丙型肝炎病毒基因重组体免疫诱发小鼠产生抗体的研究

以丙型肝炎病毒（ＨＣＶ）基因免疫的动物实验方法,将构建的ＨＣＶ基因重组体（ｐＣＤ ＨＣＶ１）,采用不同方法免疫ＢＡＬＢ／ｃ小鼠,观察其诱发小鼠抗体水平变化。结果：ｐＣＤ ＨＣＶ１经肌肉注射３次免疫小鼠后,比１、２次免疫抗体水平要高;经灌胃、腹腔注射、皮下注射和肌肉注射不同途径免疫小鼠,以肌肉注射免疫效果最佳;不同剂量（１０μｇ、５０μｇ和１００μｇ）３次免疫小鼠,以１００μｇ剂量组诱发小鼠抗体反应最强;用普鲁卡因处理小鼠后再肌肉、皮下注射同剂量ｐＣＤ ＨＣＶ１,抗体水平均比直接注射同剂量ｐＣＤ ＨＣＶ１明显增高。     

环磷酰胺免疫删减法制备细胞表面抗原单克隆抗体小鼠模型的建立

目的:观察不同剂量环磷酰胺(CP)诱导免疫耐受的作用, 建立一个免疫删减法制备细胞表面抗原单克隆抗体(mAb)的动物免疫模型.方法:雌性 BALB/c小鼠经CHO细胞免疫后分别给予不同剂量的CP诱导免疫耐受, 选择血清抗体效价最低的一组小鼠按常规方式腹腔注射CHO-TM5细胞进行免疫.ELISA测定各小鼠血清中CHO抗体及CHO-TM5抗体效价, 检测血清抗体对CHO细胞与CHO-TM5细胞结合反应的差异性.结果:BALB/c小鼠腹腔注射不同剂量的CP后, 各剂量组均出现不同程度的免疫耐受作用.CP 100 mg/kg组与CP 125 mg/kg组小鼠血清抗体效价较低, 接近阴性对照组.ELISA检测显示CP 100 mg/kg组血清抗体与CHO-TM5细胞呈阳性结合反应, 不与CHO细胞结合;而CP 0 mg/kg组血清抗体与CHO细胞与CHO-TM5细胞的结合反应均呈阳性.结论:CP 100 mg/kg具有良好诱导小鼠对CHO和CHO-TM5细胞的共同抗原表位产生免疫耐受, 相对增强对CHO-TM5细胞目的抗原hTM的免疫应答的作用, 为免疫删减法制备细胞表面抗原mAb提供了一个理想的动物免疫模型.     

肺炎链球菌重组自溶酶(Re-LytA)滴鼻免疫小鼠的实验研究

目的 评价重组肺炎链球菌(Streptococcus pneumoniae,Sp)自溶酶(autolysin,LytA)滴鼻免疫诱导小鼠抗Sp感染的保护性效果.方法 用亲和层析的方法纯化出重组Sp LytA蛋白,以CpG作佐剂,对小鼠进行滴鼻免疫(A组).对照组(B组)用无菌盐水滴鼻;多糖疫苗组(C组)用23价多糖疫苗肌肉注射.于末次免疫后2周,用Sp分别以滴鼻和腹腔注射两种方式进行攻击感染.攻击之前采集血清和鼻咽灌洗液等样品,采用ELISA测试其抗体水平.滴鼻攻击后4 d,采集小鼠鼻咽灌洗液和肺灌洗液,进行Sp细菌计数;腹腔注射攻击后7 d内观察各组小鼠死亡情况.结果 B组未检测到LytA抗体和多糖抗体,C组多糖抗体阳性;A组LytA抗体(包括tgG、IgA、sIgA)均明显高于对照组,差异具有统计学意义(P<0.05).腹腔注射攻击后,A、C组小鼠的存活率明显高于B组(P<0.05),A组和C组之间没有明显区别(P>0.05).滴鼻攻击后,A组鼻咽部灌洗液Sp细菌计数明显低于B组和C组(P<0.05).结论 Re-LytA滴鼻免疫可诱导实验小鼠产生一定的抵抗Sp感染的保护性免疫力,这种保护性的免疫力可能与sIgA相关.     

小鼠脾内一次免疫法制备抗人肝癌McAb

以人体肝癌细胞系SMMC-7721为免疫原,3×10~5细胞小鼠脾内一次注射后间接免疫荧光法检测血清抗体滴度远较同剂量腹腔内注射为高,而与1×10~7细胞腹腔注射相仿。 经脾内一次注射法免疫后88～90小时,取脾与小鼠骨髓瘤NS-1细胞系按常规方法融     

汉滩病毒S基因真核表达载体的构建及免疫小鼠的初步研究

目的研究汉滩病毒Ｓ片段编码区基因免疫小鼠的作用。方法利用基因重组技术,构建含汉滩病毒Ｓ片段编码区基因的真核表达质粒。用此质粒直接注射到ＢＡＬＢ／ｃ小鼠骨骼肌内进行ＤＮＡ免疫,用间接免疫荧光法检测免疫小鼠血清中汉滩病毒抗体。结果在初次免疫的小鼠血清中检测出有低滴度的汉滩病毒抗体。加强免疫后,抗体水平显著上升,免疫荧光抗体（ＩＦＡＴ）滴度最高达１６４０。约８７％的免疫动物发生了血清抗体阳转。抗体水平及小鼠血清抗体阳转率与注射的质粒ＤＮＡ剂量及注射次数有关。结论应用基因免疫技术能够诱导机体产生针对汉滩病毒的特异性免疫应答     

不同免疫途径对抗原肽修饰DC疫苗免疫效应的影响

目的探讨不同的免疫途径对抗原肽修饰树突状细胞(DC)疫苗免疫效果的影响。方法用鸡卵清蛋白(OVA)MHCⅠ限制性多肽SIINFEKL(OVAp257-264)包被小鼠骨髓来源的DC,通过皮下、腹腔、静脉或肌肉注射免疫小鼠,7天后行体内细胞毒性T淋巴细胞杀伤实验(InvivoCTL)分析CTL杀伤活性和细胞内IFN-γ染色(ICS)分析免疫小鼠脾脏CD8+细胞产生IFN-γ的情况。结果免疫7天后,InvivoCTL结果显示SIINFEKL修饰的DC皮下、腹腔、静脉或肌肉注射免疫小鼠其特异性CTL杀伤效应分别是37.3%±7.3%、61.0%±4.2%、56.9%±3.6%和10.8%±2.3%;ICS结果示四组小鼠产生IFN-γ的CD8+细胞占总CD8+细胞的比例分别是0.31%±0.07%、0.85%±0.12%、0.76%±0.14%和0.15%±0.04%。结论不同免疫途径可明显影响抗原肽修饰DC疫苗的免疫效应,其中腹腔注射诱发的抗原特异性CTL反应最强,静脉注射者次之,而皮下注射特别是肌肉注射较弱,提示通过腹腔注射DC疫苗可能是安全、高效的免疫途径。     

国产环硫雄醇的毒性、抑癌及对生殖影响的实验研究

国产环硫雄醇系由龙舌兰制麻废液中提取合成。小鼠腹腔注射环硫雄醇,急性毒性LD_(50)1469mg/kg。大鼠亚急性毒性,高剂量(100ms/kg)腹腔注射,子宫内膜明显增殖,卵泡成熟受抑制。300mg/kg腹腔注射,对小鼠乳腺癌有明显抑制。0.5mg/只肌肉注射,对小鼠有明显抗雌激素活性和抗着床作用。2.5mg/只肌肉注射,可增强大鼠子宫孕激素活性,组织化学显示子宫内膜上皮和腺细胞的β-葡萄糖苷酸酶、酸性磷酸酶和碱性磷酸酶反应减弱。     

PGE_2治疗鼠免疫复合物肾小球肾炎导致血清抗体和周围T淋巴细胞亚群的改变

每日给CBA雄性小鼠腹腔内注射脱铁铁蛋白4mg,诱导免疫复合物性肾小球肾炎.同时给小鼠注射不同剂量的前列腺素E_2(PGE_2),连续注射四周,然后处死小鼠,取抗凝血浆进行总免疫球蛋白和脱铁铁蛋白抗体的测定.用单克隆抗体染色检测T淋巴细胞亚群.取其肾脏用光学显微镜和免疫荧光进行检测.     

体内电穿孔在质粒介导的基因转移及DNA免疫中的应用

目的 研究体内电穿孔技术对于质粒DNA介导的体内基因表达以及HIV-1 Env DNA疫苗免疫效果的影响.方法 将携带荧光素酶Luciferase基因的表达质粒p1.0-Luc和携带HIV-1 CN54 env基因的DNA疫苗质粒p1.0-gp1455m通过单独肌肉注射或肌肉注射后加电穿孔两种不同方法 注射小鼠.用IVIS(R)活体成像系统实时检测Luciferase报告基因在体内的表达情况.用ELISA检测HIV-1 Env特异的抗体反应,用IFN-γ ELISPOT检测HIV-1 Env特异的T细胞免疫反应.结果 体内电穿孔技术可以显著提高Luciferase在小鼠体内的表达水平,幅度达35倍.HIV-1 Env DNA疫苗免疫结果 显示,8μg质粒剂量电穿孔途径诱导的体液和细胞免疫应答强于40μg质粒剂量单纯肌肉注射组;体内电穿孔途径免疫2次与单纯肌肉注射途径免疫3次诱导的体液和细胞免疫应答水平相当.结论 体内电穿孔技术可以大幅度提高报告基因在体内的表达水平和DNA疫苗的免疫应答.     

ＡＣＴＨ单克隆抗体的制备和鉴定

ＡＣＴＨ是垂体分泌的重要激素之一 ,测定血浆ＡＣＴＨ对下丘脑 垂体 肾上腺轴功能的评价具有重要意义。制备ＡＣＴＨ单克隆抗体 ,以期建立ＡＣＴＨ ＩＲＭＡ方法。ＡＣＴＨ或其片段与ＢＳＡ经碳化二亚胺偶联剂联接后制备免疫原 ,初次免疫为完全福氏佐剂乳化后在背部皮下多点注射 ,ＡＣＴＨ剂量约 4 0 μｇ ,以后每隔 4ｗ以不完全福氏佐剂乳化后在背部或腹腔交替注射 4～ 6次 ,剂量减半。融合前 3ｄ每天以无佐剂的ＡＣＴＨ联接物行腹腔注射 ,总剂量为 60μｇ。按常规方法进行细胞融合 ,每周换液 2至 3次。阳性克隆的筛选 :培养上清液抗体…     

DNA-mediated immunization of mice with plasmid encoding HBs antigen.

In order to develop an experimental DNA vaccine for the prevention and treatment of hepatitis B virus infection, hepatitis B virus surface antigen (HBsAg) DNA was subcloned into an E. coli-eukaryotic cell shuttle vector and was expressed in the Baculovirus expression system. Intramuscular, intradermal, and intraperitoneal injections of 30 microg of the plasmid DNA expressing HBsAg induced humoral and cellular immune responses in ICR mice. The first IgG antibodies were detected after ten days and specific IgG antibody titers peaked after two months of a single intramuscular DNA injection. Anti-HBs antibody titers gradually increased and peaked at four months following intradermal DNA injection, and in case of intraperitoneal injection they peaked at seven months. Generation of HBs-specific helper T lymphocytes was also investigated through the production of interleukin-2 by T helper cells. Boosting effects of HBs DNA were investigated without much results. In general, DNA-mediated HBs immunization induced humoral and cellular immune responses in mice that appears to simulate immune responses in human during the course of HBV vaccination.     

骨骼肌注射乙型肝炎表面抗原基因在小鼠体内诱发乙肝表面抗原特异的免疫反应

将含有乙型肝炎表面抗原（ＨＢｓＡｇ）基因的重组质粒ｐＡｃｔ－ＭＳ直接注入小鼠骨骼肌中，２周后加强免疫１次。免疫后４～９周间，每周检测小鼠血清中ＨＢｓ。结果表明实验组６／８只小鼠ＨＢｓ为阳性，对照组８只小鼠均未检测到ＨＢｓ。本实验证实直接注射质粒ＤＮＡ外源基因能获得表达，并能引起免疫反应。     

In vivo induction of cellular and humoral immune responses by hybrid DNA vectors encoding simian/human immunodeficiency virus/hepatitis B surface antigen virus particles in BALB/c and HLA-A2-transgenic mice

To improve the immunogenicity of epitopes derived from Gag proteins of simian immunodeficiency virus (SIV) and from the envelope (Env) protein of human immunodeficiency virus type 1 (HIV-1), we have designed hybrid DNA vaccines by inserting sequences encoding antigenic domains of SIV and HIV-1 into the hepatitis B virus envelope gene. This gene encodes the hepatitis B surface antigen (HBsAg) capable of spontaneous assembly into virus-like particles that were used here as carrier. Injections of hybrid vectors encoding B-cell epitopes from the gp41 and the gp120 envelope proteins of HIV-1 induced specific humoral responses in BALB/c mice. Furthermore, high frequencies of IFN-gamma-secreting CD8+ T cells specific for various antigenic determinants of SIV-Gag were observed after intramuscular injections of hybrid DNA vectors in BALB/c mice. Genetic immunization of HLA-A2.1-transgenic mice with HIV-Env/HBsAg-encoding DNA generated a strong CTL response and IFN-gamma-secreting CD8+ T lymphocytes specific for HIV-1 envelope-derived peptide. H-2d-restricted HBs-specific T-cell responses dominated over SIV-Gag responses in BALB/c mice whereas HLA-A2-restricted HIV-Env response was enhanced after fusion with HBsAg. These data demonstrate that different B and T-cell epitopes of vaccine-relevant viral antigens can be expressed in vivo as fusion proteins with HBsAg but that the optimal immunogenicity may differ strikingly between individual epitopes.     

In VivoInduction of Specific Cytotoxic T Lymphocytes in Mice and Rhesus Macaques Immunized with DNA Vector Encoding an HIV Epitope Fused with Hepatitis B Surface Antigen

DNA immunization offers a novel means to induce humoral and cellular immunity in inbred or in outbred animals. Here we have tested the efficiency of genetic immunization with hepatitis B virus (HBV) envelope-based vectors. In naive primates, injection of a plasmid DNA encoding HBV envelope proteins induced an HBV-specific cytotoxic response and appearance of potentially protective anti-HBs antibodies. Moreover, intramuscular and intradermal injections of a DNA expression vector encoding an epitope of the human immunodeficiency virus envelope fused to the surface protein of the hepatitis B virus (HBsAg) induced strong humoral and cytotoxic responses to antigenic determinants of both viruses in mice and nonhuman primates alike. In addition, in protein-primed Rhesus monkeys B-cell memory was successfully boosted by DNA injection of hybrid vectors and animals subsequently developed a multispecific cellular response. This suggests that DNA-based immunization could be used to boost efficiently and broaden the immune response in individuals immunized with conventional vaccines, regardless of their genetic variability. These results also indicate that it might be possible to rationally design HBsAg-based expression vectors to induce multispecific immune responses for vaccination against hepatitis B and other pathogens.     

Polyvalent DNA vaccines with bidirectional promoters

The hepatitis B surface antigen (HBsAg) and core antigen (HBcAg) were coexpressed from a synthetic bidirectional promoter with the tetracycline-inactivated transactivator (tTA). The function of this autoregulative system was evaluated following either transfer into established cell lines or intramuscular and intradermal injection of high or low doses of DNA into mice. We measured in vitro antigen expression and in vivo the induction of specific humoral and cellular immune responses. Successful regulation of antigen expression was observed in cultured cells. DNA vaccination with these constructs efficiently primed hepatitis B virus (HBV) specific immunity. However, immunogenic concentrations of the antigens were expressed even in the absence of the transactivator, indicating that low expression level is sufficient to prime an immune response. The bidirectional promoter allows coexpression of either both HBV antigens or a HBV antigen and enhanced green fluorescent protein leading to efficient priming of stable immunity against both antigens. This study demonstrates the potential of synthetic polyvalent plasmids in DNA vaccination.     

HBsAg基因疫苗诱导小鼠抗体产生的观察

将携带编码乙型肝炎病毒(HBV)表面抗原主要蛋白的S基因的质粒,直接注射小鼠肌肉中,小鼠4周后产生抗-HBs,阳转率83.3%(5/6),加强组阳转率66.7%(2/3),未加强组阳转率为100%(3/3);两组小鼠抗体持续时间可达28周以上;4周以后小鼠体内未检测到HBsAg.     

Liposome/DNA complexes coated with biodegradable PLA improve immune responses to plasmid encoding hepatitis B surface antigen

We hypothesized that the addition of polymer to the surface of liposome/DNA complexes may potentially enhance in vivo delivery of plasmid DNA to antigen-presenting cells and thereby facilitate enhanced immune responses to encoded protein. BALB/c mice were immunized subcutaneously or intramuscularly three times with a total of 50 microg of the plasmid pRc/CMV-HBs(S) (ayw subtype) encoding for the hepatitis B surface antigen. We measured transgene-specific total immunoglobulin G (IgG), IgG2a, IgG2b and IgG1 antibody responses as well as splenocyte and T-cell proliferation and cytokine production upon re-stimulation following immunization. Modification of lipid/DNA complexes by the polymer precipitation method used here for the addition of poly(d,l-lactic acid) was found to be consistently and significantly more effective than either unmodified liposomal DNA or naked DNA in eliciting transgene-specific immune responses to plasmid-encoded antigen when administered by the subcutaneous route. In addition, the polymer-modified formulations delivered by this route were more effective than naked DNA delivered by the intramuscular route in inducing antibody responses (n=5, P<0.03). Our observations provide 'proof of principle' for the use of these multicomponent formulations, which offer potential for manipulation and increased transfection efficiency in vivo for the purposes of genetic immunization.     

IL-2-preS融合蛋白表达质粒的基因枪DNA免疫研究

目的 观察基因枪肌肉人注射ＩＬ－２－ｐｅｒＳ融合蛋白表达质粒（ｐＣＷＩＩＰ）的基因免疫效率,为乙型肝炎基因免疫研究提供基础。方法 设基因枪肌内注射组和正常肌肉注射组,分别用ｐＣＷＩＩＰ质粒及对照质粒ｐＣＩＩＰ－２和ｐＣＩ免疫Ｂａｌｂ／ｃ小鼠,免疫后第４天,取肌肉组织通过免疫组化观察ＩＬ－２－ｐｒｅＳ在肌肉中的表达;另设基因枪、正常肌肉、皮下注射组分别免疫ｐＣＷＩＩＰ及对照质粒在２、４、６、８Ｗ眼后     

Differential dynamics of the peripheral and intrahepatic cytotoxic T lymphocyte response to hepatitis B surface antigen

The distribution and dynamics of the cytotoxic T lymphocyte (CTL) response to hepatitis B surface antigen (HBsAg) were studied in mice after intramuscular DNA immunization and after hepatic infection by a recombinant adenovirus that expresses the hepatitis B virus genome (Ad-HBV). CTLs specific for HBsAg accumulate preferentially in the spleen after DNA immunization but are primarily intrahepatic after Ad-HBV infection. The secondary CTL response to Ad-HBV in DNA-primed mice is characterized by rapid depletion of effector CTLs from the spleen, and their expansion in the liver where they cause hepatitis, secrete interferon gamma (IFNγ), and inhibit HBV gene expression. Suppression of HBsAg synthesis is accompanied by disappearance of intrahepatic IFNγ-producing CTLs and their reaccumulation in the spleen. The data suggest a possible explanation for the paucity and functional deficiency of HBV-specific CTLs in the periphery during chronic HBV infection, and that the severity of infection can be worsened by a preexisting CTL response if neutralizing antibody is not also present.