中华鳖嗜水气单胞菌微球缓释疫苗的研制及免疫效果

为通过免疫防治途径控制嗜水气单胞菌引起的多种中华鳖疾病,采用复乳化-溶剂挥发法制备缓释微球疫苗,通过灌胃方式免疫中华鳖(体质量150～200 g),每只灌胃0.05 g或0.1g,微球含灭活菌约为6.75×1010/g.免疫应答反应结果显示,缓释微球疫苗体外释放可持续近40 d,血清抗体效价、白细胞吞噬率、白细胞吞噬指数及淋巴细胞转化率最高分别可达512,71.25％,1.51和77.25％.结果表明:中华鳖嗜水气单胞菌微球缓释疫苗可以达到注射灭活菌液疫苗的水平,且持续性略优于注射免疫.通过灌胃方式(0.1 g/只)、注射方式分别对中华鳖进行免疫,研究攻毒后的免疫保护率,结果显示注射免疫组和微球疫苗免疫组的免疫保护率均为85％.免疫应答实验和攻毒实验结果分别证明了中华鳖口服缓释微球疫苗免疫的持续性和有效性.     

肠毒素大肠杆菌LT—B基因转基因马铃薯口服免疫原性研究

探讨马铃薯表达的产肠毒素大肠杆菌热敏肠毒素B亚基口服免疫原性,选择4周龄的昆明白小鼠。转基因马铃薯块茎经冷冻干燥后,研磨成粉状,以灌胃给药的方式对小鼠进行免疫。分0.2 g、0.4 g和0.6 g三个剂量组,免疫三次,每周一次。对照组用5μg细菌中表达的抗原蛋白腹腔注射免疫小鼠,空白组小鼠饲喂普通鼠粮。利用ELISA方法对小鼠血清、粪便特异性抗体表达进行分析。结果有45%小鼠经转基因马铃薯口服免疫后可诱导特异性的免疫应答。与细菌表达的抗原免疫反应相比,血清IgG反应和黏膜sIgA反应略强或相当。说明产肠毒素大肠杆菌转基因植物疫苗免疫动物可诱导特异的系统免疫应答和黏膜免疫应答。为利用植物生产预防大肠杆菌性腹泻可食用的口服疫苗,保护儿童免受细菌性腹泻的侵染奠定了基础。     

肠毒素大肠杆菌LT-B基因转基因

目的 探讨马铃薯表达的产肠毒素大肠杆菌热敏肠毒素B亚基口服免疫原性。方法 选择4周龄的昆明白小鼠,转基因马铃薯块茎经冷冻干燥后,研磨成粉状,以灌胃给药的方式对小鼠进行免疫,分0.2g、0.4g和0.6g三个剂量组,免疫三次,每周一次,对照组用5μg细菌中表达的抗原蛋白腹腔注射免疫小鼠,空白组小鼠饲喂普通鼠粮,利用ELISA方法对小鼠血清、粪便特异性抗体表达进行分析。结果 有45%小鼠经转基因马铃薯口服免疫后可诱导特异性的免疫应答,与细菌表达的抗原免疫反应相比,血清IgG反应和黏膜sIgA反应略强或相当。结论 产肠毒素大肠杆菌转基因植物疫苗免疫动物可诱导特异的系统免疫应答和黏膜免疫应答,这一研究结果为利用植物生产预防大肠杆菌性腹泻可食用的口服疫苗,保护儿童免受细菌性腹泻的侵染奠定了基础。     

季节性流感病毒裂解疫苗在小鼠中的 安全性和免疫原性研究

目的 评价季节性流感病毒裂解疫苗在动物体内的安全性和免疫原性。 方法 按《 中国药典》 三部( 2015 版)方法进行 BALB / c 小鼠异常毒性试验。 免疫原性试验将 BALB / c 小鼠随机分为 2 组,试验组每只小鼠肌肉注射 0. 1 mL 测试疫苗,空白对照组注射 0. 1 mL PBS。 接种后连续观察 49 d,每天记录动物的活动、饮食及体质量情况。 分别于接种后 0、14、28、35 和 49 d,采血并分离血清,红细胞凝集抑制试验( hemagglutination inhibition,HI)检测血清 抗体水平。 结果 异常毒性试验结果提示该疫苗的安全性良好,对小鼠的生长无影响,符合《中国药典》 三部( 2015 版)判定标准。 免疫原性试验结果显示,BALB / c 小鼠免疫后 28 d,血清抗 A( H1N1) pam09 和 A( H3N2) HI 抗体阳 转率均达到了 100% ;免疫后 35 d,抗 B( Victoria 系) 的抗体阳转率达到 70% 。 BALB / c 小鼠抗 A( H1N1) pam09、A ( H3N2)和 B( Victoria 系)抗体水平均达到峰值。 结果表明,该季节性流感病毒裂解疫苗对小鼠进行一次性免疫, 即能达到良好的免疫效果。 结论 该流感病毒裂解疫苗在 BALB / c 小鼠中具有良好的安全性和免疫原性。     

补中益气散对猪瘟疫苗免疫效果的影响

为了进一步提高猪瘟疫苗的免疫效果,探讨补中益气散对该疫苗的免疫增强作用,该研究以某猪场27头30日龄健康仔猪随机分为3组,对照组和试验1,2组.对照组仅注射猪瘟疫苗;试验1组用补中益气散拌料饲喂一周后注射疫苗;试验2组注射疫苗同时拌料饲喂补中益气散一周,中药用量均按0.2%加入饲料.分别于免疫当天、免疫接种后7,30d无菌操作采集血样,运用正向间接血凝试验检测血清中的抗体水平.结果表明:补中益气散可显著提高血清中的抗体水平,免疫后30d免疫保护率维持在88.9%以上,提示补中益气散具有显著增强猪瘟疫苗免疫效果的作用.     

五指毛桃拮抗环磷酰胺诱发的遗传损伤效应

目的探讨五指毛桃对环磷酰胺所致小鼠遗传物质损伤的拮抗效应.方法 30只SPF级昆明种雄性小鼠,随机分为阴性对照组、阳性对照组及五指毛桃高（20g/kg）、中（10g/kg）、低（5g/kg）3个抗突变组.抗突变组用五指毛桃水提液灌胃,阴性对照组、阳性对照组灌以等量生理盐水,1次/d,连续15d.于第14 d,除阴性对照组外,各组小鼠腹腔注射环磷酰胺40mg/kg,24h后重复注射一次.用微核试验,检测五指毛桃水提液对环磷酰胺诱发小鼠骨髓嗜多染红细胞和睾丸生精细胞微核发生率的影响.结果抗突变组的骨髓嗜多染红细胞和睾丸生精细胞微核率均明显降低,与阳性对照组比较有统计学意义（P〈0.01）,并且随着五指毛桃液浓度的增高,微核率逐渐降低.结论五指毛桃可以拮抗环磷酰胺诱发的小鼠遗传物质损伤,提示五指毛桃有一定的抗突变作用.     

重组人干扰素内皮抑制肽融合蛋白抗肿瘤药效的研究

目的 考察融合蛋白I30肽抗肿瘤的效果,为I30肽后续开发提供实验支持。方法 使用NIH小鼠,取4只小鼠腹腔注射H22瘤株,1周时处死所有小鼠,取腹水。另取40只小鼠,每只腋下注射腹水0.2 mL,细胞浓度为1×106个/mL。接种后4 h开始给药。将腋下注射小鼠随机分为4组:IFN组,皮下注射干扰素9×105IU/只;I30高剂量组,皮下注射I30 80 μg/只;I30低剂量组,皮下注射I30 30 μg/只;阴性对照组,皮下注射等体积生理盐水。连续给药16 d。停药后2 h,处死所有动物,再解剖皮下瘤块称重。结果 I30给药组具有明显的治疗效果,且具有显著性差异(P<0.05)。结论 I30肽具有抗肿瘤的作用。     

家蝇蛹凝集素对免疫低下小鼠免疫功能的影响

将家蝇蛹凝集素(MPL)分别按400μg/(kg·d)、800μg/(kg·d)、1 200μg/(kg·d)连续给小鼠灌胃10d,于第8d和第9 d给阴性对照组和3个剂量组小鼠腹腔注射环磷酰胺,造成小鼠免疫功能低下.观察家蝇蛹凝集素对免疫低下小鼠免疫器官指数、巨噬细胞的吞噬能力、脾脏淋巴细胞增殖和血清溶血值HC50的影响.结果表明,家蝇蛹凝集素能显著拮抗环磷酰胺所致免疫功能低下小鼠的免疫器官指数下降,提高巨噬细胞的吞噬能力,明显促进脾脏淋巴细胞增殖,且能使血清溶血素含量显著增加.说明家蝇蛹凝集素对环磷酰胺所致小鼠免疫功能低下有较好的预防作用.     

金翁止痢颗粒对免疫低下小鼠的免疫调节作用

为探讨金翁止痢颗粒对免疫低下小鼠的免疫调节作用,将72只小鼠随机分为空白组、模型组、阳性药物组、金翁止痢颗粒高剂量组、中剂量组、低剂量组,其中空白组小鼠给予生理盐水,模型组与各药物组小鼠腹腔注射环磷酰胺构建免疫低下模型．造模成功后,模型组给予生理盐水,阳性药物组给予2 g／kg黄芪多糖液,金翁止痢颗粒高剂量组、中剂量组、低剂量组分别灌胃20 g／kg ,16 g／kg ,12.8 g／kg的金翁止痢颗粒提取液,1次／d ,连续治疗7 d后,检测小鼠碳粒廓清指数、免疫器官指数,T／B淋巴细胞转化等指标．结果表明,金翁止痢颗粒能显著提高免疫低下小鼠碳粒廓清指数、免疫器官指数,促进 T／B淋巴细胞转化,与模型组相比差异极具有统计学意义（p＜0.01）,表明金翁止痢颗粒具有增强免疫低下小鼠免疫功能的作用．     

小白菊注射液对大鼠的亚慢性毒性试验研究

将雌雄各半的80只SD大鼠,随机分为对照组和1.5,mL/100,g、0.75,mL/100,g和0.325,mL/100,g 3种剂量的小白菊注射液给药组,每组20只,连续35,d腹腔注射,进行小白菊注射液的亚慢性毒性试验。试验结果显示,各组大鼠给药直至35,d均无死亡,3个给药组大鼠体重、进食量、脏器系数、血液学及血液生化学与对照组相比均无显著差异(P0.05)。结论提示,小白菊注射液在1.5,mL/100,g、0.75,mL/100,g和0.325,mL/100,g剂量下连续腹腔注射35,d对SD大鼠无不良影响。     

Successful vaccination with a polyvalent live vector despite existing immunity to an expressed antigen

A global vaccination strategy must take into account production and delivery costs as well as efficacy and safety. A heat-stable, polyvalent vaccine that requires only one inoculation and induces a high level of humoral and cellular immunity against several diseases is therefore desirable. A new approach is to use live microorganisms such as mycobacteria, enteric bacteria, adenoviruses, herpesviruses and poxviruses as vaccine vectors. A potential limitation of live polyvalent vaccines, however, is existing immunity within the target population not only to the vector, but to any of the expressed antigens. This could restrict replication of the vector, curtail expression of antigens, and reduce the total immune response to the vaccine. Recently acquired immunity to vaccinia virus can severely limit the efficacy of a live recombinant vaccinia-based vaccine, so a strategy involving closely spaced inoculations with the same vector expressing different antigens may present difficulties. We have constructed a recombinant vaccinia virus that expresses surface proteins from two diverse pathogens, influenza A virus haemagglutinin and herpes simplex virus type 1 (HSV-1) glycoprotein D. Mice that had recently recovered from infection with either HSV-1 or influenza A virus could still be effectively immunized with the double recombinant.     

Protection against malaria by immunization with plasmid DNA encoding cholera toxin B subunit andPlasmodium falciparum epitopes

Cholera toxin B subunit is a good carrier protein and an effective adjuvant which can boost both cellular and humoral immunity. DNA fragments encoding B cell, Th cell and CTL epitopes of P. falciparum CS, MSA-1, MSA-2 and RESA antigens were cloned down-stream of cholera toxin B subunit gene in the same reading frame. Another modification using IL2 as adjuvant was also made. High titer of anti-malaria epitopes antibodies and strong cellular immunogenicity were elicited after Balb/c mice were immunized three times with 100 μg recombinant plasmid DNA dissolved in 100 μL PBS. 200 vaccinees were challenged with mouse Plasmodium yoelli to investigate if cross protection existed. The protective efficacy was about 50%. And it is found that the protective efficacy is correlated with CTL activity which was considered to be the primary effects of anti-sporozoite protective immunity. Better results might be expected when the DNA vaccine candidates were applied to primates.     

MNV感染对乙肝疫苗效价评价的影响

目的 研究小鼠诺如病毒(murine norovirus,MNV)感染实验小鼠对乙肝疫苗效价评价的影响.方法 将BALB/c小鼠分为对照组和感染组,感染组经口灌胃MNV,对照组灌胃生理盐水,参照《中国药典》2015年第三部乙肝疫苗效价测定方法,将乙肝疫苗分为高、中、低3个剂量组,分别免疫对照组和感染组小鼠,于免疫后第7...     

小鼠结核分枝杆菌感染模型用于评价重组HBHA疫苗免疫效果的实验研究

目的通过复制小鼠结核病模型评价重组HBHA疫苗的免疫效果。方法将重组肝素结合血凝素(HBHA)疫苗接种BALB/c小鼠,观察其诱导的细胞免疫应答水平,并以MTB H37Rv毒株攻击免疫小鼠,研究重组疫苗诱导的保护力。结果重组HBHA免疫BALB/c小鼠后可诱发细胞毒作用,将体内MTB裂解。重组疫苗不仅能刺激CD4+T,CD8+T增殖、活化,还可诱导脾细胞分泌IFN-γ、IL-2等细胞因子。H37Rv毒株攻击后,被免疫小鼠的组织病理学症状减轻。结论本研究通过检测小鼠体内细胞免疫应答和肺脏组织病理学改变等指标,客观的反映了重组疫苗免疫小鼠结核模型的保护效果。     

Specific anti-tumor effect induced by attenuated Salmonella typhimurium vaccine expressing extracellular region of vascular endothelial growth factor receptor 2

The purposes of this research were to study the stable expression of exogenous gene encoding therapeutic protein in attenuated Salmonella typhimurium, observe the metabolism of oral gene vaccine carried by attenuated Salmonella typhimurium in BALB/c mouse, and investigate the feasibility of prevention and treatment of tumors by the recombinant bacteria. Recombinant plasmid pcDNA3.1 VEGFR2(n1-7) was transformed into competent attenuated Salmonella typhimurium SL3261 to develop oral DNA vaccine SL3261-pcDNA3.1 VEGFR2(n1-7). To observe whether the exogenous gene can be expressed in the recombinant bacteria, PCR was performed to amplify the CMV promoter of the eukaryotic expression vector as the proof of stable expression of exogenous protein; transmission elec- tron microscopy (TEM) was applied to observe the morphology of the recombinant bacteria to confirm that the exogenous gene has no impact on the growth of the bacteria, and then BALB/c mice were immunized with the gene vaccine. After inoculation of the gene vaccine, the recombinant bacteria SL3261 could be detected in the tissues such as small intestine, colon, liver and spleen. And then, mice in each group were challenged with tumor cells. The results of animal experiment showed that tumor growth of the mice in experimental group was inhibited and survival time of immunized mice was prolonged compared with control groups. A higher lymphocyte infiltration in tumors from animals treated with DNA vaccine was observed. Immunohistochemical analysis of tumor samples revealed an en- hanced accumulation of CD8 cytotoxic T lymphocytes, as well as an increase in CD4 cells in the tumors of animals treated with the oral gene vaccine compared to tumors from control group mice. Ultrastructure of the tumor tissue showed that tumor cells in the samples of the immunized mice were well-differentiated. Our research confirmed that the exogenous gene can be stably expressed in the attenuated Salmonella typhimurium and has no impact on the growth of the recombinant bacteria; the exogenous gene can de delivered to the host by attenuated Salmonella typhimurium to produce anti-tumor effect with no obvious cytotoxity to the host. In this study, it is established that attenuated Salmonella typhimurium could be used as a vector for oral gene vaccine, and our study provided a theoretical basis for the body distribution and the metabolism of the recombinant bacteria. This strategy may provide a simple, safe and effective way for the prevention and treatment of tumors.     

小鼠腹腔注射卡介苗加脂多糖建立急性免疫性肝损伤的实验研究

目的比较卡介苗(BCG)加脂多糖(LPS)小鼠腹腔注射和尾静脉注射建立急性免疫性肝损伤模型的优劣。方法用不同剂量BCG和LPS腹腔注射诱导昆明小鼠建立急性免疫性肝损伤模型,与BCG 0.5 mg/只和LPS 7.5μg/只联合尾静脉注射诱导昆明小鼠建立急性免疫性肝损伤模型比较;以小鼠血清丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)水平变化及肝脏病理学检查指标作为肝损伤判断标准。结果BCG+LPS小鼠腹腔注射所造急性免疫性肝损伤模型,ALT和AST随注射剂量增加均逐步增高;第6组(BCG 0.5 mg/只+LPS7.5μg/只)和第13组(BCG1 mg/只+LPS15μg/只)相对较高。与空白组比较,ALT升高约3倍,AST升高1~3倍;肝组织病理损伤则随着BCG和LPS剂量增加逐渐升高。第5组大部分小鼠出现Ⅰ级或Ⅰ级以上肝细胞损害;第13组大部分小鼠出现Ⅱ级或Ⅱ级以上肝细胞损害。但与尾静脉注射造模相比,小鼠腹腔注射造模肝细胞损伤的特异性实验室指标ALT和AST表达不高,肝组织病理损伤较轻。结论"BCG+LPS"小鼠尾静脉注射建立急性免疫性肝损伤模型优于腹腔注射建立免疫性肝损伤模型。     

TritonX-100裂解流感病毒的研究

 在流感病毒高浓度条件下,寻找TritonX-100裂解流感病毒的适宜条件.在不同条件下用TritonX-100裂解WHO推荐用2007～2008年度流感病毒疫苗毒株制备的3个亚型的流感病毒,用电镜观察裂解程度,通过蔗糖密度梯度离心纯化抗原后免疫动物,以检测疫苗的免疫效果.结果表明当流感病毒的血凝效价是1:16382,裂解时间为24h时,TritonX-100在质量分数为1%可以完全地裂解B型,质量分数达到2%才可以完全裂解流感病毒H1N1和H3N2,这说明TritonX-100对A,B亚型毒株的裂解效果不同.由于每年都要更换毒株工人生产新的流感疫苗,因此应该对使用TritonX-100的裂解不同亚型的流感病毒条件进行研究,以保证裂解疫苗的质量.     

H5亚型禽流感病毒HA基因昆虫/杆状病毒系统的表达及对BALB/c小鼠的免疫保护

经RT-PCR扩增了禽流感病毒A/Goose/Guangdong/1／96 H5N1亚型1．7kb HA基因的cDNA,将其克隆到pMD18-T中并测序。亚克隆到杆状病毒转移载体pMelBacA的蜜蜂蜂毒素分泌信号下游中,测序正确后与线性化的杆状病毒DNA(Bac-N-BlueTM DNA)共转染Sf9昆虫细胞。将重组杆状病毒感染HFive细胞,72h左右收获细胞,超声波裂解,SDS—PAGE结果表明HA基因在重组杆状病毒感染的HFive细胞中获得表达。蛋白胶薄层扫描分析显示:表达的HA蛋白占重组杆状病毒感染细胞总蛋白含量的17．1％。Western-blot 及血凝实验结果显示,表达的禽流感H5N1亚型病毒HA蛋白具有生物学活性。表达的H5 HA蛋白定量乳化后,皮下多点注射免疫SPF 级BALB/c雌性小鼠,免疫后产生了H5 HA特异抗体,并在三免前后达到并保持较高水平。用致死剂量的HPAIV H5N1攻击小鼠,免疫组小鼠提供了100％的保护力,而对照组小鼠先后发病且死亡:为研制禽流感H5N1亚型病毒亚单位疫苗,防制禽流感奠定了基础。     

Combined immunity of DNA vector and recombinant vaccinia virus expressing Gag proteins of equine infectious anemia virus

In order to develop a new vaccine candidate for equine infectious anemia virus (EIAV), gag gene of Chinese donkey leukocyte attenuated strain (EIAV DLV) and its parental virulent strain (EIAV LN) were inserted respectively into the TK region of the Tiantan strain (VV) of vaccinia virus by homologous recombination and the positive clone was confirmed by blue plaque assay. Protein expression was examined by Western blot. Prime and prime-boost procedures were used to immunize mice with two DNA vectors and two recombinant vaccinia viruses expressing EIAV Gag proteins. The results showed that the specific lysis of CTL responses in the DNA rVV groups was stronger than those in the DNA groups, amounting to 31%. Although the levels of specific antibodies were not significantly different, we could conclude that the recombinant vaccinia virus could boost the cellular responses following DNA vector priming. There was no detectable difference between the immune responses induced by DLV and LN Gag proteins. This data demonstrates that the combined immunity of DNA vector and recombinant vaccinia virus expressing EIAV gag proteins, utilizing the prime-boost procedure, can drive immunized mice to produce powerful cellular responses. These results lay an important foundation for the development of a new EIAV genetic engineering vaccine.