Immune responses against SARS-coronavirus nucleocapsid protein induced by DNA vaccine

The nucleocapsid （N） protein of SARS-coronavirus （SARS CoV） is the key protein for the formation of the helical nucleocapsid during virion assembly. This protein is believed to be more conserved than other proteins of the virus, such as spike and membraneglycoprotein, In this study, the N protein of SARS-CoV was expressed in Escherichia coli DH5alpha and identified with pooled sera from patients in the convalescence phase of SARS.     

ESAT6抗原DNA疫苗在小鼠体内诱导的免疫应答

目的:构建结核杆菌ESAT6抗原DNA疫苗,并观察其在BALB/c小鼠体内诱导的免疫应答.方法:构建结核杆菌ESAT6抗原DNA疫苗pVAX-ESAT6,并将其肌肉注射免疫BALB/c雌性小鼠,用ELISA方法检测小鼠的血清抗体(IgG、IgG2a/IgG1)水平,并在体外用特异抗原刺激小鼠脾细胞,检测其分泌细胞因子(IFN-γ、IL-4)的水平;用乳酸脱氢酶(LDH)释放法测定小鼠细胞毒性T淋巴细胞反应.结果:pVAX-ESAT6 DNA疫苗诱导了抗原特异的体液免疫应答和细胞免疫应答,抗体亚型检测显示IgG2a与IgG1的比值为(2.28±0.4),脾细胞分泌的细胞因子水平检测显示IFN-γ的水平(360士45 Pg/ml)高于IL-4的水平(124±16 Pg/m).结论:成功地构建了pVAX-ESAT6 DNA疫苗,它能在小鼠中诱导抗原特异的免疫应答,应答类型以Th1型占优势.     

猪囊尾蚴抗原cC1 DNA疫苗和蛋白质疫苗联合诱导小鼠免疫应答

目的:观察cC1 DNA疫苗和蛋白质疫苗联合免疫对诱导BALB/c小鼠免疫应答的影响.方法:雌性BALB/c小鼠随机分为4组:A组(DNA疫苗组), 质粒DNA以10 d间隔免疫3次;B组(联合免疫组),质粒DNA以10 d间隔免疫 2次后以GST-cC1融合蛋白加强免疫1次;C组(蛋白质疫苗组),分别在第0、3 周免疫接种融合蛋白;D组(pcDNA3对照组),以10 d间隔,3次免疫接种质粒pcDNA3.ELISA法检测血清样品中的特异性抗体水平以及实验小鼠脾脏淋巴细胞分泌IFN-γ和IL-4水平,以MTT法行脾脏淋巴细胞增殖实验.结果:C组可诱导相对较强的小鼠免疫应答,其特异性IgG和IgG1以及脾脏淋巴细胞分泌IL-4水平均高于其他各组(P<0.05),免疫的类型以Th2应答为主.B组诱导的特异性抗体水平和淋巴细胞分泌细胞因子水平均明显高于A组(P<0.05),B组和A组诱导的免疫应答类型均以Th1应答为主.结论:以 DNA priming-protein boost的策略可有效诱导较DNA疫苗单独使用更高的特异性抗体应答,且以Th1型免疫应答为主.     

Immune responses in wild-type mice against prion proteins induced using a DNA prime-protein boost strategy

Objective To break immune tolerance to prion (PrP) proteins using DNA vaccines.Methods Four different human prion DNA vaccine candidates were constructed based on the pcDNA3.1 vector:PrP‐WT expressing wild‐type PrP,Ubiq‐PrP expressing PrP fused to ubiquitin,PrP‐LII expressing PrP fused to the lysosomal integral membrane protein type II lysosome‐targeting signal,and PrP‐ER expressing PrP locating the ER.Using a prime‐boost strategy,three‐doses of DNA vaccine were injected intramuscularly into Balb/c mice,fol...     

Immune responses against Schistosoma japonicum after vaccinating mice with a multivalent DNA vaccine encoding integrated membrane protein Sj23 and cytokine interleukin-12

Background The vaccination of mice with DNA encoding single candidate antigens has failed to induce significant protection against Schistosoma japonicum ( S. japonicum) challenge infections. In this study, we evaluated the feasibility of using a multivalent DNA vaccine which co-expressed S.japonicum integral membrane protein Sj23 and murine cytokine IL-12 to induce protective immune responses.Methods The plasmid pVIVO2-IL12-Sj23, a eukaryotic expression vector expressing Sj23 and murine IL-12 simultaneously, was constructed, identified, and tested for expression in vitro. Its ability to protect against S. japonicum challenge infections was analyed according to worm reduction rate and egg reduction rate after vaccination of BALB/c mice. The serum levels of specific IgG antibody were determined by enzyme-linked-immuno sorbent assay (ELISA) and Western blot analysis. Using cultured spleen cells, IFN-γ and IL-4 post-stimulation were quantified by ELISA. The phenotypes of splenocyte populations were analyzed by flow cytometry (FCM).Results The plasmid DNA pVIVO2-IL12-Sj23 was proven to express well in vitro by transient transfection of HEK-293 cells. Immunization resulted in a worm reduction rate of 45. 53% and egg reduction rate of 58.35%. ELISA and Western blot analysis indicated that immunized mice generated specific IgG against Sj23. Spleen cells showed significant increases in IFN-γ but decreases in IL-4.No significant differences in CD4^ and CD8^ subgroup ratios were observed after the challenges.Conclusions The multivalent DNA vaccine pVIVO2-1L12-Sj23 is sufficient to elicit moderate but highly significant levels of protective immunity against challenge infections. Cytokine IL-12, as a gene adjuvant, was able to enhance the Thl responses and, hence, the protective immunity.     

Immune response induced by HPV18 E2 DNA vaccine combined with IL-12

Objective:To study the effectiveness of human papillomavirus type 18 E2 gene (HPV18-E2) vaccine and interleukin-12(IL-12) on their immune effects. Methods:Thirty-five Balb/c female mice aged 6-8-week-old were randomly divided into five groups (each n =7).The mice were given instramuscular injection of DNA vaccines at 2-week four 4 times(100mg/time).The tails were cut to draw blood at the 0,2,4,6 weeks,and then the mice were executed by eyeball blood letting at 8 weeks.ELISA was used for the quantitative detection of the specific lgG antibody in the srea of Balb/c mice and the cytokine IFN-γ and IL-4 in mice MTT assay.Results: after 8 weeks immunization, antibody IgA in E2 vaccine group andE2 +IL-12 vaccine group was statistically increased as compared with that of control group, and the difference was statistically significant (P<0.01). IFN-γand IL-4 levels of mice spleen lymphocyte culture supernatant inE2 group and E2 + IL-12 were significantly higher than other control groups (P<0.01). the spleen lymphopoiesis activeness of E2 + IL-12 group was significant enhanced as compared with the E2 group, there were statistically significant differences (P<0.01) . Conclusions: E2 DNA vaccine combined with IL-12 immune mice can effectively induce cell-mediated immunity and humoral immune responses, their capabilities of inducing immune response might be more stronger than that of single one.     

汉滩病毒核蛋白诱导肾综合征出血热患者的T淋巴细胞反应

目的 测定汉滩病毒(HTNV)核蛋白(NP)C-端多肽诱导肾综合征出血热(HFRS)患者的特异性T淋巴细胞反应,为HFRS发病机制、疫苗研制及抗病毒免疫反应研究提供资料.方法 采用Ficoll密度梯度离心法,分离HFRS恢复期患者外周血单个核细胞(PBMC),用IFN-y酶联免疫斑点试验(ELIspot)测试13名患者对23条多肽的T淋巴细胞反应,筛选出反应较强的肽段.结果 5名患者有不同程度的肽特异性T细胞反应.No.70肽可诱导3号和10号患者分泌IFN-y,T细胞频率分别为51和55 SFC/106 PBMC,No.51肽可诱导4、7、12号患者分泌IFN-y,T细胞频率分别为90、65、95 SFC/106PBMC.结论 No.51肽和No.70肽可诱导较强的T淋巴细胞反应,可能是NP C端的细胞毒性T淋巴细胞(CTL)袁位.     

轮状病毒DNA疫苗经不同途径接种诱导体内免疫应答研究

目的:通过研究不同途径接种轮状病毒DNA疫苗所诱导的体内免疫应答,寻找其适合的免疫途径.方法:轮状病毒基因疫苗pcDNA1/VP7经肌肉注射及鼻粘膜两种途径免疫BALB/c小鼠,利用ELISA方法对其诱导产生的体液免疫应答进行测定.结果:经肌肉注射及鼻粘膜两种途径接种轮状病毒DNA疫苗后,均在BALB/c小鼠体内诱导产生了病毒特异性IgG增高(P＜0.05),而病毒特异性IgA与对照组相比无显著性差异.结论:肌肉注射及鼻粘膜两种途径均能作为轮状病毒DNA疫苗的候选接种途径.     

Epitope DNA vaccines against tuberculosis: spacers and ubiquitin modulates cellular immune responses elicited by epitope DNA vaccine

Cell-mediated immune responses are crucial in the protection against tuberculosis. In this study, we constructed epitope DNA vaccines (p3-M-38) encoding cytotoxic T lymphocyte (CTL) epitopes of MPT64 and 38 kDa proteins of Mycobacterium tuberculosis. In order to observe the influence of spacer sequence (Ala-Ala-Tyr) or ubiquitin (UbGR) on the efficacy of the two CTL epitopes, we also constructed DNA vaccines, p3-M-S(spacer)-38, p3-Ub (UbGR)-M-S-38 and p3-Ub-M-38. The immune responses elicited by the four DNA vaccines were tested in C57BL/6 (H-2b) mice. The cytotoxicity of T cells was detected by LDH-release method and by enzyme-linked immunospot assay for epitope-specific cells secreting interferon-gamma. The results showed that DNA immunization with p3-M-38 vaccine could induce epitope-specific CD8 CTL response and that the spacer sequence (AAY) only enhanced M epitope presentation. The protein-targeting sequence (UbGR) enhanced the immunogenicity of the two epitopes. The finding that defined spacer sequences at C-terminus and protein-targeting degradation modulated the immune response of epitope string DNA vaccines will be of importance for the further development of multi-epitope DNA vaccines against tuberculosis.     

弓形虫复合黏膜疫苗滴鼻免疫小鼠诱导的脾淋巴细胞持续性免疫应答

目的观察弓形虫复合黏膜疫苗滴鼻免疫BALB/c小鼠后诱导的脾淋巴细胞的免疫应答及持续时间。方法96只BALB/c小鼠随机分为免疫组和对照组,免疫组以弓形虫复合黏膜疫苗20μl/只(20μg可溶性速殖子抗原 1μg霍乱毒素)滴鼻免疫2次(间隔2周),对照组以PBS滴鼻。分别于末次滴鼻后第1,2,3,4,6,8,10,12周处死小鼠,脾淋巴细胞计数,免疫细胞化学法检测CD4 、CD8 T细胞亚群水平。结果免疫组小鼠于第1,2周脾淋巴细胞明显高于对照组(P<0.01),其中以CD4 T细胞增生为主,第1,2,3,4,6周增生显著(P<0.01),CD8 T细胞水平在第2,3周也有增生(P<0.05),CD4 /CD8 比值第6周高于对照组(P<0.01)。结论弓形虫复合黏膜疫苗滴鼻免疫BALB/c小鼠可有效诱导脾淋巴细胞增殖性免疫应答,且可持续较长时间。     

结核分枝杆菌MPT64-ESAT6融合蛋白在小鼠体内诱导的免疫应答及其保护力

目的:测定MPT64与ESAT6融合蛋白在小鼠体内诱导的免疫应答及保护力. 方法:将MPT64与ESAT6的融合蛋白皮下免疫小鼠3次,每次间隔2 wk,ELISA测定免疫小鼠血清特异性抗体的滴度. 最后一次免疫完成后4 wk,分离部分免疫小鼠脾淋巴细胞,MTT法检测淋巴细胞刺激指数,ELISA检测悬液中IFN-γ水平. 另一部分免疫的BALB/c小鼠经尾静脉感染MTB毒株H37Rv,4 wk后计数脾脏细菌负荷数. 结果:MPT64-ESAT6融合蛋白免疫小鼠血清特异性抗体滴度1∶ 1500,免疫小鼠后淋巴细胞刺激增殖指数为2.23,而生理盐水免疫组只有0.88;脾淋巴细胞悬液中诱发的IFN-γ为(4.28±0.27)μg/L,显著高于生理盐水免疫组[(0.48±0.17) μg/L,P<0.05],但不及BCG免疫组[(5.18±0.31)μg/L]. 与生理盐水免疫组(细菌负荷6.45±0.17)相比较,融合蛋白免疫的小鼠,对攻击感染后抗MTB在脾脏中增殖有显著作用,细菌负荷为5.04±0.11,但与BCG免疫组相比脾脏细菌负荷减少甚微(细菌负荷4.38±0.22). 结论:MPT64与ESAT6融合蛋白可作为新型结核疫苗的候选组分.     

Augmented humoral and cellular immune responses of a hepatitis B DNA vaccine encoding HBsAg by protein boosting

Several reports have indicated that combinatorial regimens with DNA and protein vaccines can elicit both strong immune responses, to circumvent the limits of each vaccine. Surprisingly little was known on HBV vaccine. Here, we investigated the immunization effects of several regimens in BALB/c mice. The level of total antibody and isotypes of IgG were determined by ELISA. Cellular immune responses were assayed by measuring the production of cytokines and CTL activity after restimulation for 7 days in vitro with tumor cells CT26/S stably expressing HBsAg. The efficacy of immunoprotection against the challenge of transplanted CT26/S was also examined. The regimen involving twice priming pVAX（S） encoding HBsAg and once recombinant HBsAg protein （rHBsAg） boosting, induced strong and homogenous antibody responses. This regimen induced significant stronger responses of interleukin-12 and gamma interferon （IFN-gamma） in splenocytes, and elicited stronger CD8^＋ CTL responses and greater immunopretectional efficacy than those elicited by immunization with rHBsAg or pVAX（S） alone. Our regimen may thus provide a strategy for developing an improved immunization against HBV and many other pathogens.     

利用体内电脉冲增强治疗型HBV DNA疫苗细胞免疫反应的研究

目的：研究在初次免疫和加强免疫阶段分别使用不同的免疫模式能否增强HBV DNA疫苗的细胞免疫反应。方法：肌内注射10μg HBV DNA疫苗的Balb/c小鼠根据电脉冲实施时间的不同选择两种免疫模式,DNA＋EP或EP＋3d＋DNA,按在初次免疫和加强免疫阶段免疫模式的不同组合次序进行分组。各组小鼠在实施加强免疫后第14天取脾细胞做抗原特异的IFN-γ ELISPOT检测和FACS分析。结果：在初次免疫和加强免疫阶段使用不同免疫模式的实验组与采用相同免疫模式的实验组相比,诱导的特异性IFN-γT细胞频数和IFN-γ的CD8＋T细胞百分比均增加了4倍以上,差异有高度统计学意义（P〈0.01）,而分别在初次免疫和加强免疫阶段实施的两种免疫模式的不同次序产生的细胞免疫效果之间差异无统计学意义（P〉0.05）。结论：在初次免疫和加强免疫阶段利用体内电脉冲实施不同的免疫模式能显著增强HBV DNA疫苗在动物体内的细胞免疫效果。     

微小隐孢子虫子孢子表面蛋白基因诱导小鼠产生免疫应答的研究

目的　探讨微小隐孢子虫子孢子表面蛋白CP15重组质粒pCR3 1- 15DNA疫苗诱导机体产生体液和细胞免疫应答的效果。方法　用重组的DNA疫苗于BALB/c小鼠后腿胫骨前肌注射免疫 ,于 0、3、6w共免疫 3次 ,10 0 μg/次。免疫后不同时间检测体液和细胞免疫应答指标。并用 1× 10 6卵囊进行攻击感染试验。结果　pCR3.1- 15可诱导机体产生相应的特异性抗体 ,对C .parvum卵囊攻击具有保护作用。结论　微小隐孢子虫子孢子表面蛋白CP15重组质粒pCR3.1- 15有可能作为侯选的隐孢子虫DNA疫苗 ,值得进一步深入研究。     

嗜肺军团菌主要外膜蛋白DNA疫苗的免疫保护性研究

目的观察m ompS基因DNA疫苗对嗜肺军团菌感染的免疫保护作用。方法将18只BALB/c雌性小鼠随机分为3组,pcDNA 3.1-m ompS实验组股四头肌肌肉接种pcDNA 3.1-m om opS质粒100μg,两周后加强免疫1次,2个对照组分别肌肉注射等体积的生理盐水和空质粒,加强免疫后3周小鼠鼻腔滴注嗜肺军团菌L 1型菌液进行攻击感染,感染4周后,检查小鼠肺组织带菌量,观察肺组织病理形态学改变。结果pcDNA 3.1-m ompS免疫组肺组织带菌量少于生理盐水对照组和空质粒对照组(P<0.01);与生理盐水对照组和空质粒对照组比较,pcDNA 3.1-m ompS免疫组肺组织病理改变轻微。结论由m ompS基因构建的嗜肺军团菌DNA疫苗能诱导小鼠产生保护性免疫。     

猪囊尾蚴抗原DNA疫苗诱导的免疫保护效应

观察猪囊尾保护性原ｃＣ１ ＤＮＡ疫苗诱导的小鼠免疫应答,及对仔猪的免疫保护作用。方法：将全长ｃＣ１ ｃＤＮＡ插入真核表达质粒载体ｐｃＤＮＡ３,构建了重组质粒ｐ３－ｃＣ１,肌注小鼠,ＥＬＩＳＡ检测小鼠血清抗ｃＣ１抗体ＩｇＧ及ＩｇＧ２ａ水平,并进行仔猪的免疫保护实验。结果：免疫小鼠第３周出现特异性ＩｇＧ应答,第８周ＩｇＧ水平达到最高;小鼠血清的ＩｇＧ２ａ检测显示,第２周ＩｇＧ２ａ应答即为阳性,且长时间     

抗SARS-CoV-2 N蛋白单克隆抗体的制备与鉴定

目的 制备针对严重急性呼吸综合征冠状病毒2(SARS-CoV-2)核衣壳蛋白(N)的单克隆抗体(mAbs)并鉴定其生物学特性。方法 利用大肠杆菌原核表达重组SARS-CoV-2 N蛋白,通过杂交瘤技术将小鼠骨髓瘤细胞SP2/0与经重组蛋白免疫的BALB/c小鼠的脾细胞进行融合,采用间接ELISA法和有限稀释法筛选出阳性杂交瘤细胞株,并鉴定其亚型,腹水法制备mAbs, Protein G亲和柱纯化后通过SDS-PAGE、间接ELISA和Western blot检测其纯度、效价及特异性。结果 获得5株能稳定分泌抗SARS-CoV-2 N蛋白mAbs的杂交瘤细胞株,分别命名为N1～5。通过腹水法制备获得5个mAbs,亚型鉴定结果显示1个为IgG2a*κ型、4个为IgG1*κ型,其效价均达到2×104以上。Western blot结果表明5个mAbs都能与重组SARS-CoV-2 N蛋白结合。结论通过杂交瘤技术成功制备了5个能特异性识别重组SARS-CoV-2 N蛋白的mAbs,为开发免疫诊断试剂奠定了基础。     

SARS冠状病毒N基因重组腺病毒载体的构建及其在Vero E6细胞中的表达

目的：构建SARS冠状病毒N基因重组腺病毒载体并在Vero细胞中表达,为进一步研究SARS病毒的基因疫苗提供试验基础。方法：用化学合成的方法得到SARS病毒N基因,以PCR法使其加上CACC序列接头,经过定向拓扑克隆使目的基因连接到转移载体上,再通过LR酶促反应,将目的基因转移到腺病毒表达载体DNA上,获得N基因重组的腺病毒DNA,转染HEK293A细胞,包装、扩增后得到SARS冠状病毒N基因重组的腺病毒,进一步感染Vero E6细胞,用westem blot的方法检测目的基因表达产物。结果：目的基因能成功地在感染细胞中表达并保持了较好的免疫原性。结论：该研究为SARS冠状病毒基因疫苗的研制了提供理论和实验依据。     

乙肝病毒表面抗原基因疫苗诱导小鼠细胞及体液免疫应答的研究

为观察乙肝病毒 Ｈ Ｂｓ Ａｇ 基因疫苗免疫小鼠后的细胞及体液免疫应答,将该疫苗定向克隆于质粒ｐｃ Ｄ Ｎ Ａ３ 巨细胞病毒启动子下游,构建能在真核细胞内表达的重组基因疫苗ｐｃ Ｄ Ｎ Ａ Ｈ Ｂｓ Ａｇ 。将此疫苗通过多点肌肉注射免疫 Ｂａｌｂ／ Ｃ 小鼠后第六周,实验组小鼠均出现抗－ Ｈ Ｂｓ Ｉｇ Ｇ 阳性,其抗体水平明显高于对照组,同时 Ｔｈ１ 免疫应答相关的细胞因子 Ｉ Ｌ２ 及γ干扰素水平也明显高于对照组,表明本文构建的基因疫苗ｐｃ Ｄ Ｎ Ａ３ Ｈ Ｂｓ Ａｇ 能诱导 Ｂａｌｂ／ Ｃ 小鼠产生良好的细胞及体液免疫应答。