P277多肽融合热休克蛋白65提高抗1型糖尿病的作用

为了提高P277肽抗1型糖尿病的作用, 把P277肽融合在卡介苗热休克蛋白65的C端, 构建了pET28a- HSP65-P277高效表达载体, 在大肠杆菌中高效可溶性表达。利用硫酸铵分级沉淀、阴离子交换柱层析分离纯化了融合蛋白HSP65-P277。使用HSP65-P277在没有任何佐剂存在的情况下免疫非肥胖性糖尿病(NOD)小鼠, 通过三次腹腔注射, 每月收集被免疫动物的血清, 血糖浓度用自动生化分析仪测定。结果显示HSP65-P277免疫组小鼠血糖平均值及糖尿病的累积发病率和其余组相比均有显著差异(P<0.01), 融合蛋白HSP65-P277抗NOD小鼠糖尿病的作用显著高于单独的P277和HSP65。为进一步开发能用于临床的1型糖尿病疫苗提供了良好的设计思路, HSP65-P277极有可能进一步发展成为新的抗I型糖尿病的疫苗。     

基于热休克蛋白65的抗动脉粥样硬化蛋白疫苗的免疫效果

选取了HSP65上对自身免疫性炎症疾病有防治作用的两段功能表位P1(179-190)、P2(31-46),分别以HSP65和CTB为载体蛋白构建高效表达载体pET28a-HSP65-P1P2P1与pET28a-CTB-P1P2P1,以硫酸铵分级沉淀和包涵体洗涤、经DEAE阴离子交换柱层析分离纯化得到HSP65-P1P2P1与CTB-P1P2P1融合蛋白,并用戊二醛一步偶联法将两者偶联形成HpCp复合物。以小剂量滴鼻粘膜免疫高胆固醇膳食诱发产生动脉粥样硬化的新西兰大白兔,结果显示两组融合蛋白均表现出一定的减斑功效,与PBS对照组相比较,主动脉病斑面积分别减少了34.9%和27.9%,为进一步开发能用于临床的抗AS疫苗提供了良好的设计思路,可经进一步优化设计提高其免疫原性,研发为抗动脉粥样硬化疫苗。     

L-天冬酰胺酶表面呈现对P277肽免疫原性及抗NOD小鼠糖尿病作用的影响

将P277多肽融合在 Ｌ-天冬酰胺酶的C端,中间引入破伤毒素肽(TTP),重组嵌合酶AnsB-TTP-P277能以可溶性蛋白的形式表达于大肠杆菌周质内,且能保持大约80%的天然酶催化活力.用纯化的嵌合酶腹腔注射非肥胖性(NOD)小鼠, 可成功诱导小鼠体内产生高水平抗P277抗体.研究表明,P277肽可呈现于 Ｌ-天冬酰胺酶表面,有效地克服单独P277的弱免疫原性,激发机体产生较强烈的免疫反应.小鼠胰腺病理学切片也显示：重组嵌合酶AnsB-TTP-P277和单纯P277肽相比,能更有效地抑制NOD小鼠糖尿病的发生.     

Hsp65与IL-2融合蛋白诱导小鼠细胞免疫应答及保护力

目的研究Hsp65与hIL-2的融合蛋白在小鼠体内诱导的免疫应答及保护力。方法在大肠杆菌中诱导表达Hsp65与hIL-2的融合蛋白,通过Ni-NTA亲合柱纯化后的蛋白经鉴定后,与佐剂DDA和MPL联合免疫小鼠,连续免疫3次,每次间隔2周,最后一次免疫结束后两周,分离5只小鼠脾淋巴细胞,测定淋巴细胞增殖指数,IFN-γ和IL-2水平,以及特异性淋巴细胞杀伤功能,其余5只免疫小鼠用于MTB毒株攻击实验。结果获得融合蛋白可分别与抗Hsp65和抗hIL-2的单抗发生特异性反应。融合蛋白免疫小鼠后,小鼠脾淋巴细胞被有效活化,诱导产生的-γIFN和IL-2的水平以及CTL杀伤功能均显著高于BCG和单纯Hsp65免疫组（P〈0.05）。融合蛋白免疫组可有效抵抗MTB毒株攻击,脾脏细菌数显著减少（4.36±0.48）,提供的保护力与BCG相当（4.30±0.53）。结论Hsp65与hIL-2的融合蛋白是一种有效的亚单位疫苗,可用于TB的预防。     

胞壁表达HSP65-IL-2融合蛋白的重组耻垢分枝杆菌的构建和鉴定

目的：获得胞壁表达结核分枝杆菌热激蛋白65（HSP65）和人白细胞介素2（IL-2）融合蛋白的重组耻垢分枝杆菌。方法：将HSP65和IL-2融合基因克隆入大肠杆菌-卡介苗（E．coli-BCG）穿梭质粒pCW,构建成重组质粒HSP65-IL-2-pCW,电穿入耻垢分枝杆菌,经潮霉素抗性筛选和PCR方法选取阳性克隆;用间接免疫荧光法对重组耻垢分枝杆菌进行表型鉴定;用重组耻垢分枝杆菌免疫BALB／c小鼠,检测其诱导的抗体水平。结果：筛选获得的重组耻垢分枝杆菌增殖特性与普通耻垢分枝杆菌无明显区别;与抗人的IL-2抗体和HSP65抗体均可形成免疫印迹条带;间接荧光染色后,可见细菌表面有极强的绿色荧光;重组耻垢分枝杆菌免疫BALB／c小鼠2周后,小鼠血清中HSP65抗体平均滴度为1：4000,而生理盐水组的抗体几乎为阴性。结论：结核分枝杆菌HSP65和人IL-2融合基因在耻垢分枝杆菌胞壁获得表达,有望为结核病的预防提供有效的疫苗。     

热休克蛋白表位肽免疫对动脉粥样硬化斑块的影响

利用HSP65(heat shock protein 65,HSP65)中与炎症性自身免疫性疾病相关的两段功能表位EE14(176-189)、DL18(215-232),通过戊二醛偶联法与BSA(bovine serum albumin,BSA)制备成多肽-BSA偶联蛋白,获得可用于免疫高脂膳食动物的蛋白。与PBS(phosphate buffer saline,PBS)组比较,多肽免疫组抗HSP65抗体滴度与总胆固醇(total cholesterol,TC)含量无明显区别(P0.05),抗自身肽的抗体在两免疫组的结果不同,但两组主动脉粥样硬化斑块的面积都显著增加,两免疫组斑块面积与血管总面积百分比分别为39.61%与36.75%,为PBS组(斑块面积占血管总面积百分比为10.35%)的3.8倍和3.6倍,有显著性差异(P0.05)。实验结果表明HSP65相关B细胞表位肽,皮下免疫能显著促进AS(atherosclerosis,AS)斑块生成。在后续研究中可利用以上表位,通过改变免疫方式,设计可用于抑制AS斑块生成的免疫调节剂,进一步考察HSP65功能性B细胞表位的免疫预防及治疗效果。     

Tfh 细胞在NOD小鼠糖尿病发病过程中的作用机制的研究

目的:研究滤泡辅助性T细胞(Follicular Helper T cell,Tfh)在非肥胖性糖尿病小鼠(Non-obese Diabetic mice,NOD)发病过程中的作用机制。方法:实验动物NOD小鼠按血糖值分为胰岛炎组(血糖浓度≤9 mmol/L)及糖尿病组(血糖浓度≥20 mmol/L)。ELISA法检测各组中糖尿病自身抗体谷氨酸脱羧酶抗体(65-kda glutamate decarboxylase antibody,GAD65Ab)、抗胰岛素自身抗体(Insulin autoantibody,IAA)表达水平,Western blot检测B细胞型淋巴瘤6蛋白(B-cell lymphoma 6 protein,Bcl-6)及可诱导共刺激分子(Inducible costimulatory molecule,ICOS)表达,流式细胞仪检测各组外周血及脾脏Tfh细胞水平。结果:糖尿病组NOD鼠自身抗体GAD65Ab(1.21±0.23 nmol/L)、IAA(0.96±0.12 nmol/L)浓度较胰岛炎组(0.32±0.09 nmol/L,0.25±0.06 nmol/L)均有明显升高;糖尿病组NOD鼠Bcl-6及ICOS表达较胰岛炎组NOD鼠有明显升高,外周血和脾脏Tfh细胞水平糖尿病组NOD鼠(24.55%)较胰岛炎组NOD鼠(4.27%)升高明显。结论:NOD小鼠自发糖尿病与自身抗体浓度升高相关,Tfh细胞可能参与NOD鼠糖尿病发生及发展过程。     

LIGHT在自发Ⅰ型糖尿病小鼠中表达的分析

目的建立I型糖尿病NOD小鼠模型,探讨LIGHT在I型糖尿病模型NOD小鼠中的表达及意义。方法监测雌性NOD小鼠血糖、胰岛素水平;NOD小鼠分为4组,即未发病5周龄及11周龄组、发病1 w和2 w组。采用qRT-PCR、Western blot、免疫组化技术分析NOD小鼠胰腺组织LIGHT的mRNA、蛋白表达变化;ELISA法检测血清胰岛素、可溶性LIGHT及Th1细胞因子IFN-γ含量;HE染色检测胰岛炎,对胰岛炎评分,并对血清LIGHT含量与IFN-γ含量及胰岛炎积分进行相关性分析。结果糖尿病发病组NOD小鼠胰腺组织LIGHT基因的mRNA和蛋白表达及血清LIGHT浓度均明显高于未发病的5周龄组(P0.05),血清中Th1细胞因子IFN-γ分泌显著增加。血清LIGHT含量与IFN-γ水平及胰岛炎积分呈明显正相关(r1=0.52,r2=0.87,P0.01)。结论 LIGHT在NOD小鼠胰腺组织中的表达上调,可能影响Th1细胞因子IFN-γ的分泌及胰岛炎程度,血清LIGHT能反映I型糖尿病胰岛炎的严重程度。     

牛疱疹病毒Ⅰ型VP22和猪繁殖与呼吸综合征病毒GP5融合基因DNA疫苗的免疫效应

为了提高表达GP5的猪繁殖与呼吸综合征病毒（PRRSV）DNA疫苗的免疫效应,将具有蛋白转导功能的牛疱疹病毒1型（BHV-1）VP22基因插入到经过修饰具有更好免疫原性的PRRSV修饰型ORF5基因（ORF5M）上游,构建VP22和ORF5M融合表达的真核表达质粒pCI-VP22-ORF5M。经间接免疫荧光试验（IFA）和Westernblot检测证实体外表达后,免疫BALB/c小鼠,检测小鼠免疫后的GP5特异性ELISA抗体、抗PRRSV中和抗体和脾淋巴细胞增殖反应,并与非融合的真核表达质粒pCI-ORF5M进行比较。结果显示,融合表达VP22-GP5的DNA疫苗 pCI-VP22ORF5M诱导的体液免疫和细胞免疫反应均明显高于非融合表达的DNA疫苗pCI-ORF5M,表明蛋白转导相关蛋白BHV-1 VP22能显著增强表达GP5的PRRSV DNA 疫苗的免疫效应,有效发挥了基因免疫佐剂效应;这为研制PRRSV高效DNA疫苗奠定了基础,同时也为其它疾病的高效新型疫苗研究提供了思路。     

乙型脑炎病毒DNA初免-蛋白加强策略在小鼠模型上的免疫效应评价

为了评价基因Ⅰ型乙型脑炎病毒prM-E DNA疫苗与prM和EⅢ融合抗原亚单位疫苗采用DNA初免-蛋白加强免疫策略对小鼠的免疫效果,本研究将prM-E融合基因插入到pVAX1真核表达载体中,构建重组表达载体prM-E-pVAX1作为DNA疫苗进行初免,利用原核表达系统获得的prM和EⅢ融合抗原作为亚单位疫苗进行加强免疫。将32只4−6周龄雌性BALB/c小鼠随机分成4组,设置prM-E-pVAX1 DNA疫苗组、DNA初免-蛋白加强免疫组、prM和EⅢ融合抗原亚单位疫苗组及pVAX1载体对照组,通过ELISA检测血清中特异性抗体水平;通过噬斑减少中和试验滴定中和抗体滴度;通过细胞因子表达丰度和淋巴细胞增殖试验分析不同疫苗免疫组诱导产生的细胞免疫反应。结果表明,用DNA初免-蛋白加强策略免疫的小鼠诱导产生的中和抗体滴度略高于prM和EⅢ融合抗原亚单位疫苗免疫组,显著高于prM-E-pVAX1 DNA疫苗免疫组。DNA初免-蛋白加强策略在小鼠模型中诱导产生了有效的Th1/Th2型免疫反应,特别是显著诱导了Th1型细胞免疫反应。本研究为预防流行性乙型脑炎提供了新的免疫策略和理论参考依据。     

Oral Administration of Recombinant Lactococcus lactis Expressing HSP65 and Tandemly Repeated P277 Reduces the Incidence of Type I Diabetes in Non-Obese Diabetic Mice

Diabetes mellitus type 1 (DM1) is an autoimmune disease that gradually destroys insulin-producing beta-cells. We have previously reported that mucosal administration of fusion protein of HSP65 with tandem repeats of P277 (HSP65-6P277) can reduce the onset of DM1 in non-obese diabetic (NOD) mice. To deliver large amounts of the fusion protein and to enhance long-term immune tolerance effects, in the present study, we investigated the efficacy of using orally administrated L. lactis expressing HSP65-6P277 to reduce the incidence of DM1 in NOD mice. L. lactis strain NZ9000 was engineered to express HSP65-6P277 either constitutively or by nisin induction. After immunization via gavage with the recombinant L. lactis strains to groups of 4-week old female NOD mice for 36 weeks, we observed that oral administration of recombinant L. Lactis resulted in the prevention of hyperglycemia, improved glucose tolerance and reduced insulitis. Immunologic analysis showed that treatment with recombinant L. lactis induced HSP65- and P277- specific T cell immuno-tolerance, as well as antigen-specific proliferation of splenocytes. The results revealed that the DM1-preventing function was in part caused by a reduction in the pro-inflammatory cytokine IFN-γ and an increase in the anti-inflammatory cytokine IL-10. Orally administered recombinant L. lactis delivering HSP65-6P277 may be an effective therapeutic approach in preventing DM1.     

Vaccination with empty plasmid DNA or CpG oligonucleotide inhibits diabetes in nonobese diabetic mice: modulation of spontaneous 60-kDa heat shock protein autoimmunity

Nonobese diabetic (NOD) mice develop insulitis and diabetes through a process involving autoimmunity to the 60-kDa heat shock protein (HSP60). Treatment of NOD mice with HSP60 or with peptides derived from HSP60 inhibits this diabetogenic process. We now report that NOD diabetes can be inhibited by vaccination with a DNA construct encoding human HSP60, with the pcDNA3 empty vector, or with an oligonucleotide containing the CpG motif. Prevention of diabetes was associated with a decrease in the degree of insulitis and with down-regulation of spontaneous proliferative T cell responses to HSP60 and its peptide p277. Moreover, both the pcDNA3 vector and the CpG oligonucleotide induced specific Abs, primarily of the IgG2b isotype, to HSP60 and p277, and not to other islet Ags (glutamic acid decarboxylase or insulin) or to an unrelated recombinant Ag expressed in bacteria (GST). The IgG2b isotype of the specific Abs together with the decrease in T cell proliferative responses indicate a shift of the autoimmune process to a Th2 type in treated mice. These results suggest that immunostimulation by bacterial DNA motifs can modulate spontaneous HSP60 autoimmunity and inhibit NOD diabetes.     

Purification of a non-tagged recombinant BCG heat shock protein 65-Her2 peptide fusion protein from Escherichia coli

Bacille Calmette-Guerin (BCG)-derived heat shock protein 65 (HSP65) has been demonstrated capable of assisting a fused peptide to generate the peptide-specific cellular immunity. Various HSP65 fusion proteins have been developed as therapeutic cancer vaccines. Purifying a recombinant HSP65 fusion protein with no purification tags for human use is routinely a challenge. Here, we report a scheme for purifying a non-tagged recombinant HSP65-Her2 peptide fusion protein (HSP65-Her2) from Escherichia coli. The HSP65-Her2 is being developed as an immunotherapeutic for the treatment of Her2-positive tumors. After fermentation in a 10-L fermentor, the HSP65-Her2 expressing E. coli were harvested and lysed by sonication. The recombinant HSP65-Her2 was then purified with four successive steps including Butyl-Sepharose FF, DEAE-Sepharose FF, 1% Triton X-114 phase separation and Sephadex G-25. Results showed that HSP65-Her2 was purified up to 97% purity and was able to generate Her2-specific cytotoxic T lymphocytes (CTLs), suggesting that the scheme is efficient for purifying the non-tagged HSP65-Her2 fusion protein with biological activity.     

A Th1-recognized peptide P277, when tandemly repeated, enhances a Th2 immune response toward effective vaccines against autoimmune diabetes in nonobese diabetic mice

Subunit immunogens containing tandemly repeated copies of T and B cell epitopes have been shown to be more immunogenic than the respective immunogen containing only a single copy of the sequence. To investigate whether the increased copies of the Th2-activated peptide sequence will enhance the Th2-like immune response, we compared the cytokine secreted in mice that inoculated with two immunogens containing one or six tandemly repeated copies of a Th2-activated peptide sequence P277. Immunization of mice with a 6xP277 fusion protein elicited much higher levels of Th2-type cytokines and lower Th1-type cytokines than with a fusion protein with one P277 peptide. The data of tandemly repeated peptide P277 potentiate the anti-inflammatory in NOD mice, most likely associated with a Th1 to Th2 cytokine shift specific for the autoimmune T cells, which suggested that application of multiple tandem repeats of a Th2-activated epitope is an efficient method to enhance the anti-inflammatory immune response by shifting the immune response from Th1-like to Th2-like. The subunit immunogens containing tandemly repeated copies of peptide P277 might be effective vaccines against autoimmune diabetes in NOD mice.     

Heat shock protein 60 activates cytokine-associated negative regulator suppressor of cytokine signaling 3 in T cells: effects on signaling, chemotaxis, and inflammation

Previously, we reported that treatment of T cells with the 60-kDa heat shock protein (HSP60) inhibits chemotaxis. We now report that treatment of purified human T cells with recombinant human HSP60 or its biologically active peptide p277 up-regulates suppressor of cytokine signaling (SOCS)3 expression via TLR2 and STAT3 activation. SOCS3, in turn, inhibits the downstream effects of stromal cell-derived-1alpha (CXCL12)-CXCR4 interaction in: 1) phosphorylation of ERK1/2, Pyk2, AKT, and myosin L chain, required for cell adhesion and migration; 2) formation of rear-front T cell polarity; and 3) migration into the bone marrow of NOD/SCID mice. HSP60 also activates SOCS3 in mouse lymphocytes and inhibits their chemotaxis toward stromal cell-derived factor-1alpha and their ability to adoptively transfer delayed-type hypersensitivity. These effects of HSP60 could not be attributed to LPS or LPS-associated lipoprotein contamination. Thus, HSP60 can regulate T cell-mediated inflammation via specific signal transduction and SOCS3 activation.     

A peptide binding motif for I-Eg7, the MHC class II molecule that protects E alpha-transgenic nonobese diabetic mice from autoimmune diabetes.

The nonobese diabetic (NOD) mouse, a model of spontaneous insulin-dependent diabetes mellitus (IDDM), fails to express surface MHC class II I-Eg7 molecules due to a deletion in the E alpha gene promoter. E alpha-transgenic NOD mice express the E alpha E beta g7 dimer and fail to develop either insulitis or IDDM. A number of hypotheses have been proposed to explain the mechanisms of protection, most of which require peptide binding to I-Eg7. To define the requirements for peptide binding to I-Eg7, we first identified an I-Eg7-restricted T cell epitope corresponding to the sequence 4-13 of Mycobacterium tuberculosis 65-kDa heat shock protein (hsp). Single amino acid substitutions at individual positions revealed a motif for peptide binding to I-Eg7 characterized by two primary anchors at relative position (p) 1 and 4, and two secondary anchors at p6 and p9. This motif is present in eight of nine hsp peptides that bind to I-Eg7 with high affinity. The I-Eg7 binding motif displays a unique p4 anchor compared with the other known I-E motifs, and major differences are found between I-Eg7 and I-Ag7 binding motifs. Analysis of peptide binding to I-Eg7 and I-Ag7 molecules as well as proliferative responses of draining lymph node cells from hsp-primed NOD and E alpha-transgenic NOD mice to overlapping hsp peptides revealed that the two MHC molecules bind different peptides. Of 80 hsp peptides tested, none bind with high affinity to both MHC molecules, arguing against some of the mechanisms hypothesized to explain protection from IDDM in E alpha-transgenic NOD mice.