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《中国动物传染病学报》2015,(5)
为研究副猪嗜血杆菌PlpD基因的免疫保护性,根据Gen Bank上已公布的PlpD基因序列设计该基因的特异性引物,以本实验室保存的副猪嗜血杆菌5型菌株为模板,PCR扩增目的片段,构建重组质粒p ET28-PlpD,并将其转至表达菌BL21(DE3)中进行诱导表达。SDS-PAGE电泳检测显示,在温度为16℃时使用终浓度为1 mmol/L IPTG诱导12 h,目的蛋白可溶性表达量最高,Western blot结果表明该目的蛋白具有较好的反应原性。将获得的融合蛋白PlpD进行纯化后制备成亚单位疫苗,经腹腔注射免疫小鼠,3免后用高于副猪嗜血杆菌的致死剂量1.5×109CFU进行攻毒,结果显示PlpD基因对小鼠具有一定的免疫保护性。 相似文献
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运用PCR方法扩增副猪嗜血杆菌SC-1株荚膜多糖输出蛋白(CPEP)基因,克隆至pMD-19T载体。经鉴定测序验证后,将获得的CPEP基因克隆至pET-32a(+)原核表达载体,构建原核表达质粒pET-32a-CPEP,转化大肠杆菌BL21(DE3),IPTG诱导表达,进行SDS-PAGE以及Western blot分析免疫原性,纯化CPEP,并用纯化的蛋白免疫小鼠,检测其特异性血清IgG抗体水平及攻毒保护率。结果显示,成功构建原核表达质粒pET-32aCPEP;SDS-PAGE分析结果显示,得到约35ku的蛋白;Western blot分析显示,CPEP能与Hps SC-1阳性血清发生抗原抗体的特异性反应,证实该重组CPEP蛋白具有反应原性。以致死剂量的强毒株SC-1攻毒CPEP免疫小鼠,重组蛋白的免疫能够减缓发病,并表现出40%的保护率。重组蛋白CPEP的部分保护作用表明其可以作为免疫候选因子,为未来副猪嗜血杆菌标准化疫苗的研制奠定基础。 相似文献
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副猪嗜血杆菌血清5型TbpA蛋白的表达及其免疫原性分析 总被引：1，自引：0，他引：1
为原核表达副猪嗜血杆菌血清5型TbpA蛋白,本研究利用特异性引物扩增tbpA基因,并将其克隆到pMD 18-T载体中进行序列测定.再将其亚克隆于pET-28a中构建重组表达质粒pET-tbpA.将其转化受体菌E.coli BL21 (DE3),经IPTG诱导后,SDS-PAGE电泳和western blot分析表明,表达的重组蛋白约106 ku,并以包涵体形式存在.表达的重组蛋白经纯化后,免疫6周龄昆明小鼠制备免疫血清,ELISA分析表明制备的血清抗体效价在1∶3 200以上,表明TbpA具有良好的免疫原性. 相似文献
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根据GenBank中登录的副猪嗜血杆菌外膜蛋白P5(outer membrane protein P5,OMP5)基因序列设计1对特异性引物,以江西分离株NC0807基因组DNA为模板,扩增出OMP5基因。将其克隆到pET-28a(+)中,构建重组表达质粒pET-28a-OMP5,质粒转化大肠杆菌BL21(DE3),通过SDS-PAGE和Western blotting分析重组蛋白的表达情况和反应原性。重组蛋白经镍柱亲和层析纯化后免疫豚鼠,测定其免疫原性和保护效率。结果表明,重组蛋白在大肠杆菌中获得了高效表达。表达的蛋白分子质量约为43 ku,能被副猪嗜血杆菌阳性血清识别。动物试验结果表明,重组蛋白免疫后能诱导产生高水平的OMP5特异性抗体,并可显著保护豚鼠抵抗副猪嗜血杆菌强毒菌株的攻击,提示OMP5是副猪嗜血杆菌的保护性抗原。 相似文献
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《中国兽医学报》2020,(9)
提取南京地区经鉴定确诊为犬吉氏巴贝斯虫感染病犬血液中的DNA,设计引物采用PCR方法扩增犬吉氏巴贝斯虫HSP70基因,克隆至pET-32a质粒中,转化Rosetta-gami(DE3)pLyS大肠杆菌,利用IPTG诱导表达得到重组热休克蛋白(heat shock protein 70,HSP70)。用Ni柱纯化重组HSP70蛋白免疫BALB/c小鼠制备抗血清,并分析重组蛋白的免疫原性及反应原性。本研究成功构建了pET-32a-hsp70重组质粒,IPTG诱导后表达出可溶的重组HSP70蛋白。经过Western blot分析,表达的重组蛋白不但能够免疫小鼠产生抗体,还可以和天然犬吉氏巴贝斯虫抗血清发生特异性反应,为进一步建立犬巴贝斯虫免疫血清学诊断和疫苗抗原的开发奠定基础。 相似文献
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为原核表达副猪嗜血杆菌(H.parasuis)CDT毒素并检测其在体外培养时的分泌表达情况,本实验将切除信号肽的CDT毒素的3个亚基基因分别克隆于pET-28a载体中在大肠杆菌进行表达,并将重组蛋白经亲和层析纯化后,免疫兔制备抗血清,用于鉴定H.parasuis体外培养时CDT分泌表达情况。结果表明CDT毒素3个亚基均在Rosetta菌株中得到表达,其中cdtB约为28.2 ku,符合预期大小,而cdtA和cdtC亚基比预期的23.5 ku和17.4 ku略大。表达的3个重组蛋白可以被自然感染猪血清识别,表明其具有良好的反应原性,同时也表明H.parasuis在猪体内定殖或感染过程中分泌CDT。制备的抗血清可以与体外培养H.parasuis的分泌蛋白中的CDT亚基反应,表明重组蛋白具有良好的免疫原性,同时也表明H.parasuis体外培养时也分泌CDT。 相似文献
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实验通过将副猪嗜血杆菌(Haemophilus parasuis,HPS)外膜蛋白(Outer membrane protein,OMP)P5基因克隆至pET-28a(+)中,构建pET-OMP5原核表达质粒,再将其转化至大肠杆菌Rosetta(DE3),通过IPTG诱导重组菌,SDS-PAGE显示pET-OMP5重组表达质粒在大肠杆菌中实现了高效表达,Western-blot表明融合蛋白能被阳性血清识别。昆明小鼠试验结果表明OMP5重组蛋白具有一定的诱导免疫保护反应的能力。 相似文献
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猪链球菌-副猪嗜血杆菌二联亚单位疫苗在小鼠的免疫效力分析 总被引：1，自引：0，他引：1
本研究旨在开发一种用于防制猪链球菌病和副猪嗜血杆菌病的新型二联基因工程疫苗,并对其免疫效应进行评价.选择猪链球菌2型蛋白溶血素SLY、HP0197和副猪嗜血杆菌外膜蛋白D15和HPS06257 4种蛋白,重组表达并纯化,添加一定比例的佐剂制成含有不同组分的亚单位疫苗.研究表明该二联亚单位疫苗及各单苗能够引起小鼠良好的体液免疫,并能保护机体分别抵抗致死剂量猪链球菌2型(SS2)和副猪嗜血杆菌(HPS)强毒株的攻击.此外,调理吞噬试验表明疫苗高免血清能够有效杀死细菌,并赋予机体被动抵抗SS2和HPS感染的能力.研究证实制备的猪链球菌-副猪嗜血杆菌二联亚单位疫苗在小鼠具备良好的免疫保护效力. 相似文献
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Full-length canine HSP70 cDNA was sequenced and the expression of HSP70 mRNA was investigated. The full-length cDNA sequence of the HSP70 gene (2322 bp) contained a single long open reading frame (1920 bp) coding a protein of 640 amino acids. The amino acid sequence of the canine HSP70 gene shared about 90-95% sequence similarity with bovine, human and mouse HSP70 proteins. Southern blot analysis with HSP70 probe gave three distinct bands of 9.4 kb, 5 kb and 4.4 kb in BamHI digests and two distinct bands of 19 kb and 4 kb in EcoRI digests. Canine HSP70 mRNA was detectable in canine peripheral blood mononuclear cells and stomach but not in liver, kidney, spleen, small intestine, large intestine and skin of dogs. 相似文献
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热应激蛋白 (heatstressprotein,HSP)或热休克蛋白 (heatshockpro tein,HSP)是机体受到应激原的刺激后产生的几族蛋白质 ,具有高度保守性 ,对维持细胞生存和内环境稳定起重要作用 [1]。多种应激原如高热、重金属、饥饿、缺氧、缺血等都可诱导HSP的表达 ,但人们习惯上仍称其为热应激蛋白或热休克蛋白 ,有时也称为应激蛋白 (stressprotein,SP)。一般来说 ,根据HSP的同源性、功能和分子量 ,主要HSP可分成4个家族 :小分子量HSP家族、HSP70家族、HSP90家族和大分子量HSP家族。HSP70家族是HSP中最保守和最重要的一族 ,在大多数生物… 相似文献
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Tian Z Liu G Zhang L Yin H Wang H Xie J Zhang P Luo J 《Veterinary parasitology》2011,181(2-4):282-290
A Haemaphysalis longicornis heat shock protein 70 (HLHsp70) was identified from a cDNA library synthesized from tick eggs. The HLHsp70 cDNA is 2311 bp in length and encodes 661 amino acid residues with the predicted molecular weight of 72.5 kDa and an isoelectronic point (pI) of 5.2. It also contains the highly conserved functional motifs of the Hsp70 family and a specific endoplasmic reticulum (ER) retention signal "KDEL" that is common among ER-localized proteins. The HLHsp70 exhibits 90% amino acid identity to the putative Hsp70 of Ixodes scapularis, and 85% to Gallus gallus 78 kDa glucose-regulated protein precursor. Real time RT-PCR analysis showed that the expression levels of the Hsp70 in ovaries and salivary glands were significantly higher than in other tested tissues in partially fed females. Although the expression level of the HLHsp70 was constantly low in unfed ticks, it was significantly induced by blood-feeding. Further, the expression was positively correlated to the temperature (4-37°C, tested). Western blot analysis showed that the rabbit antiserum against the recombinant HLHsp70 protein (rHLHSP70) recognized bands of approximately 100, 72, and 28 kDa from egg lysates, as well as a 72kDa fragment in protein extracts from partially fed larvae. Immunization of rabbits with the rHLHSP70 did not result in a statistically significant reduction of female tick engorgement and oviposition. These results suggest that although HLHSP70 plays a role in the physiological activities of ticks, as a constitutive protein it was not suitable for selection as a candidate vaccine antigen against ticks. 相似文献
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热激蛋白在动物应激中的应用 总被引：4，自引：0，他引：4
热应激蛋白(HSPs)存在于所有动物细胞中，是动物受到应激原刺激后诱导产生的一组应激蛋白，与机体的应激关系密切，在进化上高度保守。本文在对HSP发现、分类及功能简要介绍的基础上，对HSP70在动物应激中的作用及其调控机制进行了详细的综述，指出HSP将对阐明动物应激机理有重要的意义。 相似文献
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Fusion expression of major antigenic segment of JEV E protein-hsp70 and the identification of domain acting as adjuvant in hsp70 总被引：3，自引：0，他引：3
Ge FF Qiu YF Gao XF Yang YW Chen PY 《Veterinary immunology and immunopathology》2006,113(3-4):288-296
Hsp70 potentiates specific immune responses to some antigenic peptides fused to it. A recombinant hsp70 protein expression vector in methylotrophic yeast, Pichia pastoris, was developed that fused the major antigenic segment of Japanese encephalitis virus (JEV) E protein to the amino terminus of Mycobacterium tuberculosis hsp70. The C-terminal peptide binding domain of hsp70 stimulated Th1-polarizing cytokines, CC chemokines and an adjuvant effect. However, the N-terminal ATPase domain (hsp70 1-358) failed to stimulate any of these cytokines or chemokines. Based on these data, a vector was constructed that permits the fusion of major antigenic segment of E protein to the amino terminus of peptide binding domain of hsp70. Antibody titers, lymphocytes proliferation, the level of mIL-2 or mIFN-gamma and neutralizing antibodies in immunized mice showed that antigenicity of E-binding domain fusion protein was almost as effective as E-hsp70 fusion protein and more effective than carrier protein hsp70 alone. In eliciting a humoral and cellular immune response, both fusion proteins were more powerful than the major antigenic segment of E protein alone, but less effective than the segment administered with Freund's adjuvant. 相似文献