猪细小病毒核酸疫苗的构建及其对小鼠免疫原性的研究

将猪细小病毒VP2基因克隆至pCI-neo真核表达载体中,构建了pCIneo-VP2重组质粒,转染至PK-15细胞中,利用免疫荧光方法检测在体外表达情况;并以小鼠为动物模型,将pCIneo-VP2、pCI-neo重组质粒、猪细小病毒活疫苗和对照组通过肌肉注射进行免疫,检测免疫小鼠的淋巴细胞转化功能,特异性CTL杀伤活性和血清抗体滴度。结果显示,pCIneo-VP2在体外能够诱导PK-15细胞表达VP2蛋白,小鼠注射pCIneo-VP2质粒 1周后能够诱导机体产生抗体,4周时达到峰值,与活疫苗对照组产生的抗体滴度、诱导T淋巴细胞增殖和诱导强的细胞毒性基本一致。试验表明,构建的pCIneo-VP2能够有效诱导机体产生体液免疫和细胞免疫,为研制出高效、新型猪细小病毒疫苗提供了科学依据和试验依据。     

IL-2与猪细小病毒VP2基因双表达载体的构建及免疫原性的研究

将白细胞介素-2基因和猪细小病毒VP2基因主要抗原区克隆至pCI-neo真核表达载体中,构建了pCIneo-IL2-VP2重组质粒,用脂质体将其转染到PK-15细胞中,利用免疫荧光方法检测在体外表达情况。并以小鼠为动物模型,将pCIneo-IL2-VP2重组质粒、对照组pCI-neo和猪细小病毒活疫苗通过肌肉注射进行免疫,检测免疫小鼠的淋巴细胞转化功能,特异性CTL杀伤活性和血清抗体滴度。结果显示,pCIneo-IL2-VP2在体外能够诱导PK-15细胞表达VP2蛋白,小鼠注射pCIneo-IL2-VP2质粒1周后能够诱导机体产生抗体,4周时达到峰值,与活疫苗对照组产生的抗体滴度、诱导T淋巴细胞增殖和诱导强的细胞毒性基本一致。试验表明,构建的pCIneo-IL2-VP2能够有效诱导机体产生体液免疫和细胞免疫。     

新型呼肠病毒 S基因 DNA 疫苗在小鼠体内的免疫原性

目的：探讨新型呼肠病毒R4株S片段免疫小鼠后引发的免疫应答。方法构建4个不同S基因节段的重组真核表达质粒,并免疫小鼠;ELISA检测血清以研究R4特异性抗体升高水平,并对其抗体亚型进行鉴定;ELISPOT检测小鼠淋巴细胞INF-γ的表达情况。结果与对照组相比,4个重组质粒免疫的小鼠血清都有明显的R4特异性抗体升高,尤其以S1和S3基因免疫后抗体水平较高,且均以IgG2a占绝对优势;S1基因免疫组小鼠的细胞免疫应答最强。结论 S1基因重组质粒免疫小鼠后可同时引发较强的体液免疫和细胞免疫应答,是较为理想的疫苗备选基因片段。     

共表达H5N1流感病毒M1和HA基因的DNA疫苗与腺病毒载体疫苗的小鼠免疫评价

为评价在小鼠体内表达流感病毒M1和HA基因诱导的免疫反应,制备共表达H5N1亚型禽流感病毒 (A/Anhui/1/2005) 全长基质蛋白1 (M1) 基因和血凝素 (HA) 基因的重组DNA疫苗pStar-M1/HA和重组腺病毒载体疫苗Ad-M1/HA,将其按初免-加强程序免疫BALB/c小鼠,共免疫4次,每次间隔14 d。第1、3次用DNA疫苗,第2、4次用重组腺病毒载体疫苗,每次免疫前及末次免疫后14 d采集小鼠血清用于检测体液免疫应答,末次免疫后14 d采集小鼠脾淋巴细胞用于检测细胞免疫应答。血凝     

HIV-1 p24 DNA和P24蛋白联合免疫小鼠的效果

DNA疫苗能够诱导机体产生特异的细胞免疫和体液免疫反应,在肿瘤和感染性疾病的疫苗开发中显示出巨大的潜能。以HIV-1核心蛋白P24为抗原基因,构建pVAX1-p24 DNA,经Western blotting和动物活体成像检测证明,pVAX1 DNA携带的外源基因可以在293T 细胞和小鼠肌肉组织有效表达。采用不同的免疫策略免疫BALB/c小鼠 (DNA/DNA,DNA/Protein),实验结果表明：pVAX1-p24单独免疫BALB/c小鼠,可诱导明显的体液免疫及细胞免疫反应;pVAX1-p24与P24蛋白联合免疫诱导的体液免疫反应高于pVAX1-p24单独免疫,所获得的抗体滴度是单独免疫的7.3~8.0倍,但细胞免疫反应则不及单独免疫组。研究结果表明采取不同的免疫策略可以诱导产生不同的免疫反应,根据具体情况调整免疫策略将获得更好的免疫效果。这些研究为艾滋病疫苗的研发提供了实验依据。     

口服型HCV融合抗原DNA疫苗在小鼠诱导免疫应答

将编码一个外源信号肽、一个通用型辅助性T淋巴细胞抗原表位和HCV核心 包膜蛋白E2融合抗原基因的真核表达质粒pST CE2t(DNA疫苗 )转化到减毒鼠伤寒沙门菌SL72 0 7.将该重组菌口服接种BALB c小鼠 3次 .小鼠的抗HCV核心和E2抗体阳转率分别达 6 0 %和 70 % .体外以重组HCV核心或E2抗原刺激小鼠脾细胞 ,均使之发生明显的增殖反应 ,且小鼠脾细胞能有效杀伤表达HCV核心抗原的同系骨髓瘤细胞SP2 0 .这为研制高效免疫、成本低廉、接种方便的HCV疫苗提供了一个新的可行途径     

Selection and identification of a new adhesion protein of Cryptosporidium parvum from a cDNA library by ribosome display

In this study, we described a novel display method to identify surface adhesion proteins of Cryptosporidium parvum. A cDNA library of the sporozoite and oocyst stages of C. parvum was expressed on ribosome and selectively and specifically screened with intestinal epithelial cells (IECs) from newborn Cryptosporidium-free Holstein calves. Proteins were then enriched using a multi-step panning procedure. A new surface adherence protein of C. parvum was selected, named Cp20. Sequence analyses showed that Cp20 has a N-terminal signal peptide and four transmembrane regions. Indirect immunofluorescence assay (IFA) using an antibody specific for rCp20 demonstrated that the antibody specifically bound to the surface of sporozoites and oocysts. The recombinant plasmid pVAX1-Cp20 was constructed to examine the potential of the Cp20 gene as a target for specific preventive and therapeutic measures for cryptosporidiosis. The in vivo efficacies of the DNA vaccine was tested in BALB/c mice. The results indicated that the DNA vaccine elicited significant antibody responses and specific cellular responses when compared to control mice that received vector only or PBS. The DNA vaccine induced strong protective immune response against C. parvum and lower level of the oocysts shedding after challenge infection. This study suggested that Cp20 could serve as an effective target for specific preventive and therapeutic measures for cryptosporidiosis.     

Dietary nucleosides and nucleotides reduce Cryptosporidium parvum infection in dexamethasone immunosuppressed adult mice.

Numerous studies have demonstrated that dietary sources of nucleosides and nucleotides are important for the maintenance of cellular and humoral immune responses. To determine the immunological effects of feeding a nucleoside-nucleotide mixture to dexamethasone-immunosuppressed C57BL/6 adult mice infected with Cryptosporidium parvum, we examined fecal oocyst shedding, lymphoproliferative responses to concanavalin (Con) A, and C. parvum antigen, interleukin (IL-2), and gamma-interferon (IFN-gamma) production by cultured spleen cells. Mice were fed a nucleotide-free 20% casein diet (control group) or this diet supplemented with a 0. 5% nucleoside-nucleotide mixture before and after inoculation with C. parvum. Spleens from mice receiving the supplemented diet had higher (P < 0.05) Con A and antigen-specific induced cell proliferation than those from control mice. In addition to the increased cell proliferation, the spleen cells from the supplemented mice produced significantly more IL-2 (P < 0.002) and significantly more IFN-gamma (P <; 0.004) than cells from the control mice. Mice fed the supplemented diet excreted fewer (P < 0.05) C. parvum oocysts in the feces than control mice. The cumulative survival rate in the nucleoside-nucleotide mixture-fed group was higher compared with the control group (P < 0.05). We conclude that nucleosides and nucleotides may partially counteract the immunosuppressive effects of dexamethasone in C. parvum-challenged mice.     

Plasmid DNA vaccine-elicited cellular immune responses limit in vivo vaccine antigen expression through Fas-mediated apoptosis

Particularly potent cellular or humoral immune responses are needed to confer protection in animal models against such pathogens as HIV/SIV, Mycobacterium tuberculosis, and malarial parasites. Persistent, high-level vaccine Ag expression may be required for eliciting such potent and durable immune responses. Although plasmid DNA immunogens are being explored as potential vaccines for protection against these pathogens, little is known about host factors that restrict long-term plasmid DNA vaccine Ag expression in vivo. We observed rapid damping of transgene expression from a plasmid DNA immunogen in wild-type, but not in T cell-deficient mice. This damping of Ag expression was temporally associated with the emergence of Ag-specific cellular immune responses. A requirement for Fas and the appearance of apoptotic nuclei at the site of vaccine inoculation suggest that T cells induce Fas-mediated apoptosis of plasmid DNA vaccine Ag-expressing cells. These studies demonstrate that high levels of in vivo Ag expression are associated with high-frequency cellular immune responses that in turn rapidly down-regulate vaccine Ag expression in vivo. These findings argue that it may not be possible to maintain persistent, high-level production of vaccine Ag in vivo to drive persistent immune responses as long as vaccine Ag production can be limited by host immune responses.     

MAGE-3 DNA疫苗的构建及其免疫效果的观察

通过RT PCR方法扩增MAGE 3cDNA ,以pcDNA3 1+为载体 ,构建重组表达质粒pcDNA3 1 MAGE 3。重组质粒用脂质体转染鼠B16细胞 ,经RT PCR、细胞免疫染色及免疫印迹法鉴定转化细胞中MAGE 3的表达。以 10 0 μg质粒剂量肌肉注射接种小鼠 ,间隔 10天 ,共 3次 ,以空载体和PBS为对照。结果 ,重组质粒免疫的小鼠 ,其脾淋巴细胞对MAGE 3阳性靶细胞的杀伤活性为 51 0 8± 7 41% ,与空载体组 (8 44± 1 89% )及PBS组 (5 76± 1 75% )相比 ,差异有显著性 (P <0 0 1) ,而对MAGE 3阴性靶细胞的杀伤活性分别为 8 2 1± 1 65% ,7 68± 1 56%和 5 13±1 42 % ,其差异无显著性 ;MAGE 3DNA疫苗组免疫血清 1∶15稀释时能检测到抗MAGE 3抗体 ,脾细胞培养上清中Th1类细胞因子IFN γ、IL 2水平明显升高 ,外周血中CD4+、CD8+T细胞也提高 ,小鼠肿瘤的生长速度明显减慢 ,与对照组相比 ,差异显著 (P <0 0 1)。说明MAGE 3重组质粒免疫小鼠可以诱导小鼠产生特异性的体液和细胞免疫应答     

Potent T cell responses induced by single DNA vaccine boosted with recombinant vaccinia vaccine

Plasmid DNA, an effective vaccine vector, can induce both cellular and humoral immune responses. However, plasmid DNA raises issues concerning potential genomic integration after injection. This issue should be considered in preclinical studies. Tiantan vaccinia virus (TV) has been most widely utilized in eradicating smallpox in China. This virus has also been considered as a successful vaccine vector against a few infectious diseases. Potent T cell responses through T-cell receptor (TCR) could be induced by three injections of the DNA prime vaccine followed by a single injection of recombinant vaccinia vaccine. To develop a safer immunization strategy, a single DNA prime followed by a single recombinant Tiantan vaccinia (rTV) AIDS vaccine was used to immunize mice. Our data demonstrated that one DNA prime/rTV boost regimen induced mature TCR activation with high functional avidity, preferential T cell Vβ receptor usage and high sensitivity to anti-CD3 antibody stimulation. No differences in T cell responses were observed among one, two or three DNA prime/rTV boost regimens. This study shows that one DNA prime/rTV boost regimen is sufficient to induce potent T cell responses against HIV.     

A 23-kDa recombinant antigen of Cryptosporidium parvum induces a cellular immune response on in vitro stimulated spleen and mesenteric lymph node cells from infected mice

In the present study, we focused on a 23-kDa antigen, Cp23, which has been shown to be a major target of humoral immune responses in Cryptosporidium parvum infections and is present in both the sporozoite and merozoite stages. Recombinant Cp23 antigen was shown to stimulate a specific proliferative response by splenocytes and mesenteric lymph node cells from infected interferon gamma knockout BALB/c mice. Cp23 stimulation also induced TNF-alpha, IL-2, and IL-5 mRNA production by spleen cells from infected animals. In contrast, IL-12 mRNA was decreased by Cp23 stimulation compared with unstimulated splenocytes. These data suggest that, as with humoral responses, Cp23 is an important target of cellular immune responses in experimental C. parvum infections. The potential role of this antigen in conferring protective immunity is also discussed.     

汉滩病毒84Fli株DNA疫苗诱导小鼠免疫应答的初步研究

为了加强我国病毒性出血热的防治,本研究将汉滩病毒84Fli株核蛋白S和糖蛋白M编码片段分别克隆至pcDNA3.0载体,构建了pcDNA3/84S和pcDNA3/84M重组质粒,等量混合采用肌肉注射途径免疫C57BL/6小鼠,免疫3次,每次间隔2周,同时与双价出血热病毒灭活疫苗进行对比。ELISA及免疫荧光(IFA)分别检测小鼠血清中汉滩病毒核蛋白及糖蛋白特异性抗体,流式细胞仪和ELISPOT方法分析小鼠免疫后的细胞免疫水平。微量中和试验检测小鼠血清抗体的的中和活性。结果显示,DNA疫苗免疫组C57BL/6小鼠在初次免疫2周后即能检测到汉滩病毒核蛋白与糖蛋白的特异性抗体,与灭活疫苗组相比,重组质粒诱导的抗体滴度高,产生时间早,产生的抗体具有中和活性;同时可诱导产生特异性细胞免疫应答。研究表明,汉滩病毒pcDNA3/84S和pcDNA3/84M重组质粒能有效刺激小鼠产生特异性体液免疫和细胞免疫应答。     

乙肝病毒DNA疫苗的构建及其诱导小鼠的免疫应答

构建含adr亚型HBV表面抗原基因的核酸疫苗 ,考察人白细胞介素II基因及重组白细胞介素II的免疫佐剂作用。用含有人白细胞介素II基因的真核表达质粒及基因重组白细胞介素II蛋白作为佐剂 ,将编码乙型肝炎病毒表面抗原的重组真核表达质粒 pVAX/HBS免疫BALB/C小鼠 (试验组 ) ,同时设置注射质粒pVAX的阴性对照组 ,并分别于第 2 ,4周后加强免疫各 1次。试验组在第 4周时开始有HBsAb产生 ,阴性对照组未测到HbsAb ,试验组和对照组均未检测到HBsAg。乙肝病毒DNA疫苗能引起小鼠特异性体液免疫应答 ,白细胞介素II的真核表达质粒的佐剂作用不明显 ,基因重组白细胞介素II蛋白具有提高小鼠对乙肝病毒核酸疫苗免疫应答水平的佐剂活性。     

携带HBV包膜大蛋白DNA疫苗的减毒沙门菌在小鼠诱导免疫应答

探索一种简便、有效的乙型肝炎病毒DNA疫苗免疫方法。将编码绿色荧光蛋白的真核表达质粒pEGFPN1转化到减毒鼠伤寒沙门菌SL7207,灌胃饲服BALB/c小鼠,流式细胞术检测出小鼠脾细胞内表达的绿色荧光蛋白;构建编码HBV包膜大蛋白的DNA疫苗pCIS1S2S,分别以SL7207为载体的口服途径或直接肌肉注射途径免疫BALB/c小鼠,检测小鼠的血清抗体、T细胞增殖和细胞毒性T淋巴细胞反应,结果表明两种免疫途径均能在小鼠体内诱生细胞和体液免疫应答,但口服途径诱导免疫应答的强度明显强于肌肉注射途径。口服携带HBV DNA疫苗的减毒伤寒沙门菌可能代表一种简便、有效的治疗乙型肝炎的新方法。      

Enhancing immune responses against SARS-CoV nucleocapsid DNA vaccine by co-inoculating interleukin-2 expressing vector in mice

The immunogenicity of SARS-CoV nucleocapsid DNA vaccine and the immunoregulatory activity of interleukin-2 (IL-2) were investigated. DNA vaccine plasmids, pcDNA-N and pcDNA-IL2, were constructed and inoculated into BALB/c mice with or without pcDNA-IL2 by intramuscular injection. Cellular and humoral immune responses were assessed by indirect ELISA, lymphocyte proliferation assays, ELISPOT and FACS. The nucleocapsid DNA vaccine had good immunogenicity and can induce specific humoral and cellular immunity in BALB/c mice, while IL-2 plays an immunoadjuvant role and enhances specific immune responses. This study provides a frame of reference for the design of DNA vaccines against SARS-CoV.     

Recombinant thioredoxin peroxidase from Cryptosporidium parvum has more powerful antioxidant activity than that from Cryptosporidium muris

Cryptosporidium parvum can survive exposure to harsh environmental conditions, various disinfectants, and high doses of γ-irradiation. In an animal study, more than 25kGy of γ-irradiation was necessary to eliminate C. parvum infectivity from mice. In contrast, Cryptosporidium muris (murine Cryptosporidium), which lives in stomach epithelium, lost its infectivity in mice with 1kGy of γ-irradiation. Recently, it was found that thioredoxin peroxidase was highly expressed in C. parvum oocysts irradiated with high doses of γ-irradiation. Therefore we hypothesize that antioxidant activity of the thioredoxin peroxidase is involved in the radioresistance of C. parvum. To verify this, thioredoxin peroxidases of C. parvum (CpTPx) and C. muris (CmTPx) were expressed in Escherichia coli cells, and their antioxidant activities were compared. Both CpTPx and CmTPx belong to the 2-Cys family of peroxiredoxins. Hydrogen peroxide consumption was approximately 2- to 12-fold greater in recombinant CpTPx (rCpTPx) than in recombinant CmTPx (rCmTPx) in the presence of 0.2mM dithioerythritol or glutathione (GSH), respectively. The peroxidase activity of rCpTPx was highly enhanced by GSH, but that of rCmTPx was not. The minimum dose of rCpTPx required to protect supercoiled plasmid DNA from damage by metal-catalyzed oxidation was only 12% of that required with rCmTPx. The results showed that rCpTPx has more powerful antioxidant activity than rCmTPx. Further investigations on the role of CpTPx in the radioresistance of C. parvum are warranted.     

用体内激活的启动子调控HCV核心抗原表达可增强重组鼠伤寒沙门氏菌的免疫原性

为提高抗原表达质粒在重组伤寒沙门氏菌中的稳定性以增强重组伤寒沙门氏菌诱导的免疫应答 ,克隆鼠伤寒沙门氏菌pagC基因启动子 ,以其为转录调控元件构建HCV核心抗原表达质粒 ,转化到减毒鼠伤寒沙门氏菌中。体外培养时 ,Mg2 能够剂量依赖性抑制该重组菌表达HCV核心抗原。将该重组菌和组成性表达的重组菌分别口服接种BALB/c小鼠 ,观察质粒的稳定性和小鼠的免疫应答。结果表明 ,体内激活的pagC基因启动子能明显提高质粒在重组鼠伤寒沙门氏菌中的稳定性和增强重组菌诱导的体液和细胞免疫应答 ,这为发展高效免疫、成本低廉的口服丙肝疫苗提供了一个新思路     

Multiple oral inoculations with Cryptosporidium parvum as a means of immunization for production of monoclonal antibodies

Abstract Oral immunization of suckling mice with Cryptosporidium parvum results in a humoral response to a limited set of antigens. Six-day-old BALB/c mice were each inoculated orally with 1 × 10⁶ viable oocysts and subsequently administered oral inoculations of 2 × 10⁶ viable oocysts at 30 and 60 days following the primary infection. After 45 days, mice were boosted with 1 × 10⁶ oocysts orally, plus soluble extracts equivalent to 2 × 10⁶ and 1 × 10⁶ oocysts given intravenously and intraperitoneally, respectively. Four days later, splenic lymphocytes were fused to Ag8 myeloma cells. Using this method, we have been able to select for monoclonal antibodies that predominately recognize sporozoite surface and apical complex antigens.