硫酸化人参总皂苷的制备及其对鸡外周血淋巴细胞增殖的影响

用氯磺酸-吡啶试剂制备了硫酸化人参总皂苷衍生物，采用红外光谱仪检测到制备的硫酸化人参总皂苷衍生物中在1230cm^-1和810cm^-1有硫酸酯键的特征吸收峰。为了初步探讨硫酸化人参总皂苷的免疫学活性．以MTT法测定了人参总皂苷及其衍生物对鸡外周血淋巴细胞增殖的影响。结果表明，150μg／m1人参总皂苷和衍生物均能显著抑制鸡外周血淋巴细胞的增殖（P〈0．05），10μg／ml的衍生物能极显著促进淋巴细胞增殖，与同浓度人参总皂苷相比活性极显著增强（P〈0．01）。实验结果提示，对人参总皂苷进行硫酸化修饰是可行的，经硫酸化后人参总皂苷衍生物的免疫活性明显增强。     

硫酸化人参总皂苷体外对免疫活性细胞功能的调节

采用MTT法及形态学观察法研究4种硫酸化人参总皂苷衍生物对体外ConA促淋巴细胞增殖、转化的影响,并采用乳酸脱氢酶释放法探讨了其对NK细胞杀伤活性的影响.结果表明,人参总皂苷对淋巴细胞增殖影响不明显,较高质量浓度(>150 mg/L)能抑制淋巴细胞增殖(P<0.01);硫酸化人参总皂苷衍生物2、3在一定质量浓度下都能明显或极明显增加淋巴细胞(鸡的外周血淋巴细胞,小鼠脾淋巴细胞)增殖活性(P<0.05,P<0.01),且所试质量浓度硫酸化人参总皂苷衍生物的D值显著或极显著高于同质量浓度人参总皂苷(P<0.05.P<0.01);人参总皂苷、衍生物2、3都能提高小鼠NK细胞的活性(P<0.01),且衍生物2、3的作用比人参总皂苷更强(P<0.05,P<0.01).结果提示,人参总皂苷经硫酸化修饰后,在体外能明显增强免疫活性细胞功能.     

人参皂苷Rh2硫酸化衍生物抗炎作用的分子机制

通过构建的细菌脂多糖（LPS）诱导小鼠巨噬细胞（RAW264．7）的体外炎症模型,研究了不同质量浓度人参皂苷Rhz硫酸化衍生物B2（Rh2-B2）对TNF-a、IL-6、IL-1β和IL-10合成的影响,以及对NF-kB信号通路的影响。结果显示,1mg／L和5mg／L的人参皂苷Rh2-B2能显著抑制RAW264．7合成TNF-a、IL-6和IL-18（P〈0．01）,同时显著促进IL-10的合成（P〈0．01）,并且通过显著抑制IkBa的降解而阻止NF-kB／p65易位入核（P〈0．01）。结果表明,人参皂苷Rh2—B2可能通过阻断NF-kB信号通路,抑制LPS诱导的炎性细胞因子的产生,进而发挥其抗炎作用。     

CpG ODN对仔猪淋巴细胞增殖和细胞因子诱生的影响:在体和离体试验

将CpGODN、non CpG ODN分别接种初生仔猪，同时分离仔猪的外周血单核细胞，以CpGODN、non CpG ODN对外周血单核细胞体外培养，分别检测仔猪Th1型细胞因子分泌情况。结果表明，与non CpG ODN和对照组相比，CpG ODN能在体内显著提高仔猪的淋巴细胞白介素-2诱生活性、淋巴细胞增殖、血清中IFN-γ和IL-12水平（P〈0．05），亦能在体外刺激仔猪的外周血单核细胞分泌IFN-γ和IL-12（P〈0．01）。这显示CpG—ODN能显著增强动物的免疫应答能力。     

硫酸化人参总皂苷对人工感染MDV鸡淋巴细胞活性的调节

鸡马立克氏病病毒（MDV）经腹腔注射感染8日龄雏鸡,攻毒15 d后检测硫酸化人参总皂苷对鸡外周血白细胞数和淋巴细胞增殖活性的影响,并利用半定量RT-PCR方法分析外周血淋巴细胞IFN-γmRNA的表达水平。结果表明,MDV感染能引起鸡淋巴细胞百分比相对增多（P〈0.01）,人参总皂苷及其硫酸化人参总皂苷不能改变MDV所致变化（P〉0.05）,且硫酸化人参总皂苷与人参总皂苷之间没有差异（P〉0.05）;MDV感染极显著抑制淋巴细胞增殖（P〈0.01）,人参总皂苷能明显改善MDV对感染鸡淋巴细胞增殖的抑制（P〈0.01）,硫酸化人参总皂苷促进了MDV所致的抑制状态（P〈0.01）;人参总皂苷及其硫酸化人参总皂苷均能增强MDV所致鸡淋巴细胞IFN-γmRNA的表达,与健康组相比,硫酸化人参总皂苷极显著增加IFN-γmRNA表达（P〈0.01）。结果提示,硫酸化人参总皂苷对人工感染MDV鸡的淋巴细胞活性具有调节作用,其作用比人参总皂苷的作用更强。     

人参皂苷-Rh2的硫酸化衍生物鉴定及其对小鼠腹腔巨噬细胞功能的影响

为了评价硫酸化修饰后人参皂苷-Rh2的活性变化,利用薄层色谱、红外光谱及质谱鉴定所用Ⅱ a、Ⅱb为人参皂苷-Rh2的硫酸化衍生物,并探讨了人参皂苷-Rh2及其硫酸化衍生物Ⅱa、Ⅱ b对小鼠的巨噬细胞吞噬中性红和在LPS刺激下分泌TNF-α量的影响.结果显示,衍生物Ⅱ a、Ⅱ b在1 mg/L时均能显著促进小鼠腹腔巨噬细胞吞噬活性(P＜0.05),Rh2在0.2mg/L和5mg/L亦可显著促进巨噬细胞吞噬中性红(P＜0.05).Ⅱa 0.2、5 mg/L时,Ⅱb在为5 mg/L时其在LPS的协同作用下能极显著促进小鼠腹腔巨噬细胞分泌TNF-α(P＜0.01),而Rh2在5mg/L表现出极显著的抑制 LPS促进小鼠腹腔巨噬细胞分泌TNF-α作用(P＜0.01).结果提示,Rh2硫酸化修饰后对巨噬细胞的功能有显著增加作用.     

硫酸化20(S)-人参皂苷Rh_2对小鼠脾淋巴细胞分泌IL-4和IFN-γ的影响

为了进一步评价硫酸化修饰后人参皂苷Rh2的免疫活性变化,试验分别采用MTT法和ELISA法对比研究了浓度为0.2 mg/L、1 mg/L、5 mg/L的人参皂苷Rh2及其硫酸化衍生物(S-Rh2-1和S-Rh2-2)对ConA刺激小鼠的脾淋巴细胞增殖和分泌IL-4、IFN-γ的影响。结果表明:S-Rh2-1为0.2 mg/L、5 mg/L,S-Rh2-2为1 mg/L,Rh2为5 mg/L时能显著或极显著抑制由ConA引起的脾淋巴细胞增殖(P<0.05或P<0.01);S-Rh2-2为1 mg/L、5 mg/L,Rh2为0.2 mg/L、5 mg/L时能显著或极显著抑制由ConA引起的脾淋巴细胞分泌IL-4的量(P<0.05或P<0.01);S-Rh2-1、Rh2的3个试验浓度以及S-Rh2-2为0.2 mg/L、1 mg/L时都能极显著抑制由ConA引起的脾淋巴细胞分泌IFN-γ的量(P<0.01)。说明Rh2进行硫酸化修饰后其免疫活性在一定程度上得到了增强。     

蕨麻多糖对小鼠淋巴细胞增殖和一氧化氮分泌的影响

为探讨蕨麻多糖（Potentilla anserine polysaccharide，PAP）免疫调节的作用机理，观察了PAP对小鼠脾淋巴细胞体外增殖和分泌一氧化氮的影响。结果显示，50mg／L PAP配合ConA或LPS（lipopolysaccharide，脂多糖）能使小鼠脾淋巴细胞在体外显著增殖（P〈0．05）；100、200及400mg／L PAP配合ConA或LPS可使小鼠脾淋巴细胞在体外极显著增殖（P〈0．01）；与对照组相比，PAP以不同浓度（50～400mg／L）处理小鼠脾淋巴细胞后一氧化氮分泌量均显著升高（P〈0．05）。试验结果表明，蕨麻多糖能明显促进体外培养的小鼠脾淋巴细胞增殖和分泌一氧化氮，与ConA或LPS有协同作用。     

日本大耳白兔红细胞调控淋巴细胞和NK细胞活性研究

应用红细胞促淋巴细胞转化和促NK细胞活性试验,对不同生理发育时期日本大耳白兔红细胞调控淋巴细胞和NK细胞活性进行研究。结果表明：青年组日本大耳白兔红细胞调控淋巴细胞能力明显高于幼年兔和老年兔（P〈0．01）;老年组红细胞调控NK活性能力明显高于青年组和幼年组（P〈0．01）,青年组高于幼年组（P〈0．01）。日本大耳白兔红细胞调控淋巴细胞能力在青年时期达到了高峰．随后逐渐降低：而红细胞调控NK活性随年龄增长而显著增强,这提示老年兔的免疫活性细胞较多地处于预激活状态,并对刺激显示良好的免疫应答能力。     

CpG ODN对鸡新城疫LaSota活疫苗的免疫增强效应

将3种不同的未甲基化CpGODN分别与新城疫LaSota活疫苗混合后，经滴鼻和点眼免疫鸡，通过检测鸡血清中HI抗体、外周血T淋巴细胞增殖活性、诱导巨噬细胞分泌NO含量，以及刺激外周血淋巴细胞表达IFN-γ、IL-6与IL-1β mRNA量，分析各CpGODN对新城疫LaSota活疫苗免疫效果的影响。结果表明，经2次免疫后，含GTCGTT核心基序的CpG ODN1组，鸡血清平均HI抗体效价最高达8．2log2、淋巴细胞刺激指数达9．836、NO分泌量达35．833μmol／L，分别比疫苗单独免疫组高出2个滴度（P〈0．05）、4．4（P〈0．01）、27．6μmol／L（P〈0．01）；含GACGTT核心基序的CpG ODN2组增强作用不明显．与疫苗单独免疫组无差异；而CpGODN3的免疫刺激活性由于受其侧翼序列的影响，作用明显减弱甚至丧失。对细胞因子的影响，CpGODN3组IFN-γ mRNA表达量稍高于CpG ODN1组，而其余细胞因子均以CpG ODN1组表达水平最高（P〈0．05）。由此证明CpG ODN1能显著增强鸡对新城疫LaSota活疫苗的体液和细胞免疫反应，可以作为高效的免疫增强剂。     

Study on the Activity Estimation Methods of Recombinant Chicken Interferon-α/Interleukin-2 Fusion Protein in vitro

In order to establish a stable,simple and rapid detection method to estimate the activities of the recombinant chicken interferon-α/interleukin-2 fusion protein (rChIFN-α-Linker-ChIL-2,recombinant fusion protein) in vitro,the activities of rChIFN-α-Linker-ChIL-2 were estimated by detecting its specific immune response to monoclonal antibody (MAb) against ChIFN-α and ChIL-2 by ELISA assay.The antiviral activities of rChIFN-α-Linker-ChIL-2 protein were tested by inhibiting the 50% appearance of cytopathic effect (CPE) of vesicular stomatitis virus (VSV) and infectious bursal disease virus (IBDV) on the passage cell lines DF1.The promoting proliferation activities of lymphocytes in the chicken peripheral blood and spleen of recombinant fusion protein were tested by MTS method.The results showed that rChIFN-α-Linker-ChIL-2 protein had the ability of specific immune response to anti-ChIFN-α MAb and anti-ChIL-2 MAb,respectively.The antiviral activity of rChIFN-α-Linker-ChIL-2 protein inhibiting the reproduction of VSV on DF1 cell line was higher than IBDV,and both of the antiviral activities of recombinant fusion protein against VSV and IBDV were much higher than the recombinant ChIFN-α protein (rChIFN-α) control.The recombinant fusion protein had apparent promoting proliferation activity of lymphocytes in the chicken peripheral blood and spleen,which were much higher than that of the rChIFN-α control.The study suggested that the activities detection and estimation methods of the recombinant fusion protein in vitro were successfully established,which laid the foundation for the further study of the synergy activity of recombinant fusion protein in vivo.     

重组鸡α干扰素/白细胞介素-2融合蛋白体外活性评价方法研究

为建立稳定、便捷的重组鸡α干扰素/白细胞介素-2融合蛋白(rChIFN-α-Linker-ChIL-2蛋白,重组融合蛋白)体外活性评价方法,本研究分别采用ChIFN-α和ChIL-2 ELISA方法检测重组融合蛋白与抗ChIFN-α单抗和抗ChIL-2单抗发生特异性免疫反应的活性;采用细胞病变抑制法检测重组融合蛋白在DF1细胞上抑制水疱性口炎病毒(VSV)和传染性法氏囊病病毒(IBDV)增殖活性;采用MTS法分别测定重组融合蛋白促鸡外周血T淋巴细胞(PBLC)和脾淋巴细胞增殖活性。结果表明,重组融合蛋白可以与抗ChIFN-α单抗和抗ChIL-2单抗发生特异性免疫反应;重组融合蛋白在DF1细胞上具有明显抗病毒活性,其抗VSV活性高于抗IBDV活性,且均明显高于rChIFN-α蛋白对照;不同浓度的重组融合蛋白均具有明显的促鸡PBLC和脾淋巴细胞增殖活性,且其促增殖活性明显高于rChIFN-α蛋白对照。本研究成功建立了重组融合蛋白体外活性检测评价方法,为进一步探究重组融合蛋白在鸡体内协同作用奠定基础。     

Production and characterization of monoclonal antibodies detecting chicken interleukin-2 and the development of an antigen capture enzyme-linked immunosorbent assay

Eleven monoclonal antibodies (mAbs) which are specific for chicken interleukin-2 (chIL-2) were produced and characterized by enzyme-linked immunosorbent assay (ELISA), Western blotting and neutralizing assays. These mAbs were used to develop a mAb-based antigen capture ELISA specific for chicken IL-2 detection. Anti-IL-2 mAbs bound specifically to E. coli-derived rchIL-2 in ELISA and identified a 16 kDa IL-2 polypeptide band in Western blot. Several mAbs were shown to neutralize the biological activities of both rchIL-2 and native chicken IL-2 as measured by concanavalin A (ConA)-induced lymphocyte proliferation assay, IL-2 bioassay, and natural killer cell assay. Among the neutralizing mAbs, the mAb chIL-2/11 was most potent in neutralizing IL-2 activity. To develop a sensitive ELISA for the detection of chicken IL-2, an antigen capture ELISA was developed using the mAb chIL-2/16 as the antigen capture antibody and rabbit anti-IL-2 peptide antibody as the detection antibody. Using the mAb-based antigen capture ELISA, significant correlation between the level of IL-2 detected in bioassays and in ELISA was observed. These results showed that the mAb-based antigen capture ELISA is less time-consuming and more reliable compared to a conventional IL-2 bioassay for chicken IL-2. These neutralizing mAbs will facilitate basic immunobiological studies of the role of IL-2 in normal and disease states in chickens.     

Response to mitogens of chicken splenocytes determined by three methods of measurement

The aim of the present study was to examine whether the 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide (MTT) colorimetric assay can be applied to measurement of mitogen‐induced chicken splenocyte activation. Activation was also measured by a ³H‐thymidine uptake assay and a viable cell count assay. Optimal concentrations of mitogens and incubation periods required for maximal responses to mitogens differed between the MTT assay and the viable cell count and thymidine uptake assays. This probably reflects differences in the activities measured by the MTT assay which detects mitochondrial enzyme activity, and the thymidine uptake and viable cell count assays which detect cellular proliferation activity. The validity of the MTT assay was supported by the observation that the mitogen‐induced increase in succinate dehydrogenase activity paralleled the level of mitogen‐induced MTT formazan production. Mitogen concentrations inducing maximal formazan formation in chicken splenocytes were higher than those for chicken peripheral blood lymphocytes reported previously. Results of the present study indicate that mitogen‐induced chicken splenocyte activation could be measured by the MTT colorimetric assay, although mitogen concentrations and incubation periods required for maximal splenocyte activation differed between the MTT assay and the other two assays used in this study.     

3种植物多糖对鸡外周血和脾脏淋巴细胞体外增殖作用的影响

本试验旨在比较3种植物多糖对鸡外周血T淋巴细胞和脾脏B淋巴细胞体外增强免疫活性的研究.通过酶学检测法测定3种植物多糖对鸡胚成纤维细胞的安全浓度,MTT法比较3种植物多糖单独刺激和PHA协同刺激对鸡外周血T淋巴细胞增殖指数的影响,同时比较3种植物多糖单独刺激及与LPS协同刺激对鸡脾脏B淋巴细胞增殖率的影响.结果显示,黄芪多糖和桑叶多糖具有较高的细胞安全浓度,在64~512 μg/mL时,3种植物多糖对鸡外周血T淋巴细胞和脾脏B淋巴细胞的增殖作用较强.结果表明,3种植物多糖对鸡外周血和脾脏淋巴细胞都有不同程度的体外增殖作用,其中黄芪多糖的增殖作用最强,桑叶多糖其次,山药多糖最差.     

牛蜱龙岩分离株的分子鉴定

为了解福建省龙岩地区牛场体表蜱的种类,试验采集龙岩地区牛体表寄生蜱样本,基于蜱的核糖体rDNA内转录间隔区(internal transcribed spacer,ITS)序列PCR扩增方法进行分子鉴定,获得ITS1和ITS2基因序列,进行blastn相似性搜索,应用MegAlign和MEGA 7.0软件完成同源性分析及种系发育分析。结果表明,该蜱ITS1序列与中国甘肃微小扇头蜱分离株同源性达99.32%(JQ737124.1、JQ737125.1),与中国湖南微小扇头蜱HAI2分离株同源性达99.16%(MK224531.1);ITS2序列与中国河南、南非豪登微小扇头蜱分离株同源性均为100%(KX450287.1、KY457506.1);种系发育分析结果显示,龙岩地区牛体表分离的牛蜱ITS基因序列与扇头蜱属ITS基因序列聚类,与革蜱属和璃眼蜱属处于两个不同的分支。说明福建龙岩牛蜱分离株为微小扇头蜱。     

Lymphocyte proliferation response during Eimeria tenella infection assessed by a new, reliable, nonradioactive colorimetric assay

The application of a tetrazolium salt, WST-8,2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt to the lymphocyte proliferation assay in the chicken system was evaluated. Proliferation of concanavalin (Con A)-induced splenic lymphocytes and peripheral blood lymphocytes (PBL) was evaluated with WST-8 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Coefficients of correlation (r) between these two reagents were 0.98 and 0.97 in splenic lymphocytes and PBL, respectively. In general, the sensitivity of the WST-8 assay was significantly higher than that of the MTT assay, and the standard deviations of the WST-8 assay were significantly lower than those of the MTT assay. The WST-8 assay was fast and highly reproducible and provided a good indication of mitogen-induced proliferation of spleen cells induced by Con A. With the use of the WST-8 assay, splenic mitogenic response of chickens infected with Eimeria decreased transiently at 7 days but increased significantly at 10 days after primary infection compared with that of uninfected chickens. Additionally, the measurement of interleukin (IL)-2 production with WST-8 was highly reproducible and showed a significant increase in IL-2 production upon stimulation of Eimeria tenella-immune spleen cells with Con A. After E. tenella infection, splenic IL-2 production increased significantly at 7 days post-primary and at 2 days post-secondary infection. The WST-8 assay is fast, simple, and more reproducible and sensitive than the MTT assay. This study demonstrates the effectiveness of the WST-8 assay to assess cell-mediated immune response of chickens in normal and disease states.     

Protection of chicken against very virulent IBDV provided by in ovo priming with DNA vaccine and boosting with killed vaccine and the adjuvant effects of plasmid-encoded chicken interleukin-2 and interferon-��

The aim of this study was to examine the efficacy of in ovo prime-boost vaccination against infectious bursal disease virus (IBDV) using a DNA vaccine to prime in ovo followed by a killed-vaccine boost post hatching. In addition, the adjuvant effects of plasmid-encoded chicken interleukin-2 and chicken interferon-γ were tested in conjunction with the vaccine. A plasmid DNA vaccine (pcDNA-VP243) encoding the VP2, VP4, and VP3 proteins of the very virulent IBDV (vvIBDV) SH/92 strain was injected into the amniotic sac alone or in combination with a plasmid encoding chicken IL-2 (ChIL-2) or chicken IFN-γ (ChIFN-γ) at embryonation day 18, followed by an intramuscular injection of a commercial killed IBD vaccine at 1 week of age. The chickens were orally challenged with the vvIBDV SH/92 strain at 3 weeks of age and observed for 10 days. In ovo DNA immunization followed by a killed-vaccine boost provided significantly better immunity than the other options. No mortality was observed in this group after a challenge with the vvIBDV. The prime-boost strategy was moderately effective against bursal damage, which was measured by the bursa weight/body weight ratio, the presence of IBDV RNA, and the bursal lesion score. In ovo DNA vaccination with no boost did not provide sufficient immunity, and the addition of ChIL-2 or ChIFN-γ did not enhance protective immunity. In the ConA-induced lymphocyte proliferation assay of peripheral blood lymphocyte collected 10 days post-challenge, there was greater proliferation responses in the DNA vaccine plus boost and DNA vaccine with ChIL-2 plus boost groups compared to the other groups. These findings suggest that priming with DNA vaccine and boosting with killed vaccine is an effective strategy for protecting chickens against vvIBDV.     

Immune response to Leishmania infantum in healthy horses in Spain

Leishmania infantum infection has recently been described in horses in Europe. We report the results of a study on the immune response to L. infantum in horses living in an area endemic for leishmaniosis in NE Spain. Two ELISAs using protein A and anti-horse IgG conjugates were adapted to measure specific antibodies to L. infantum in horse sera. A lymphocyte proliferation assay (LPA) of peripheral blood mononuclear cells to L. infantum antigen was also performed to detect specific cellular immune response to Leishmania. Anti-L. infantum antibodies were detected in the serum of 16 of the horses studied (n=112) using the protein A assay but not in the assay using the anti-horse IgG conjugate. Specific lymphocyte proliferation was observed in 20 out of 55 horses. This study shows that horses in the area studied mount specific immune responses to L. infantum, and must therefore be considered among the species exposed to the parasite in this region. The infrequency of leishmaniosis in horses suggests that the immune response in this species is effective in controlling the infection.