was also higher(P<0.05);more HSP70 was released outside of the cancer cells in the moxibustion group.Conclusion:Moxibusti on and in fra red treatme nt inhibit the gastric can cer growth in the gastric cancer-bearing rats,up-regulate the HSP70 expression in gastric cancer tissues,and promote the production and extracellular release of HSP70,and the effect of moxibustion is more obvious.

Prokaryotic expression and purification of fusion protein HSP70-EGFP and its application in the study of dendritic cells internalization antigen

To study the endocytic activity of dendritic cells(DCs)by obtaining fusion protein HSP70-EGFP as exogenous antigen and loading it with DCs derived from human peripheral blood.Fusion protein HSP70-EGFP was prokaryotically expressed,isolated and purified.DCs were isolated and cultured from human peripheral blood.The DCs were divided into 3 groups in the endocytic experiment.There were 106 DCs in each group.Group 1 and 2 were respectively incubated for 30 min.with HSP70-EGFP and EGFP.Group 3 was incubated with HSP70 for 30 min,and then incubated for 30 min.with HSP70-EGFP.Subsequently,3 groups were placed in an incubator at 37℃for 0.5,1,2 and 24 h.Flow cytometry(FCM)was adopted to detect the amount of DCs with EGFP inside.IL-12 Eli-spot was adopted to detect the amount of DCs which secreted IL-12.There were 5 types in the experiment:LPS,inactive LPS,HSP70-EGFP,EGFP and no antigen.Fusion protein HSP70-EGFP was successfully obtained and its molecular weight was 97000.It accounted for 35.32%of the total protein.Under irradiation of an ultraviolet lamp,the protein solution sent out viridescent fluorescence.The result detected by FCM indicated that after incubation for 0.5 h at 37℃,the positive rate in group 1 was 63%,while the other 2 groups were negative.After incubation for 1,2 and 24 h at 37℃,the positive rates in the 3 groups were above 80%.The IL-12 Eli-spot examination shows that with HSP70-EGFP being loaded,the amount of DCs secreting IL-12 was 134.09±31.78/10^(5)cells,a little lower than that of DCs with LPS loaded(with the average point of 156.36±15.73).There was no significant difference between the 2 groups(P<0.01).By contrast,both of them were significantly higher than inactive LPS-(33.78±1.40)/10^(5)cells and EGFP-loaded(23.13±4.57)/105 cells DC groups in the amount of DCs secreting IL-12(P<0.01).The results suggest that receptor-mediated phagocytosis plays a main role in the preliminary stage of DCs internalizing HSP70-EGFP.With increasing incubation time,pinocytosis begins to dominate.HSP70-EGFP may promote DCs to secret cell factor IL-12.

短指软珊瑚(Sinularia acuta)热休克蛋白HSP70家族特征及进化分析

热休克蛋白70(heat shock protein 70, HSP70)在生物细胞或组织免受热或氧化应激等方面起着关键作用,是已知高度保守的蛋白质之一。由于全球环境持续升温,珊瑚大面积白化、死亡,珊瑚如何应对持续升温的抗逆机制是科学研究热点。本研究从高温胁迫短指软珊瑚测序蛋白序列数据库分析鉴定出了28个HSP70蛋白家族成员,均为酸性亲水蛋白,大部分蛋白质结构较为稳定。亚细胞定位表明HSP70蛋白主要分布在珊瑚细胞核、细胞质中,在线粒体、内质网上也有少量分布。信号肽预测表明, 28个HSP70蛋白成员中26个没有信号肽,大部分不属于分泌蛋白,不存在跨膜结构。系统进化树结果表明短指软珊瑚HSP70蛋白家族成员聚成5大类。短指软珊瑚HSP70蛋白家族结构和保守基序分析中预测到了10条保守基序motif分为5个亚族。短指软珊瑚HSP70蛋白家族二级结构主要以α-螺旋和无规则卷曲为主,α-螺旋的含量占比大。28个HSP70家族蛋白中有25个预测到了N-糖基化位点,且位点个数在1~9范围内。28个HSP70家族蛋白均预测到磷酸化位点和O-糖基化位点,总个数分别在41~96和1~23范围内。本研究HSP70家族蛋白结果为今后珊瑚在应对全球升温胁迫中的适应机制等方面研究奠定了基础。

白背飞虱Hsp70基因的克隆及其对非生物胁迫的响应

【目的】探明热休克蛋白Hsp70基因在白背飞虱适应非生物[杀虫剂、高温/低温和紫外线A(UV-A)]胁迫中的作用,为白背飞虱绿色防控提供参考。【方法】基于白背飞虱的基因组和转录组数据,克隆白背飞虱热休克蛋白Hsp70基因并对其进行鉴定,同时采用RT-qPCR测定其在杀虫剂、高温/低温和UV-A胁迫下的转录水平。【结果】克隆获得1个Hsp70基因,命名为SfHsp70-1,其全长序列为2322 bp,开放阅读框为2001 bp,编码666个氨基酸残基,分子量为72.91 kDa,理论等电点为5.11。SfHsp70-1氨基酸序列中包含3个Hsp70家族蛋白的保守结构域,且在C端具有内质网Hsp70的保守序列“HDEL”。噻虫嗪亚致死浓度表现出显著的剂量效应,LC_(10)胁迫能够诱导SfHsp70-1基因的转录水平显著升高,为对照(丙酮处理)的2.59倍;而LC_(25)胁迫对SfHsp70-1基因的转录水平无显著影响。10℃(30 min)、20℃(30,60 min)和40℃(10,30,60 min)胁迫均能诱导SfHsp70-1基因的转录水平显著提高,依次较对照(25℃)显著提高77.24%、62.00%、85.89%、138.96%、75.67%和61.10%。UV-A(300μW/cm~2)胁迫显著抑制SfHsp70-1基因的转录水平,分别较对照(照射0 min)显著降低87.85%、88.74%、91.91%、89.78%、93.04%和91.16%,且随处理时间延长其表达水平呈逐渐降低趋势。【结论】SfHsp70-1基因的过表达有助于白背飞虱对噻虫嗪和高温/低温胁迫的适应。研究结果可为进一步明确Hsp70基因在白背飞虱适应非生物胁迫中的作用奠定基础,并为制订白背飞虱绿色防控措施提供参考。

Immunogenicity and protective efficacy of recombinant M2e.Hsp70c(Hsp70_(359–610)) fusion protein against influenza virus infection in mice

New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for universal vaccine development against influenza A viruses. Mycobacterium tuberculosis Hsp70(mHsp70) is known to cultivate the function of immunogenic antigen-presenting cells, stimulate a strong cytotoxic T lymphocyte(CTL) response, and stop the induction of tolerance. Thus, in this study, a recombinant protein from the extracellular domain of influenza A virus matrix protein 2(M2e), was fused to the C-terminus of Mycobacterium tuberculosis Hsp70(Hsp70c), to generate a vaccine candidate. Humoral immune responses, IFN-γ-producing lymphocyte, and strong CTL activity were all induced to confirm the immunogenicity of M2 e.Hsp70c(Hsp70359–610). And challenge tests showed protection against H1N1 and H9N2 strains in vaccinated groups. Finally these results demonstrates M2 e.Hsp70c fusion protein can be a candidate for a universal influenza A vaccine.

脊尾白虾热休克蛋白HSP70基因的克隆及其表达分析

克隆了脊尾白虾热休克蛋白基因全长cDNA,并进行了序列分析。该基因由2 250 bp的碱基组成,开放阅读框长1 959 bp,编码由652个氨基酸组成的蛋白,基因两翼分别存在83 bp(5'端)和208 bp(3'端)的非翻译区,将该基因命名为Ec-HSP70。与其它物种HSP70s氨基酸序列进行同源性比较发现,与甲壳动物的同源性都在90%以上,表明该蛋白属于热休克蛋白HSP70家族。聚类分析表明,脊尾白虾热休克蛋白氨基酸序列与中国明对虾和凡纳滨对虾紧密聚为一支,之后聚类顺序依次为斑节对虾、日本囊对虾、刀额新对虾等。通过荧光定量RT-PCR对该基因在肝胰腺、肌肉的表达分析表明,温度、pH和氨氮胁迫都可以引起该基因的高表达,而且肝胰腺中的高表达时间相对肌肉中出现的较早。试验结果表明,温度、pH和氨氮胁迫对脊尾白虾HSP70基因表达有一定的诱导效果,但胁迫的时间过长则抑制其表达,肝胰腺对胁迫比肌肉较敏感。

松材线虫Hsp70基因的克隆与原核表达

热激蛋白70（Hsp70）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,提高对环境胁迫或伤害的耐受性。采用RACE-PCR技术,从松材线虫（Bursaphelenchus xylophilus）中克隆了Hsp70基因的全长cDNA序列（共2 061 bp）（GenBank登录号为：DQ785812）。其编码一个分子量为70 kD的642个氨基酸的蛋白序列,含有3段Hsp70家族的签名序列。同源性分析表明,氨基酸序列与其它真核生物的Hsp70序列具有很高的相似性,并与秀丽隐杆线虫（Caenorhabditis elegans）热激蛋白70家族中的hsp-1基因编码的氨基酸序列更为相似。因此,将克隆的松材线虫Hsp70基因命名为Bx-hsp-1。构建了一个原核表达载体Bx70pEASY-E1,当IPTG终浓度为0.4-0.8 mmol/L时,能诱导表达融合蛋白。Bx-hsp-1基因的克隆和表达,将为松材线虫的生态适应性机理研究奠定基础。

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2182bp,包含1959bp的开放阅读框,编码653个氨基酸,理论分子量为70.9kDa,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℃)1h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。

基于Hsp70基因的马梨形虫分类学定位分析

【目的】为了进一步确定马泰勒虫的分类学定位。【方法】根据GenBank上登录的马巴贝斯虫Hsp70基因序列(AB248743.1),设计引物TeHsp70F,TeHsp70R,以马泰勒虫基因组DNA为模板进行扩增,获得全长为1 920 bp的核酸片段。将该基因序列与GenBank中23种已知虫种的相应序列进行分析比较。【结果】该片段编码639个氨基酸,其疏水性氨基酸达到208个,极性氨基酸167个。同一性分析显示,该基因片段与报道的马巴贝斯虫Hsp70基因(B.equi BAF02625.1)亲缘关系最近,其次是小泰勒虫(T.parva XP764717.1)和环形泰勒虫(T.annulata AAA30130.1),而与感染马属动物的另一个虫种驽巴贝斯虫(B.caballi BAF02619.1)亲缘关系较远。【结论】马泰勒虫Hsp70基因的克隆及系统发育分析显示,之前被定名为马巴贝斯虫(B.equi)的虫种隶属于泰勒虫虫种。本试验为马巴贝斯虫正确更名为马泰勒虫(T.equi)的分类地位提供了又一佐证。

维氏气单胞菌感染对西伯利亚鲟血清指标和hsp70基因表达的影响

为研究西伯利亚鲟Acipenser baerii在维氏气单胞菌Aeromonas veronii感染过程中的生理免疫反应,进行了维氏气单胞菌攻毒试验,将体质量为(260.2±37.8)g的西伯利亚鲟从背部肌肉注射菌液浓度为2.0×107cfu/mL的维氏气单胞菌,注射后40h后开始死亡,62h后停止死亡,累积死亡率为46.7%。结果表明:40h对照组和53h处理正常鱼组中的血清皮质醇含量最低,与不再出现死亡后的处理组和对照组无显著性差异(P>0.05),与处理患病鱼各组有显著性差异(P<0.05);62h处理组血清超氧化物歧化酶(SOD)活力最低,与40h对照组、53h处理组、53h处理正常鱼组、93h对照组、93h处理组有显著性差异(P<0.05);93h处理组血清丙二醛(MDA)含量最低,与40h处理组、44h处理组、53h处理正常鱼组、62h处理组有显著性差异(P<0.05),其他各组之间无显著性差异(P>0.05);各处理组血清溶菌酶(LZM)活力从44h开始升高,直到62h,各个处理组均显著高于40h的处理组和对照组以及93h和168h的对照组和处理组(P<0.05);实时定量了肝、鳃、脾、肠组织中hsp70基因表达的变化。研究表明:西伯利亚鲟感染维氏气单胞菌后,血清皮质醇含量和溶菌酶活力升高可作为机体免疫状态的参考指标;热休克蛋白hsp70基因的诱导表达较血清指标变化更为灵敏,hsp70基因在鳃组织最先有较高的诱导表达。