SARS冠状病毒S基因重组DNA诱导的体液免疫反应

目的 以严重急性呼吸综合征冠状病毒（SARS-CoV）密码子优化的S、S1和S2基因分别构建其真核表达质粒，免疫BALB／c小鼠，以初步评价其诱导特异性体液免疫的效果。方法将人工合成密码子优化的S、S1和S2基因克隆入pcDNA4／HisMax-TOPO表达载体，重组质粒转染293T细胞，Western blot和免疫组化检测其真核表达，重组质粒免疫BALB／c小鼠，酶联免疫（ELISA）检测抗S蛋白抗体，伪病毒中和试验、细胞融合抑制试验检测中和抗体。结果3种重组质粒均可在真核细胞中获得表达，免疫小鼠后可诱导针对S蛋白的特异性抗体，抗体在12周观察期内呈持续上升趋势；其中，仅S和S1蛋白重组质粒能够诱导中和抗体的产生，以S蛋白的效价为高。结论密码子优化S和S1蛋白重组质粒可以有效诱导BALB／c小鼠产生中和抗体，其抗体可能具有阻断SARS-CoV侵袭易感细胞的能力。该结果为进一步研究SARS-CoV DNA疫苗提供了参考依据。     

DNA mediated immunization with encoding the nucleoprotein gene of porcine transmissible gastroenteritis virus

The immune response to a naked plasmid DNA encoding the nucleoprotein (N protein) of porcine transmissible gastroenteritis virus (TGEV) was investigated in this study. A complementary DNA of the entire N gene was amplified by RT-PCR, and inserted into a mammalian expression vector (pcDNA3.1) to construct a recombinant plasmid (pcDNA/N). To evaluate the immunogenicity of the construct, BALB/c mice were intramuscularly immunized with different doses (50, 100 and 200 μg/mouse) of pcDNA/N twice at a 5-week interval. An optimal antibody response was achieved with 100 μg of pcDNA/N. The response lasted at least 11 weeks after primary immunization. By western blotting analysis, the antibodies specifically recognized a 47 kDa protein corresponding to the viral N protein, but they did not reveal neutralizing activity against infectious TGEV in vitro. Immunoglobulin G2a was predominant among these antibodies, which was indicative of Th1 type cell activation in pcDNA/N immunized mice. Moreover, spleen cells from these mice showed stronger immune responses than those from live vaccine or parental vector immunized mice. These results suggest that the construct can elicit both humoral and cell-mediated immune (CMI) responses against TGEV N protein in mice.     

Tumours can act as adjuvants for humoral immunity

Tumour cells transfected with cDNAs encoding non-self proteins were used to investigate the ability of the immune system to respond to immunogenic antigens expressed by tumours. Secreted, intracellular and surface proteins were used as model antigens, as these reflect the potential forms of tumour antigens. Syngeneic BALB/c mice injected with viable line 1 lung carcinoma or EMT6 mammary tumour cells secreting ovalbumin (OVA) or prostate-specific antigen (PSA) produced very high immunoglobulin G (IgG) antibody titres, equivalent to those of mice injected with protein in Freund's complete adjuvant (FCA). Secretion of the antigens was not necessary as tumour cells expressing a cell-surface antigen (HER-2/Neu) or an intracellular antigen - green fluorescence protein (GFP) - also generated high-titre antigen-specific IgG antibodies. In interleukin-4 (IL-4)-deficient mice, both IgG1 and IgG2a were produced in response to OVA administered in FCA, whereas in response to tumour-produced antigen, the antibodies switched from predominantly IgG1 to IgG2a, indicating that the mechanisms responsible for antibody induction differed between these forms of immunization. In contrast to the line 1 and EMT6 tumours, which are of BALB/c origin, OVA- or PSA-producing B16 melanoma cells, which are of C57BL/6 origin, failed to elicit antibody production. This was not the result of strain differences, as a similar finding was observed when the tumours were grown in (BALB/c x C57BL/6)F1 mice, but appeared to be caused by intrinsic differences in the tumours. Furthermore, co-injection of both B16/OVA and line 1 tumours resulted in production of anti-OVA antibody, indicating that B16 tumours were not immunosuppressive, but instead line 1 tumours appear to exert an adjuvant effect.     

Transmissible gastroenteritis virus (TGEV)-based vectors with engineered murine tropism express the rotavirus VP7 protein and immunize mice against rotavirus

A coronavirus vector based on the genome of the porcine transmissible gastroenteritis virus (TGEV) expressing the rotavirus VP7 protein was constructed to immunize and protect against rotavirus infections in a murine model. The tropism of this TGEV-derived vector was modified by replacing the spike S protein with the homologous protein from mouse hepatitis virus (MHV). The rotavirus gene encoding the VP7 protein was cloned into the coronavirus cDNA. BALB/c and STAT1-deficient mice were inoculated with the recombinant viral vector rTGEV_S-MHV-VP7, which replicates in the intestine and spreads to other organs such as liver, spleen and lungs. TGEV-specific antibodies were detected in all the inoculated BALB/c mice, while rotavirus-specific antibodies were found only after immunization by the intraperitoneal route. Partial protection against rotavirus-induced diarrhea was achieved in suckling BALB/c mice born to dams immunized with the recombinant virus expressing VP7 when they were orally challenged with the homotypic rotavirus strain.     

含有丙型肝炎病毒核心基因表达质粒的构建及其基因免疫

目的：研究丙型肝炎病毒（ＨＣＶ）核心（Ｃ）基因免疫诱生特异性免疫应答的可行性。方法：将ＨＣＶ Ｃ基因片段插入真核表达载体ｐｃＤＮＡ３质粒ＣＭＶ启动子的下游,构建真核表达载体ｐｃＤＮＡＨＣＶ－Ｃ,分别转染小鼠骨髓瘤细胞ＳＰ２／０和人肝癌细胞７７２１进行瞬时表达,用免疫荧光法和Ｗｅｓｔｅｒｎ－ｂｌｏｔ检测表达产物,将重组质粒注射,ＢＡＬＢ／ｃ（Ｈ－２＾ｄ）小鼠股四头肌,ＥＬＩＳＡ法检测血清中抗体产生水     

Fine specificity of the murine immune response to SV40 large tumour antigen utilizing synthetic peptides that define selected epitopes.

Baculovirus-derived recombinant simian virus 40 large tumour antigen (SV40 T-ag) was used to immunize BALB/c, C57Bl/6 and CB6/F1 mice and their anti-SV40 T-ag antibody responses were examined for the ability to bind synthetic peptides representing six predicted B cell epitopes on SV40 T-ag. In C57Bl/6 mice, anti-SV40 T-ag antibodies failed to bind any of the six SV40 T-ag peptides. However, the antibody responses induced in both BALB/c and CB6/F1 mice recognized synthetic peptides corresponding to two distinct epitopes (amino acids 690-708 and 660-679, respectively) associated with the carboxyl-terminal half of SV40 T-ag. In addition, murine MoAbs (BALB/c) generated to native SV40 T-ag, and previously characterized as recognizing the carboxyl-terminus of SV40 T-ag by deletion mutant analysis, also bound the synthetic peptide (residues 690-708) defining the carboxyl-terminus of SV40 T-ag. These data indicate that the antibody responses induced in BALB/c and CB6/F1 mice by immunization with baculovirus-derived recombinant SV40 T-ag are capable of recognizing sequential carboxyl-terminal epitopes on SV40 T-ag defined by peptides 690-708 and 660-679, respectively. No statistically significant differences in anti-SV40 T-ag antibody titres were observed between the three inbred mouse strains. These data suggested that the fine specificities of the anti-SV40 T-ag responses as assessed by synthetic peptide binding were different in the three inbred strains of mice examined. Finally, in vivo tumour challenge studies comparing recombinant SV40 T-ag with the two carboxyl-terminus peptide epitopes indicated that some tumour immunity was induced in BALB/c, but not CB6/F1 mice, by immunization with peptide 690-708 conjugated to a carrier protein. These studies suggest that the carboxyl-terminal region of SV40 T-ag represents a continuous sequential epitope involved in both the antibody response to SV40 T-ag and tumour immunity in BALB/c mice.     

Characterization of the murine immune response to type 6 pneumococcal polysaccharide.

The antibody response to type 6 (Danish type 6A) pneumococcal capsular polysaccharide (S6) was determined in various strains of mice. S6 elicited a very low plaque-forming cell (PFC) response in most strains of mice. BALB/c mice responded better than most other strains, and the response to S6 was further characterized in BALB/c mice. Immunization with 1 to 5 micrograms of S6 induced a plaque-forming cell response of short duration which consisted totally of immunoglobulin M-producing PFC. Athymic nude mice produced three- to fivefold more PFC in response to S6 than their euthymic littermates, suggesting that thymus-derived (T) cells negatively influence the S6 response. Regulation of the response by T cells was further suggested by the ability of antilymphocyte serum to enhance the S6 PFC response and by the suppressed response induced by priming with a low dose of S6 3 days before immunization with an optimal dose of S6 (low dose paralysis). When concanavalin A was given 2 days after immunization with S6, the response was enhanced two- to sevenfold, suggesting that the response is also positively influenced by T amplifier cells. When the avidity of the antibody produced by S6-specific PFC was measured by a plaque inhibition test, the avidity of the anti-S6 antibody was found to be very low. These results suggest that S6 is a poor immunogen because the affinity of the S6-specific antibody and B cell surface receptors is low.     

Autologous anti-idiotypic antibody response is regulated by the level of circulating complementary idiotype.

BALB/c mice injected with lyophilized vaccine from Streptococcus pneumoniae R36a (Pn) predominantly responded with antibody molecules the vast majority of which expressed the public idiotype T15 and were directed to the immunodominant epitope phosphorylcholine (PC). However, after a single immunization with Pn vaccine young (3-month-old) BALB/c mice did not produce any specific anti-T15 antibody response. In contrast, young D1.LP mice were able to mount a specific anti-T15 response upon primary immunization with pneumococcal vaccine. The anti-PC response in the two mouse strains differed in that the proportion of antibody molecules that expressed the T15 idiotype for Pn-primed D1.LP mice showed a smaller proportion of PC-specific antibody expressing the T15 idiotype. Neonatal injection of anti-T15 monoclonal antibodies led to a long-term suppression of the PC-specific T15+ B-cell clones but at young/adult age these mice maintained the ability to produce a normal amount of PC-specific antibody. Interestingly, the idiotypically-suppressed BALB/c mice mounted a significant anti-T15 response during the primary response to Pn. We interpreted these data as showing that the level of circulating idiotype may regulate the production of the complementary anti-idiotypic antibody. In addition, in vitro experiments demonstrated that the lack of the anti-T15 response during primary antibody response in BALB/c mice is probably because of a state of tolerance that is regulated by T cells.     

酵母表达的戊型肝炎病毒结构区ORF2蛋白的免疫原性研究

目的 研究巴氏毕赤酵母表达系统表达的戊型肝炎病毒（Hepatitis Evirus,HEV)结构区ORF2蛋白的免疫原性。方法 将重组蛋白免疫BALB/c小鼠。首先通过亲和捕获反转录PCR鉴定免疫小鼠抗血清是否能结合感染HEV恒河猴粪便及胆汁中的病毒颗粒,其次将重组蛋白分别以不同免疫途径,不同佐剂免疫小鼠,通过检测小鼠血清中抗-HEVORF2抗体阳转率及抗体滴度,观察其免疫原性。结果 亲和捕获反转录PCR阳性,表明免疫鼠抗血清可以捕获感染恒河猴粪便及胆汁样品中的HEV。在小鼠免疫实验中,肌内免疫优于腹腔免疫,抗原联合铝佐剂及CpG佐剂及CpG佐剂优于抗原联合铝佐剂,以抗原联合铝佐剂和CpG佐剂经股四头肌免疫小鼠,4周后加强一次的免疫效果最佳,ED500为0.023μg,实验表明,巴氏毕赤酵母表达的重组HEVORF2蛋白刺激小鼠产生的抗体,不仅可以特异性结合天然HEV,而且该蛋白在小鼠体内可以有效地诱发体液免疫反应。结论 巴氏毕赤酵母表达系统表达的HEVORF2蛋白具有良好的免疫原性,这为新型戊型肝炎疫苗的研制奠定了基础。     

Studies using a recombinant vaccinia virus expressing the circumsporozoite protein of Plasmodium berghei.

A recombinant vaccinia virus was constructed which expressed the circumsporozoite protein of Plasmodium berghei. Four different strains of mice belonging to different haplotypes were immunized with the recombinant virus. The antibody response to the circumsporozoite protein as well as to vaccinia virus varied among the strains, independently of each other. The anti-circumsporozoite protein titers were comparable to that obtained on immunization with irradiated sporozoites. Spleen cells from H2d mice immunized with P. berghei sporozoites showed a significant proliferative response when cultured in vitro with a low multiplicity of the recombinant vaccinia virus. A weak cytotoxic T lymphocyte response specifically targeting the circumsporozoite protein could be identified in spleens of BALB/c (H2d) mice immunized with vaccinia virus when BALB 3T3 cells transformed with a plasmid expressing the circumsporozoite protein under control of the simian virus 40 promoter were used as target cells in the cytotoxic T lymphocyte assay. However, none of the recombinant virus-immunized animals could be protected from a challenge of sporozoites even at the lowest dose of parasite used.     

两株登革2型病毒NS1基因真核重组质粒免疫BALB/c小鼠产生特异性抗体水平的比较

目的 利用登革2型病毒(dengue type 2 virus,DENV2)M株和NGC株NS1基因部分扇列PcDNA3.1重组质粒初次和加强免疫BALB/c小鼠,观察免疫小鼠体液免疫应答的差异.方法 分别构建两株DENV2 NS1基因部分序列(1-413 bp)的PcDNA3.1真核重组质粒和pET28a(+)质粒,进行原核蛋白的表达、鉴定、纯化和定量;并用pcDNA3.1重组质粒免疫BALB/c小鼠,初次免疫及第7天、14天分别加强免疫1次,共免疫3次.收集初次免疫后第7、14、28和56天外周血标本,间接ELISA法测定小鼠血清特异性IgM/IgG类抗体水平,细胞病变抑制法检测特异保护性抗体水平.结果 构建了pET28a(+)-NS1m/pET28a(+)-NS1n原核表达重组质粒,SDS-PAGE分析表明,NS1基因部分序列获得表达,其相对分子质量均约22.3×103;Western blot表明该目的 蛋白可与抗His标签单克隆抗体结合;经Ni柱亲和层析法得到纯度达92%的表达蛋白,对C6/36细胞有毒性,并可用于ELASA检测.不同DENV2毒株NS1基因部分序列的pcDNA3.1重组质粒初次和加强免疫BALB/c小鼠诱导特异性IgM、IgG类和中和抗体的产生存在差异,M株重组质粒加强免疫小鼠后特异性抗体效价水平较高并持续较长时间.结论 DENV2两毒株NS1基因部分序列重组质粒免疫小鼠后诱生的特异性抗体类别、水平存在差异.

Abstract：

Objective To compare the humoral immune response of BALB/c mice immunized by recombinant plasmids PeDNA3.1-M-NS1 and pcDNA3.1-N-NS1.Methods Dengue type 2 virus(DENV2)NS1 gene were constructed two partial sequences(1-413 bp)of the pcDNA3.1 eukaryotic plasmids and pET28a(+)plasmid for prokaryotic expression,identification,purification and quantification.The BALB/c mice were immunized by pcDNA3.1-M-NS1,pcDNA3.1-N-NS1 recombinant plasmids with adjuvant.Each animal received a primary inoculation and two boosts at 1-week intervals.Then the blood samples of BALB/c mice were collected from different experiment groups at day 7,14,28 and 56,respectively after first immunization.The specific IgM/IgG antibodies for NS1 protein in serum were confirmed by indirect ELISA.And then the activities of the specific protective antibody were determined by cytopathic effect inhibition(CPEI).Results Construction of the pET28a(+)-NS1 m/pET28a(+)-NS1n prokaryotic expression plasmid,SDS-PAGE analysis showed that,NS1 gene partial sequence was expressed,both the relative molecular weight of about 22.3×103:Western blot showed that the protein can bind anti-His tag monoclonal antibody;byNi affinity chromatographywith apurity of 92% protein,on the C6/36 cell toxicity,and can be used ELASA detection.The results showed that the levels of specific IgM/IgG antibody and neutralizing antibody activities were increased in pcDNA3.1-M-NS1 booster immunization group than other groups.The result had been observed longer duration of antibody level in peDNA3.1-M-NS1 booster immunization group.Conclusion Humoral immune response were significantly different between pcDNA3.1-M-NS1 and pcDNA3.1-N-NS1 recombinant plasmid immunized mice groups.     

柯萨奇病毒B3 VP1蛋白、rAd/VP1和pcDNA3/VP1的免疫效果比较

目的 观察柯萨奇病毒B3(Coxsackievirus B3,CVB3)衣壳蛋白VP1、表达VP1蛋白的重组腺病毒rAd/VP1和重组质粒pcDNA3/VP1的免疫效果.方法 用原核细胞表达VP1蛋白并纯化、扩增重组腺病毒rAd/VP1,扩增并提取真核表达质粒pcDNA3/VP1.BALB/c小鼠随机分为4组,每组18只,分别在股四头肌注射VP1蛋白、rAd/VP1、pcDNA3/VP1和PBS.VP1蛋白组和pcDNA3/VP1组免疫3次,间隔3周;rAd/VP1组免疫2次,间隔2周.VP1蛋白、pcDNA3/VP1和rAd/VP1每次每只注射剂量分别为50μg、100μg和1.2×107PFU.用ELISA法和微量中和试验法检测各次免疫后血清CVB3特异性IgG抗体和中和抗体滴度;末次免疫后3周,CCK-8法检测脾脏淋巴细胞的CTL杀伤活性;用致死量CVB3攻击小鼠后,检测血中病毒滴度并观察动物的存活情况.结果 VP1蛋白组血清特异性IgG抗体和中和抗体滴度明显高于其他实验组(P＜0.05),而脾脏淋巴细胞CTL杀伤活性低于rAd/VP1组(P＜0.05);致死量病毒攻击后,VP1蛋白组血中病毒滴度低于pcDNA3/VP1和rAd/VP1组(P＜0.05),生存率明显高于这两组(P＜0.05).结论 VP1蛋白疫苗能诱导较高水平的体液免疫应答,对动物有明显的免疫保护作用,免疫效果优于质粒pcDNA3/VP1和重组腺病毒rAd/VP1.

Abstract：

Objective To compare the immune effects of Coxsackievirus B3 (CVB3) capsid protein VP1 expressed bacterially, recombinant adenovirus rAd/VP1 and recombinant plasmid pcDNA3/VP1which express VP1 protein in mice. Methods After expressed in prokaryotic cells, VP1 protein was purified. Recombinant adenovirus rAd/VP1 and recombinant plasmid pcDNA3/VP1 were amplified and extracted. Six to 8-week-old, male BALB/c mice were divided into four groups randomly. Each group contained 18 mice. The mice of pcDNA3/VP1 group or VP1 protein group were immunized intramuscularly with three injections at three weeks apart, of recombinant plasmid pcDNA3/VP1 at a dose of 100 μg/mouse or recombinant protein VP1 at a dose of 50 μg/mouse. The mice of rAd/VP1 group were immunized intramuscularly twice at two weeks interval with rAd/VP1 at a dose of 1.2 × 107 PFU. The control group was mock-immunized with 100 μl of PBS intramuscularly. Mice were bled from the retroorbital sinus plexus every two weeks after each immunization. ELISA and micro-neutralization test were used to detect levels of CVB3-specific IgG antibody and neutralizing antibody titers in the sera of immunized mice. Three weeks after the last immunization, the cytotoxic T lymphocyte(CTL) killing activity of spleen lymphocytes was detected with CCK-8 assay. Subsequently, virus titers in the sera of immunized mice were determined by the 50% cell culture infective dose( CCID50 ) assay on HeLa cell monolayers and percentage of animals surviving were observed after lethal CVB3 attack over a period of 21 days. Results The titers of specific IgG antibody and neutralizing antibody in sera of VP1 protein immunized mice were higher than other groups( P ＜0.05 ). While CTL killing activity of spleen lymphocytes of VP1 protein immunized mice was lower than mice in rAd/VP1 group( P ＜0. 05). Virus titers in sera of VP1 protein immunized mice were lower than the mice in pcDNA3/VP1 or rAd/VP1 groups ( P ＜ 0.05 ), while survival rate was significantly higher than these two groups ( P ＜ 0.05 ).Conclusion VP1 protein induced higher level of humoral immune response and acquired obvious immune protection effects in mice. The immunizing potency of VP1 protein vaccine surpassed plasmid pcDNA3/VP1or recombinant adenovirus rAd/VP1. It appeared to be a promising candidate among the three different vaccines.     

Murine immune responses to liver-stage antigen 1 protein FMP011, a malaria vaccine candidate, delivered with adjuvant AS01B or AS02A

Liver-stage antigen 1 (LSA1) is expressed by Plasmodium falciparum only during the intrahepatic cell stage of the parasite's development. Immunoepidemiological studies in regions where malaria is endemic suggested an association between the level of LSA1-specific humoral and cell-mediated immune responses and susceptibility to clinical malaria. A recombinant LSA1 protein, FMP011, has been manufactured as a preerythrocytic vaccine to induce an immune response that would have the effect of controlling parasitemia and disease in humans. To evaluate the immunogenicity of FMP011, we analyzed the immune response of three inbred strains of mice to antigen immunization using two different adjuvant formulations, AS01B and AS02A. We report here the ability of BALB/c and A/J mice, but not C57BL/6J mice, to mount FMP011-specific humoral (antibody titer) and cellular (gamma interferon [IFN-gamma] production) responses following immunization with FMP011 formulated in AS01B or AS02A. Immunization of BALB/c and A/J mice with FMP011/AS01B induced more antigen-specific IFN-gamma-producing splenocytes than immunization with FMP011/AS02A. A slightly higher titer of antibody was induced using AS02A than AS01B in both strains. C57BL/6J mice did not respond with any detectable FMP011-specific IFN-gamma splenocytes or antibody when immunized with FMP011 in AS01B or AS02A. Intracellular staining of cells isolated from FMP011/AS01B-immunized BALB/c mice indicated that CD4(+) cells, but not CD8(+) cells, were the main IFN-gamma-producing splenocyte. However, inclusion of blocking anti-CD4(+) antibody during the in vitro restimulation ELISpot analysis failed to completely abolish IFN-gamma production, indicating that while CD4(+) T cells were the major source of IFN-gamma, other cell types also were involved.     

Biological and serological comparison of syngeneic and allogeneic anti-idiotypic antibodies

The majority of BALB/c antibodies with specificity for the (1→3)-glucosidic linkage of dextran B1355, fraction S (-dextran), share an idiotype crossreactive with the -dextran-binding BALB/c myeloma proteins J558 and MOPC104E. This crossreactive idiotype (IdX558) is denned by an allogeneic antiidiotypic antiserum raised in mice of strain A/HeJ against J558 myeloma protein (Carson & Weigert, 1973). Allogeneic anti-IdX558 antisera induce a long lasting complete suppression of the total anti--dextran immune response when given neonatally to BALB/c mice. Mice which recover from suppression fail to express the IdX558. In contrast, when BALB/c mice are immunized by use of J558 myeloma protein, no measurable effect on the subsequent total anti--dextran immune response or on the expression of IdX558 is found in mice making anti-J558 themselves nor in those mice which received the syngeneic anti-J558 idiotype antibody neonatally. In addition, no significant alteration of the anti--dextran response can be detected in BALB/c mice which were immunized against MOPC104E protein prior to -dextran immunization. This different suppressive capacity may be explained by different specificities of the allogeneic and syngeneic antiidiotype antisera. Hemagglutination and isoelectric focusing studies of the antisera show that BALB/c mice fail to produce antibodies against the crossreactive idiotype whereas in the allogeneic situation the anti-IdX558 is the overwhelming one. Syngeneic anti-idiotypic antibodies react only with the corresponding myeloma protein used for immunization, thus defining individual idiotypes (IdI558 and IdI104). IdI558 and IdI104 are expressed by every BALB/c mouse in response to -dextran but as a minor component of their idiotypic spectrum.     

Various factors (allergen nature,mouse strain,CpG/recombinant protein expressed) influence the immune response elicited by genetic immunization

BACKGROUND: Genetic immunization is a very promising therapeutic approach for allergy treatment. In the present study we investigate the influence of the nature of the allergen, the mouse strain, and the relative amount of CpG to expressed recombinant protein on immune responses using two major peanut allergens, Ara h 1 and Ara h 4. METHODS: The cDNA of Ara h 1 and of an isoform of Ara h 4 were cloned and inserted in pcDNA3. Antigen specific IgG1, IgG2a and IgE were followed after genetic immunization with 100 microg of these clones in mouse strain SKH-Hr1 or BALB/c and with 1 microg of the clones+99 blank plasmid in SKH-Hr1. RESULTS: Genetic immunization in SKH-Hr1 with Ara h 1 elicited a classical Th1 type response, but Ara h 4 elicited a mixed Th1/Th2 response with high IgG1 and even IgE in some mice. In BALB/c both plasmids produced a high IgG1 level. Decreasing the amount of plasmid injected did not change the immune response profile. However, increasing the amount of CpG administered relative to the recombinant Ara h 4 protein expressed reversed the Th1/Th2 response pattern in SKH-Hr1 mice. CONCLUSIONS: Immune responses after genetic immunization are strongly influenced by the nature of the allergen, the mouse strain, and the ratio of CpG to recombinant protein expressed.     

Identification and characterization of a mouse mammary tumor virus protein uniquely expressed on the surface of BALB/cV mammary tumor cells

A unique subline of BALB/c mice, designated BALB/cV, exhibits an intermediate mammary tumor incidence (47%) and harbors a distinct milk-transmitted mouse mammary tumor virus (MMTV). The BALB/cV subline was used to study the molecular basis of potential virus-host interactions involving cell surface-expressed MMTV proteins. Cell surface iodination identified virus-specific proteins expressed on BALB/cV primary mammary tumor cells grown in culture. In contrast to (C3H)MMTV-producing cell lines which expressed MMTV gp52, BALB/cV tumor cells lacked gp52 and expressed instead a 68K, env-related protein. The 68Kenv protein was also detected on the surface of metabolically labeled BALB/cV tumor cells by an external immunoprecipitation technique. The expression of 68Kenv was restricted to mammary tissues of BALB/cV mice that also expressed other MMTV proteins. Biochemical analysis established that 68Kenv was not modified by N-linked glycosylation. 125I-labeled 68Kenv was rapidly released into the media of tumor cell cultures and was recovered both in the form of a soluble protein and in a 100,000 g pellet. The biologic function of this cell surface-expressed viral protein remains unknown.     

小鼠IL-1α多克隆抗体的制备及应用

目的:构建小鼠白细胞介素-1α原核表达载体,表达并纯化IL-1α蛋白,制备兔抗鼠IL-1α多克隆抗体,并对抗体特性进行初步的鉴定。方法:利用RT-PCR技术,从BALB/c小鼠脾脏cDNA中扩增出IL-1α的全长基因,酶切后连接至pET32a(+)原核表达载体,重组载体测序正确后转化至BL21(DE3)大肠杆菌。利用蛋白质原核表达自动诱导方案成功表达重组蛋白。重组蛋白经电洗脱纯化后用以免疫新西兰大白兔,获得了抗小鼠IL-1α的多克隆抗体,ELISA检测抗体效价。Western blot和流式细胞术(FCM)检测抗血清的特异性。结果:成功构建了重组表达载体pET32a(+)-IL-1α,表达的重组蛋白纯化后免疫新西兰大白兔,得到的多克隆抗体ELISA显示抗体效价可达1∶25 600。Western blot和FCM分析该抗体能特异性结合IL-1α。结论:利用重组的IL-1α蛋白成功制备了高效价、高特异性的兔抗IL-1α抗体,为进一步研究IL-1α的生物学功能奠定了基础。     

HIV DNA疫苗与重组腺病毒伴随病毒联合免疫效果的研究

目的 构建含HIV-1B亚型中国株gagV3基因的DNA疫苗及重组腺病毒伴随病毒(rAAV)疫苗，并研究DNA疫苗和rAAV联合免疫的免疫效果。方法 将HIV-1B亚型中国株gagV3基因克隆入真核表达载体pCI-neo上，构建了含HIV-1 gagV3基因的DNA疫苗pCI-gagV3。采用电击法将pCI-gagV3质粒转染p815细胞，用G418压力筛选，得到转入重组质粒的细胞系p815-gagV3，用免疫酶法检测细胞系中HIV-1基因的表达。用该DNA疫苗进行小鼠免疫实验，检测免疫效果；用该DNA疫苗初次免疫，含同样gagV3基因的重组腺病毒伴随病毒rAAV-gagV3加强免疫，采用免疫酶法检测免疫小鼠血清中HIV-1特异性的抗体水平，用乳酸脱氢酶法检测免疫小鼠的HIV-1特异性CTL水平。结果 pCI-gagV3可以在p815细胞中表达HIV-1的基因，免疫BALB／c小鼠后可以在小鼠体内诱发HIV-1特异性的细胞和体液免疫反应。HIV-1特异性抗体滴度为1：20；效靶比为50：1时，CTL平均杀伤率为41．7％。pCI-gagV3与rAAV-gagV3联合免疫并不能明显提高抗体水平，但可以提高CTL反应，效靶比为50：1时，CTL平均杀伤率为61.3％，高于单独用DNA疫苗或重组AAV疫苗免疫后产生的CTL活性。结论 DNA疫苗与重组腺病毒伴随病毒联合免疫可以提高免疫小鼠产生的HIV-1特异性CTL反应。     

Oral immunization of mice with recombinant <Emphasis Type="Italic">Lactococcus lactis</Emphasis> expressing porcine transmissible gastroenteritis virus spike glycoprotein

Lactococcus lactis NZ9000 was selected as an antigen delivery vehicle for mucosal immunization against porcine transmissible gastroenteritis virus (TGEV) infection. An approximately 70 kDa fragment of the N-terminal globular domain of the spike (S) protein (S_N protein) from the coronavirus TGEV was used as the transmissible gastroenteritis virus antigen model. Recombinant L. lactis, expressing the S_N protein, was constructed with the pNZ8112 plasmid. Expression and localization of the transcribed S_N protein from the recombinant L_NZ9000-rTGEV-S_N were detected via SDS-PAGE, Western blot, and immunofluorescence. BALB/c mice, orally immunized with L_NZ9000-rTGEV-S_N, produced local mucosal immune responses against TGEV. The induced antibodies demonstrated neutralizing effects on TGEV infection. These data indicated that the recombinant L. lactis could be a valuable tool in the development of future vaccines against TGEV.     

Relevance of differential immunogenicity of human and mouse recombinant desmoglein-3 for the induction of acantholytic autoantibodies in mice

Pemphigus vulgaris (PV) is an antibody-mediated autoimmune disease of the skin and mucous membranes. Desmoglein-3 (dsg-3) expressed in the suprabasal layer of the skin serves as an autoantigen in PV. Passive transfer of sera, either from patients with PV or from experimental animals immunized with a recombinant human dsg3 (hdsg3) into neonatal BALB/c mice results in blister formation, suggesting strongly that there is significant cross-reactivity between the mouse dsg3 (mdsg3) and the hdsg3. However, efforts to induce disease in adult mice through active immunization using hdsg-3 have not been successful, suggesting that the epitopes required for the induction of pathogenic antibodies in adult mice might not be present in hdsg3. Therefore, in this study, we expressed a full-length mdsg3 in insect cells and compared its serological reactivity with that of the hdsg3 using species specific polyclonal sera and a panel of seven monoclonal antibodies (MoAbs) with unique binding specificities to hdsg3. Studies using sera demonstrated a considerable cross-reactivity, while studies using MoAbs exhibited specific epitope differences between the two proteins. Because of these differences, we reasoned that immunization with mdsg3 might induce disease in adult mice. Immunization of four strains of mice (i.e. BALB/c, DBA/1, HRS/J and SJL/J) with mdsg3 resulted in considerable antibody response, but failed to induce lesions. However, sera from immunized BALB/c mice induced acantholysis of neonatal mouse skin in vitro. These studies indicated that our inability to induce lesions in adult mice through active immunization is not due to differences in the ability of mouse and human dsg3 to induce acantholytic antibodies, but due probably to structural differences between adult and neonatal mouse skin. Alternatively, immunization with a combination of dsg3 protein along with other proteins might be necessary to induce pemphigus disease in adult mice. Nevertheless, our current studies show that molecular mechanisms leading to the production of acantholytic antibodies in mice can now be studied using homologous mdsg3.