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1.
目的:用大肠杆菌表达系统表达人巨细胞病毒(HCMV)pp65蛋白和gB蛋白的优势抗原表位基因,制成HCMV亚单位疫苗,探究其在Balb/c小鼠体内的体液免疫和细胞免疫活性。方法:选取pp65蛋白的490~508aa和gB蛋白的607~621aa的基因片段,经PCR扩增目的片段,连接表达载体pET-32a(+),转化BL21(DE3)plys菌株,十二烷基硫酸钠-聚丙烯酰胺凝胶(SDSPAGE)分析蛋白质表达,金属螯合亲和镍柱法纯化目的蛋白。重组蛋白免疫Balb/c小鼠,Western blotting法和间接ELISA法检测重组蛋白的体液免疫活性;通过流式细胞仪和双抗夹心ELISA法检测重组蛋白的细胞免疫活性。结果:获得分子质量约为22kDa的融合蛋白,Western blotting检测显示抗体有特异性,间接ELISA法检测其效价为1∶102 400。与对照组相比,实验组小鼠外周血中CD4~+T细胞和CD8~+T细胞的数量,以及IFN-γ、IL-2、IL-12的含量有显著提高(P0.01)。结论:制备的具有免疫优势抗原表位的重组蛋白能诱导小鼠产生较强的体液免疫和细胞免疫反应。  相似文献   

2.
为在真核细胞中表达并纯化I型单纯疱疹病毒(HSV I)包膜糖蛋白gB,并分析其抗原性和免疫原性,化学合成了包膜糖蛋白gB1胞外区基因片段,构建真核表达载体,并转染至HEK293细胞,表达的蛋白用羊抗HSV1+HSV2血清作为一抗,用ELISA检测其抗原性;用纯化的gB1蛋白免疫昆明小鼠,观察诱发抗体产生的时间及其效价,并用ELISA和Western blot检测小鼠抗gB1多克隆抗体特异性识别重组gB1抗原的能力,评价其免疫原性。结果显示在HEK293细胞中成功表达重组gB1蛋白,ELISA证实羊抗HSV1+HSV2多抗能够识别重组gB1蛋白;重组gB1蛋白免疫小鼠7周后,小鼠血清中多克隆抗体效价达到5×103,表明在真核细胞中高效表达并纯化的重组gB1蛋白具有良好的抗原性和免疫原性,为HSV检测试剂和疫苗研究提供了理论基础。  相似文献   

3.
为在真核细胞中表达并纯化I型单纯疱疹病毒(HSV I)包膜糖蛋白gB,并分析其抗原性和免疫原性,化学合成了包膜糖蛋白gB1胞外区基因片段,构建真核表达载体,并转染至HEK293细胞,表达的蛋白用羊抗HSV1+HSV2血清作为一抗,用ELISA检测其抗原性;用纯化的gB1蛋白免疫昆明小鼠,观察诱发抗体产生的时间及其效价,并用ELISA和Western blot检测小鼠抗gB1多克隆抗体特异性识别重组gB1抗原的能力,评价其免疫原性。结果显示在HEK293细胞中成功表达重组gB1蛋白,ELISA证实羊抗HSV1+HSV2多抗能够识别重组gB1蛋白;重组gB1蛋白免疫小鼠7周后,小鼠血清中多克隆抗体效价达到5×103,表明在真核细胞中高效表达并纯化的重组gB1蛋白具有良好的抗原性和免疫原性,为HSV检测试剂和疫苗研究提供了理论基础。  相似文献   

4.
目的几丁质酶是昆虫重要的防御物质,本研究采用DNA-蛋白质联合免疫策略制备荒漠昆虫小胸鳖甲几丁质酶(MpCHI786)的抗血清,用于检测小胸鳖甲在外界不良条件下几丁质酶的变化。方法从小胸鳖甲成虫中提取总RNA,反转录后RT-PCR扩增Mpchi786 cDNA,构建原核表达载体pET30a-Mpchi786,在大肠杆菌BL21中用IPTG诱导表达获得融合蛋白His-MpCHI786。对融合蛋白切胶纯化后与弗氏佐剂混合作为抗原。另外,构建真核表达质粒pcDNA3.0-Mpchi786,对小鼠尾静脉高压注射,8 h后RT-PCR检测其在肝脏的瞬时表达。以pcDNA3.0-Mpchi786肌肉注射免疫小鼠3次后,再用His-MpCHI786融合蛋白加强免疫2次,DNA免疫和免疫结束后收集免疫小鼠的血清,ELISA和Western blot法检测抗血清效价和特异性。结果 RT-PCR结果显示尾静脉高压注射8 h后,pcDNA3.0-Mpchi786质粒在小鼠肝脏有特异性表达。Western blot检测表明,用His-MpCHI786融合蛋白作为抗原,pcDNA3.0-Mpchi786DNA质粒免疫3次的抗血清和His-MpCHI786融合蛋白加强免疫2次后的抗血清都显示了特异性反应条带。ELISA检测结果显示抗血清效价高达1∶204 800以上。结论利用DNA-蛋白质联合免疫法能够获得效价高、特异性的小胸鳖甲几丁质酶MpCHI786的抗血清。  相似文献   

5.
目的:通过原核细胞表达人免疫缺陷病毒(HIV)Nef抗原,制备特异抗血清,为Nef抗原检测提供技术方法。方法:以HIVBotswana毒株基因组为模板,用PCR法获得Nef蛋白编码基因,将其克隆到pET30a载体中,在大肠杆菌中表达Nef融合蛋白;用纯化的融合蛋白免疫BALB/c小鼠获得抗血清,用真核表达的Nef抗原对其特异性进行分析。结果:构建的Nef融合基因在大肠杆菌中获得表达,相对分子质量约为36x103,免疫BALB/c小鼠获得针对融合蛋白的高效价抗血清,ELISA抗体滴度为1:6400;免疫荧光和Westemblot检测表明,该抗血清能特异地与重组痘苗病毒表达的Nef抗原反应。结论:在大肠杆菌中表达了HIVNef融合蛋白,制备了Nef融合蛋白的高效价小鼠免疫血清,该血清能特异性识别HIVNef抗原,为HlVNef抗原检测提供了技术方法。  相似文献   

6.
为研究DEAF1在小鼠胰淋巴结中的内源性表达及调控外周组织抗原基因表达的机制,将DEAF1的原核表达质粒转化至E.coli BL21中,诱导获得重组DEAF1蛋白;经尿素梯度裂解包涵体纯化获得的重组DEAF1蛋白分期免疫Balb/C小鼠,获取鼠抗DEAF1的多克隆抗血清;采用ELISA、Western blot、免疫荧光、免疫沉淀分别鉴定其效价、特异性、敏感性和亲和力。结果表明,抗血清可与抗原发生特异性的免疫反应,可用于检测DEAF1的表达及荧光定位,抗血清与抗原有较高的亲和力。结果证明所制备的多克隆抗血清具有较高的特异性、敏感性和亲和力。  相似文献   

7.
本研究旨在制备和鉴定小鼠抗Ⅱ型登革病毒(DENV‐2)10种蛋白的抗体,为后续相关研究提供实验材料。利用真核表达载体pReceiver构建DENV‐210种蛋白的重组质粒,提取质粒 DNA ,肌内注射免疫小鼠,共免疫4次。末次免疫后2周取小鼠血清,利用DENV‐2感染的Vero细胞和DENV‐2各蛋白的稳定表达细胞,通过酶联免疫吸附试验(ELISA)、间接免疫荧光法(IFA)和蛋白免疫印迹法评价免疫效果,分析抗体的特点。DNA免疫小鼠后获得抗DENV‐210种蛋白的抗血清,抗体效价波动于1∶400~1∶16127之间,以抗E蛋白抗体效价最高,达1∶16127,而抗NS3、NS4b、NS5蛋白抗体效价较低,仅为1∶400。利用DENV‐2感染的Vero细胞和稳定表达病毒蛋白的EAhy926细胞进行IFA染色,抗DENV‐2各蛋白的抗血清均可特异性识别DENV‐2抗原。蛋白免疫印迹结果显示,抗E、NS1、NS4b和NS5蛋白抗体能识别热变性蛋白,其他抗血清未呈现阳性反应条带。本研究提示,DNA免疫小鼠所获得的抗DENV‐2各蛋白抗体能特异性识别自然感染或模拟自然感染状态下的DENV‐2蛋白,可为后续相关研究提供工具,也表明DNA免疫法可作为抗体制备的一种策略。  相似文献   

8.
构建Ezrin原核重组表达载体pET-28a(+)-ezrin,将其转化至大肠杆菌BL21(DE3)中,诱导获得Ezrin蛋白,将经镍柱亲和层析纯化后的蛋白分别免疫新西兰大耳兔和昆明小鼠(KM),获得抗血清,采用ELISA和Western blotting,免疫荧光测定其效价和特异性。ELISA对抗血清进行效价测定表明抗血清可与抗原发生特异性免疫反应;通过Western blotting对抗血清进行鉴定表明抗血清可以识别几种细胞株内的特异性条带,其相对分子量为82 kD,与预测分子量相符;免疫荧光试验表明抗血清可以识别细胞内的Ezrin蛋白,且定位情况与文献报道相符。最后通过protein G对抗血清进行了纯化。结果表明用该方法制备的Ezrin多克隆抗体有较高的特异性和灵敏度。  相似文献   

9.
探讨流式细胞术检测人巨细胞病毒活动性感染检测方法的可行性及效果评价。分离人外周血白细胞,以商品化的小鼠抗人巨细胞病毒pp65抗原单克隆抗体为一抗,FITC标记的羊抗小鼠IgG抗体为二抗,采用流式细胞术对外周血人巨细胞病毒pp65抗原进行检测。同时采用间接免疫荧光法对外周血人巨细胞病毒pp65抗原进行检测。采用配对χ2检验对两种方法的检测效果进行评价。临床送检的65份疑似为人巨细胞病毒感染病人外周血标本中,间接免疫荧光法检出阳性9份,流式细胞术检出阳性11份,两种方法阳性检出率差异无统计学意义(P0.05)。采用流式细胞术可定量检测外周血人巨细胞病毒pp65抗原,与间接免疫荧光法检测结果无统计学差异,可在临床推广使用。  相似文献   

10.
为了构建人巨细胞病毒(HCMV)截短UL83基因真核表达重组体,实现其在Hep-2细胞中的稳定表达,研究该截短UL83基因真核表达重组体免疫效力,采用基因重组的方法,将HCMV AD169株截短UL83基因定向克隆到带有绿色荧光蛋白(GFP)作为报告基因的真核表达载体pEGFP-C1上,构建真核重组表达质粒pEGFP-C1-UL83;脂质体转染至Hep-2细胞中,G418筛选获得稳定表达pp65细胞表达系。经基因测序显示,重组体中截短UL83基因完全正确,RT-PCR和Western blot检测证实其可在Hep-2细胞中稳定表达。用该重组体和其表达产物在HCMV先天性感染小鼠模型上进行免疫保护试验显示,母鼠血清可检测到特异性抗HCMV pp65抗体,效价为:1∶2.51~1∶50.79;子鼠脑组织内未分离出病毒,亦未检测出病毒pp65蛋白抗原表达。初步结果表明,pEGFP-C1-UL83具有较好的免疫原性,可作为DNA疫苗刺激机体产生有效抗体,并具有阻止病毒垂直传播的保护性作用。  相似文献   

11.
To purify the protein encoding the small capsid protein (SCP) of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi's sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E. coli containing pQE-80L-orf65 was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and the fusion protein was purified by chromatography. The expressed protein and its purified product were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and showed that 9 kDa was the expected size of the purified orf65 protein. The antiserum was produced in rabbit which was immunized by purified orf65 protein. An ELISA assay was established to analyze the immunogenicity of the purified orf65 protein. The ELISA analysis demonstrated that orf65 protein has strong immune activity, and the immune activity of polyclonal antibody against orf65 was more than 4 fold higher than that in the serum of the non-immunized rabbit. These results demonstrate that purified orf65 protein has very strong immunogenicity and can be used in screening KSHV infection in the general population using ELISA.  相似文献   

12.
在分析鸭瘟病毒gB蛋白抗原性的基础上,设计一对引物克隆gB蛋白N端抗原性较好的抗原域编码基因.将克隆的基因定向插入pET-32a的EcoR Ⅰ和HindⅢ之间,构建了gB蛋白主要抗原域原核表达载体pET-gB1.将pET-gB1质粒转化BL(21)宿主菌后,对培养和表达条件进行了优化,实现了DPV gB蛋白主要抗原域的高效表达.免疫印迹试验表明获得的表达产物具有良好的反应原性.应用His·Bind亲和层析柱纯化重组DPV gB蛋白,以纯化的重组gB1蛋白作为检测抗原,初步建立了检测鸭瘟病毒抗体的igB1-ELISA.结果表明,抗原的最佳包被浓度为6.5μg/mL,血清的最佳稀释度为1∶80,阳性标准初步定为:待检血清OD490>0.4,且待检血清OD490/阴性血清OD490>2.应用igB1-ELISA对鸭血清样品进行检测,结果表明igB1-ELISA与全病毒包被的iDPV-ELISA符合率达到95.6%.  相似文献   

13.
人巨细胞病毒(HCMV)是广泛感染人类的重要病原体之一,在人群中有较高的感染率,胎儿、婴儿、器官移植及免疫缺陷者等更易感染.现有的抗HCMV药物治疗及被动免疫由于毒性大、价格昂贵及短效性等原因,应用大受限制,为此研制安全、有效的疫苗即成为防治HCMV疾病的重要手段.HCMV具有潜在的致癌作用,不宜发展活疫苗或含完整病毒DNA的死疫苗;亚单位疫苗虽然比较安全,但所用抗原不能在宿主细胞内表达,只能诱导体液免疫,这对于胞内感染的HCMV预防效果较差;通过新佐剂的应用,虽可引发细胞免疫,但免疫原性较差,且成本较高.因此,目前倾向于研制具有免疫原性的HCMV DNA疫苗.  相似文献   

14.
重组人谷氨酸脱羧酶65的表达及活性研究   总被引:2,自引:1,他引:1  
目的:获得有活性的人谷氨酸脱羧酶65(human glutamate decarboxylase 65,hGAD65)用于Ⅰ型糖尿病(type 1 diabetes mellitus,T1DM)检测。方法:将hGAD65基因插入质粒pET32a(+)中,与硫氧还蛋白(thioredox)及六聚组氨酸(hexahistidine)融合,由IPTG诱导融合蛋白(Trx-hGAD65)表达,经两次镍离子亲和层析纯化,通过薄层层析和ELISA研究hGAD65及Trx-hGAD65的活性。结果:在大肠杆菌中实现hGAD65的可溶性表达,经亲和层析得到较纯hGAD65和Trx-hGAD65蛋白,二者具有相同酶学活性,但Trx-hGAD65稳定性更高,并能检测出T1DM患者血清中的hGAD65抗体(hGAD65-Ab)。结论:Trx-hGAD65能在大肠杆菌中可溶性表达,从而为Trx-hGAD65用于T1DM诊断、预防和治疗打下基础。  相似文献   

15.
The nucleolus is a nuclear domain involved in the biogenesis of ribosomes, as well as in many other important cellular regulatory activities, such as cell cycle control and mRNA processing. Many viruses, including herpesviruses, are known to exploit the nucleolar compartment during their replication cycle. In a previous study, we demonstrated the preferential targeting and accumulation of the human cytomegalovirus (HCMV) UL83 phosphoprotein (pp65) to the nucleolar compartment and, in particular, to the nucleolar matrix of lytically infected fibroblasts; such targeting was already evident at very early times after infection. Here we have investigated the possible effects of rRNA synthesis inhibition upon the development of HCMV lytic infection, by using either actinomycin D or cisplatin at low concentrations, that are known to selectively inhibit RNA polymerase I activity, whilst leaving RNA polymerase II function unaffected. Following the inhibition of rRNA synthesis by either of the agents used, we observed a significant redistribution of nucleolar proteins within the nucleoplasm and a simultaneous depletion of viral pp65 from the nucleolus; this effect was highly evident in both unextracted cells and in nuclear matrices in situ. Of particular interest, even a brief suppression of rRNA synthesis resulted in a very strong inhibition of the progression of HCMV infection, as was concluded from the absence of accumulation of HCMV major immediate‐early proteins within the nucleus of infected cells. These data suggest that a functional relationship might exist between rRNA synthesis, pp65 localization to the nucleolar matrix and the normal development of HCMV lytic infection. J. Cell. Biochem. 108: 415–423, 2009. © 2009 Wiley‐Liss, Inc.  相似文献   

16.
Rasmussen’s encephalitis (RE) is a rare and severe progressive epileptic syndrome with unknown etiology. Infection by viruses, including human cytomegalovirus (HCMV), has been speculated to be a potential trigger for RE. However, no viral antigens have been detected in the brains of patients with RE; thus, a possible clinical linkage between viral infections and RE has not been firmly established. In this study, we evaluated the expression of HCMV pp65 antigen in brain sections from 26 patients with RE and 20 non-RE patients by immunohistochemistry and in situ hybridization, and assessed the associations between HCMV infection and clinical parameters. Elevated expression of HCMV pp65 protein and DNA was observed in 88.5% (23/26) and 69.2% (18/26) of RE cases, respectively. In the non-RE group, HCMV pp65 antigen was detected only in two cases (10%), both of which were negative for DNA staining. Additionally, the intensity of HCMV pp65 staining was correlated with a shorter duration of the prodromal stage, younger age of seizure onset, and more severe unilateral cortical atrophy. Elevated expression of HCMV pp65 was observed in RE brain tissue and was correlated with the clinical features of RE disease. In summary, our results suggested that HCMV infection may be involved in the occurrence and progression of RE disease. Thus, further studies are needed to determine whether early treatment with anti-HCMV antibodies could modulate the course of RE.
  相似文献   

17.
The cellular distribution of the human cytomegalovirus (HCMV)-specific UL83 phosphoprotein (pp65) and UL123 immediate-early protein (IEp72) in lytically infected human embryo fibroblasts was studied by means of indirect immunofluorescence and confocal microscopy. Both proteins were found to have a nuclear localization, but they were concentrated in different compartments within the nuclei. The pp65 was located predominantly in the nucleoli; this was already evident with the parental viral protein, which was targeted to the above nuclear compartment very soon after infection. The nucleolar localization of pp65 was also observed at later stages of the HCMV infectious cycle. After chromatin extraction (in the so-called in situ nuclear matrices), a significant portion of the pp65 remained associated with nucleoli within the first hour after infection, then gradually redistributed in a perinucleolar area, as well as throughout the nucleus, with a granular pattern. A quite different distribution was observed for IEp72 at very early stages after infection of human embryo fibroblasts with HCMV; indeed, this viral protein was found in bright foci, clearly observable in both non-extracted nuclei and in nuclear matrices. At later stages of infection, IEp72 became almost homogeneously distributed within the whole nucleus, while the foci increased in size and were more evenly spread; in several infected cells some of them lay within nucleoli. This peculiar nuclear distribution of IEp72 was preserved in nuclear matrices as well. The entire set of data is discussed in terms of the necessity of integration for HCMV-specific products into the pre-existing nuclear architecture, with the possibility of subsequent adaptation of nuclear compartments to fit the needs of the HCMV replicative cycle.  相似文献   

18.
将口蹄疫病毒 (FMDV)结构蛋白基因P1的完整cDNA序列插入原核表达性载体pGEX KG中 ,使P1基因与GST融合 ,获得融合表达质粒pKG P1,转化E .coliBL21 (DE3) ,经IPTG诱导 ,SDS PADE结果表明GST P1融合蛋白获得高效表达 ,Western blot检测证实表达的融合蛋白具有免疫学活性 ,表达产物主要存在于细菌裂解液上清中。进一步采用GST纯化试剂盒纯化P1蛋白并作为诊断抗原 ,建立了P1 ELISA诊断方法 ,与FMD间接血凝 (IHA)检测方法平行检测 86 4份血清样品 ,总的符合率达87%。  相似文献   

19.
实验以两种不同的表达策略构建了两个以大肠杆菌DE3为宿主的原核表达载体,由T7启动子启动外源基因的转录,在诱导剂IPTG诱导下成功地进行了戊肝病毒ORF3蛋白的原核表达。并通过SDS-聚丙烯酰胺凝胶电泳、免疫印迹、竞争抑制法酶联免疫等一系列实验对两种表达产物进行了鉴定和分析。综合分析两种表达结果发现,在融合型表达中ORF3蛋白与其融合标签蛋白(谷胱甘肽S一转移酶)之间存在免疫交叉反应,而且这种融合标签蛋白在空间结构上可能对ORF3蛋白中的抗体结合位点有掩盖作用。  相似文献   

20.
目的 :为研制预防结核病疫苗 ,选取结核杆菌HSP65蛋白为免疫抗原 ,将结核分枝杆菌HSP65抗原基因在大肠杆菌中表达、纯化。方法 :将HSP65的全长cDNA插入到原核表达载体pGEX5T中 ,构建成pGEX5T HSP65重组质粒。将质粒转化到E .coli.K80 2细菌 ,用IPTG诱导HSP65表达 ,然后用亲和层析的方法进行纯化 ,最后用Western blot方法确认表达蛋白的特异性。结果 :获得了pGEX5T HSP65重组子 ,HSP65蛋白在k80 2菌中获得了表达 ,表达的蛋白条带大小约 86kDa ,与预期的结果相符。表达产物经亲和层析后获得了较单一的蛋白条带 ;表达及纯化的蛋白在纯化前后均可被结核病患者血清特异地识别。为进一步研究其在结核病诊断和防治中的应用打下了基础。  相似文献   

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