猪繁殖呼吸综合征弱毒疫苗的研制

PRRSV—SА弱毒株免疫仔猪,无不良临床表现,不排毒;免疫怀孕母猪,母猪精神、食欲、体温均正常,并在预产期顺利产仔,表现出良好的安全性。SА弱毒免疫猪后2周即产生抗体反应,免疫仔猪4～6个月后,能抵抗强毒攻击;母猪在配种前2周免疫,在怀孕90-96d时,用强毒攻击,不表现任何临床症状,并顺利产仔,表现出坚强的免疫力。用SА弱毒制备成冻干疫苗,免疫猪表现出良好的安全性和免疫力。证明PRRSV—SА弱毒疫苗能预防猪繁殖呼吸综合征。     

雷帕霉素对稀有鮈鲫卵子发生的影响

[目的]为鱼类生殖生理学和繁殖生物学研究提供参考.[方法]以稀有鮈鲫为试验动物,采用mTOR通路抑制剂雷帕霉素处理3月龄雌性个体至4月龄,正常饲养至5月龄,以同时注射等量不合雷帕霉素的稀释剂和二甲基亚砜(DMSO)为对照,测定不同阶段性腺指数(GSI)和卵巢发育情况,Real time PCR检测卵子发生相关基因的表达...     

雷帕霉素产生菌吸水链霉菌NRRL5491接合转移系统的建立

对标准的链霉菌接合转移方法进行优化建立起适用于吸水链霉菌NRRL5491的接合转移系统。40℃温浴20 min为孢子的最佳热激处理条件;整合型质粒pSET152的接合转移效率是穿梭型质粒pOJ446的3~6倍;当阿泊拉霉素覆盖浓度为1μg/mL时,pSET152的接合转移效率是50μg/mL时的4.6倍,而pOJ446接合转移效率相应提高了2.4倍;MS培养基上接合转移效率是SY琼脂培养基上的3.4倍。采用最适优化条件时,接合转移频率可达到2×10-6,足够用于基因置换等遗传操作。     

乙型肝炎失代偿期肝硬化患者血清内毒素、 IL-12、IL-18水平的检测及其意义

目的探讨乙型肝炎肝硬化血清内毒素(LPS)、白介素-12(IL-12)、白介素-18(IL-18)水平变化及临床意义.方法采用鲎试剂(Ⅱ)-偶氮显色法、ELISA双抗体夹心法,分别测定80例肝硬化患者血清LPS、IL-12、IL-18水平.另设正常对照组(32例).结果与正常对照组比较,肝硬化患者血清LPS、IL-12、IL-18水平均明显升高,且随着肝功能越差,升高越明显(P＜0.01);腹水组的LPS及IL-12、IL-18水平较无腹水组也明显增高(P＜0.01).结论LPS、IL-12、IL-18均参与了乙型肝炎肝硬化的病理生理过程,并与病情的变化密切相关.检测这些指标有助于判断肝硬化患者的肝功能状况及疾病的严重程度.     

板蓝根多糖对PRRS弱毒苗免疫猪抗体和T细胞亚群的影响

研究板蓝根多糖对PRRSV弱毒苗免疫抗体和猪外周血T细胞亚群的影响。35日龄断奶仔猪16只随机分为4组,将多糖分别以高、低两个剂量和猪PRRSV弱毒苗同时注射,于免疫后不同的时间点采血,ELISA方法检测猪PRRSV抗体水平,流式细胞术检测猪外周血中T淋巴细胞亚群的变化。弱毒苗免疫后7d,板蓝根多糖高剂量组即可检测到PRRSV阳性抗体,而其它组在免疫后14d,均可检测到PRRS阳性抗体,板蓝根多糖对PRRS弱毒苗产生抗体的影响是和剂量密切相关的,高剂量能提高抗体水平,低剂量会阻碍PRRSV抗体的产生。板蓝根多糖和PRRS弱毒苗联合使用对猪外周血CD3^＋和CD4^＋细胞数量的影响不显著,在早期可促进猪外周血CD8^＋细胞的增殖。合适剂量的板蓝根多糖可以作为免疫增强剂与PRRSV弱毒苗联合使用。     

PCV2和猪伪狂犬病弱毒疫苗体外共接种对猪PBMC中IL-2及IL-6 mRNA表达的影响

将猪圆环病毒2型（PCV2）与猪伪狂犬病弱毒疫苗（PRV）体外单接种和共接种仔猪外周血单个核细胞（PB-MC）,采用real-time PCR技术定量检测接种后不同时间PBMC细胞因子IL-2和IL-6的mRNA表达水平,以分析PCV2对PRV免疫反应的影响。结果显示,PRV接种组PBMC中IL-2与IL-6的mRNA表达均出现显著上调（P〈0.05）,PCV2接种组IL-2与IL-6的mRNA表达在接种后的部分时间也出现显著上调,但PCV2与PRV共接种组的IL-2与IL-6的mRNA表达较PRV组均出现显著下调,表明PCV2能够抑制PRV促进猪PBMC中IL-2与IL-6的mRNA表达上调作用,提示PCV2可能会对PRV的免疫反应产生不利影响。     

中药复方多糖对鸡IFN-γ、IL-4和L-12质量浓度的影响

通过观察和比较盐酸左旋咪唑、黄芪多糖和对鼠免疫增强效果显著的中药复方多糖对雏鸡细胞因子IFN-γ、IL-4和IL-12质量浓度的动态变化,筛选出可显著提高雏鸡细胞因子质量浓度的中药复方多糖的最佳剂量。将300羽雄性良凤青脚麻鸡随机分为6组,即生理盐水组(对照)、黄芪多糖组(APS)、盐酸左旋咪唑组(LM)、高剂量中药复方多糖组(cCHMPSH)、中剂量中药复方多糖组(cCHMPSM)和低剂量中药复方多糖组(cCHMPSL),每组50只。于8日龄时分别皮下注射生理盐水、25g/L APS、50g/L LM、50g/L cCHMPS、25g/L cCHMPS、12.5g/L cCHMPS,每只注射0.2mL,连续注射7d,在免疫后第8、14、21、28、35、42天采血,测定外周血中IFN-γ、IL-4和IL-12的质量浓度。结果表明,黄芪多糖和中药复方多糖均能显著提高外周血中IFN-γ、IL-4和IL-12的质量浓度,调节Thl/Th2免疫平衡,并且中剂量中药复方多糖免疫效果优于黄芪多糖和高、低剂量中药复方多糖,提示中药复方多糖具有开发成疗效确切、稳定、无毒的免疫增强剂的潜力。     

应用非脂佐剂提高鸡新城疫(ND)Ⅱ系弱毒疫苗效力的研究

2,4日龄雏鸡接种非脂佐剂苗进行首免后,HI抗体滴度的升高不受母源抗体的干扰,注射该苗雏鸡的HI抗体滴度极显著地高于Ⅱ系苗接种组(p>0.01);HI抗体滴度下降至有效滴度以下的时间也优于Ⅱ系苗组。人工感染IBD造成免疫缺陷的雏鸡接种非脂佐剂苗,能够使其HI抗体滴度达到健康雏鸡单独应用Ⅱ系苗的水平(p>0.05)。非脂佐剂苗的保护力明显高于Ⅱ系苗(p>0.05),并且非脂佐剂苗具有Ⅱ系苗同样的安全性。当母源抗体降为零或给雏鸡二免时,非脂佐剂苗无明显提高HI滴度的作用,与Ⅱ系苗组相比较,差异不显著(P>0.05)。非脂佐剂苗免疫雏鸡和Ⅱ系苗免疫雏鸡具有相同的细胞免疫水平(p>0.05)。     

局部应用IL-12对小鼠S180实体瘤的抗瘤效应及机制初探

目的研究白细胞介素-12（Interleukin 12,IL-12）对小鼠S180实体瘤的抗肿瘤作用并初步探讨其机制.方法将小鼠左后肢皮下接种S180细胞荷瘤后的BaLB/c小鼠随机分为生理盐水组和IL-12组,分别给予生理盐水和IL-12.接种第28天,眼球取血,流式细胞仪分析外周血T细胞亚群的变化;解剖取瘤称重,计...     

鸡新城疫Ⅰ系疫苗诱导鸡IL-2基因转录及cDNA克隆的研究

应用RT-PCR方法从鸡新城疫 系疫苗接种鸡胚的脾淋巴细胞中扩增出IL-2基因cDNA。结果表明,以鸡新城疫 系疫苗稀释50倍或100倍接种10日龄鸡胚,培养48h的脾脏提取mRNA的扩增效果最好。测序结果显示,所扩增片段的核苷酸序列及其所编码的氨基酸序列,与已发表的IL-2基因的同源性分别为97.7%～99.8%和95.1%～99.3%。其中第8位、68位和130位氨基酸(分别是L,V和S)的变异是其他品种鸡所没有的。     

猪繁殖与呼吸综合征弱毒苗阴道免疫对母猪子宫IgA和IgG分泌细胞的影响

用猪繁殖与呼吸综合征 (PRRS) 弱毒苗分别通过生殖道黏膜免疫和肌肉注射免疫母猪 (各 4头 ), 另 4头经生殖道接种生理盐水作为对照, 运用免疫组化技术显示子宫局部IgA分泌细胞和IgG分泌细胞的分布。结果表明, 对照组子宫中IgA分泌细胞和IgG分泌细胞主要分布于黏膜固有层浅层, 子宫颈中的IgA分泌细胞数量多于子宫角, 而子宫角中IgG分泌细胞数量多于子宫颈。生殖道免疫后, 与对照组相比, 子宫角IgA分泌细胞数量极显著增多 (P<0 01), 子宫颈IgA分泌细胞数量无显著增加; 子宫角IgG分泌细胞数量有所增加, 但差异不显著, 子宫颈IgG分泌细胞数量无显著变化。肌肉注射后, 子宫中IgA和IgG抗体分泌细胞数量与对照组间无明显变化。表明用PRRS弱毒苗经生殖道黏膜免疫可增加子宫局部抗体分泌细胞数量, 而全身免疫对子宫抗体分泌细胞无显著影响。     

Study on the production of IL-12, IL-10 in early stage after infection with Babesia microti and Babesia rodhaini in mice

The serum levels of IL-12 and IL-10 in mice after infected with Babesia microti(B. microti) and Babesia rodhaini (B. rodhaini) were examined. Collected the mice serum and examined the concentration of IL-12 and IL-10 by using ELISA after infection with B. microti and B. rodhaini at 0, 3, 6, 9, 12, 18, 24, 36, 72, 96 h in mice. The results showed that B. microti infection resulted in IL-12 increasing, which peaked at 3 and 24 h after the infection, while same infection did not induce a significant change in IL-10 compared to uninfected mice. When mice were infected with B. rodhaini, any significant changes were not decteted both in IL-12 and IL-10 in comparison with uninfected animals during the period of 3-72 h after infection. Instead, a significant decline in IL-12 and IL-10 was found compared to uninfected mice 96 h after infection with B. rodhaini. It indicates that the mutagenetic cytokine is IL-12 in the serum of mice after infection with B. microti, and no any significant changes were detected in both IL-12 and IL-10 from 0 to 72 h after infected with B. rodhaini.     

IL-2真核质粒对乳酸杆菌载体口蹄疫VP1 DNA疫苗猪体免疫反应的分子佐剂效应

为研究猪IL-2真核表达质粒对乳酸杆菌载体口蹄疫VP1 DNA疫苗(Lactobacillus acidophilusSFMD-1)猪体免疫的分子佐剂效应,通过RT-PCR方法从猪的肠系膜淋巴结克隆猪IL-2基因,基于pRc/CMV2载体,构建编码猪IL-2的真核表达质粒pCSIL2,以RT-PCR方法分析了该质粒转染PK15细胞以后的mRNA表达。结果显示预期的429 bp IL-2带仅出现在pCSIL2转染细胞。试验猪单独免疫L.acidophilusSFMD-1或者联合免疫pCSIL2质粒,后者能够增强FMDV-VP1特异的T细胞和B细胞反应,免疫后5周,单独免疫组和联合免疫组的抗体水平分别为1.50和1.66;免疫后6周,抗体水平分别上升到1.83和2.28;在T细胞增殖试验中,L.acidophilusSFMD-1单独免疫组和pCSIL2联合免疫组的刺激指数(SI)分别为2.00和2.70。结果表明,联合应用IL-2真核表达质粒是改进乳酸杆菌载体口蹄疫VP1 DNA疫苗免疫原性和效能的有效方法。     

分子佐剂鞭毛素对弓形虫SAG1蛋白抗体应答的影响

将沙门氏菌鞭毛素和弓形虫免疫优势表面抗原1(SAG1)融合表达,研究其在小鼠上激发的体液免疫应答.首先,采用PCR扩增SAG1片段.其次,将SAG1连接到原核表达载体pET-28a中构建表达质粒pET-28a-SAG1;连接到实验室已有的pET-28a鞭毛素质粒中构建表达质粒pET-28a-F-SAG1;质粒转化至大肠杆菌BL21(DE3)菌株中并进行表达.将纯化后的蛋白免疫小鼠,每隔2周免疫一次,共免疫3次,第3次免疫后15 d取小鼠血清.最后,采用蛋白印迹检测蛋白的大小;用酶联免疫吸附方法检测血清中多克隆抗体的效价.结果表明,诱导出的SAG1蛋白大小为30 ku,F-SAG1大小为70 ku.酶联免疫吸附方法检测的D450 nm均为阴性对照的两倍,表明该多克隆抗体具有较高效价.可见,鞭毛素可以作为分子佐剂促进弓形虫亚单位疫苗的体液免疫应答.     

Evaluating the efficacy of an attenuated Streptococcus equi ssp. zooepidemicus vaccine produced by multi-gene deletion in pathogenicity island SeseCisland_4

Streptococcus equi ssp. zooepidemicus(SEZ) is a pathogen associated with a wild range of animal species. Frequent outbreaks have occurred in recent years in pigs, horses, goats and dogs which is liable to infect humans. There is a lack of efficient vaccines against this disease and the occurrence of antibiotic resistance may render drug therapies ineffective. In this study, gene deletion mutant(ΔSEZ) in pathogenicity islands SeseCisland_4 was constructed. The mutant ΔSEZ had a 52-fold decrease in 50% lethal dose(LD_(50)) and had less capacity to adhere epithelial cells. Importantly, immunization of mice with attenuated vaccine ΔSEZ at the dose of 10~2 colony-forming units(CFU) mL~(–1) elicited a significant humoral antibody response, with an antibody titer of 1:12 800. Therefore, 10~2 CFU mL~(–1) might be used as the appropriate immune dose for the attenuated vaccine ΔSEZ, which provided mice with efficient protection against virulent SEZ. In addition, the hyperimmune sera against 10~2 CFU m L–1 attenuated vaccine ΔSEZ could confer significant protection against virulent SEZ infection in the passive immunization experiment and exhibited efficient bactericidal activity in the whole blood assay. Meanwhile, no viable bacteria was detected in blood when mice were immunized with ΔSEZ at the dose of 10~2 CFU mL~(–1) via hypodermic injection. Thereafter, the mutant ΔSEZ at the dose of 10~2 CFU mL~(–1) could confer significant protection in mice and had less negative effects on host, which could be an effective attenuated vaccine candidate for the prevention of SEZ.     

一株瑞士乳杆菌的分离鉴定及其产VB_(12)的影响因素研究

对市售的乳酸饮品的乳酸菌进行了分离纯化,得到菌株HYWZ-6,结合16S r RNA基因序列分析,确定为瑞士乳杆菌(Lactobacillus helveticus),并以温度、Na Cl、胆盐、p H及柠檬酸三钠作为影响因素,对其产VB_(12)进行研究,并利用酶联免疫分析法对该菌在不同因素条件下培养100 h后产VB_(12)的含量进行测定。结果表明,HYWZ-6菌株在不同条件下产VB_(12)的差异性较大,35℃VB_(12)的含量最高,25℃最低,35℃时含量比25℃时高出175.2%;Na Cl质量分数与VB_(12)的含量呈反比,Na Cl质量分数为1%时VB_(12)含量最高,7%时最低,相差224.4%;胆盐质量分数为0.1%时VB_(12)含量达到最高;p H为4时VB_(12)含量最高,在p H为7时VB_(12)含量最低,前者是后者的5.8倍;柠檬酸三钠质量分数为2.5%时VB_(12)的产量最高,比最低产量高出143.2%。     

Cellular immune responses of BALB/c mice induced by intramuscular injection of PRRSV-ORF5 DNA vaccine with different doses

BALB/c mice were immunized with 50 μg, 100 μg, 200 μg of pcDNA-PRRSV-ORF5 DNA vaccine respectively by intramuscular injection, with PBS and pcDNA3.1(+) as controls. Fluorescence activated cell Sorter (FACS) was used to detect the number of CD₄ ⁺ and CD₈ ⁺ T-lymphocytes. T-lymphocyte proliferation test was used to detect proliferation of the T-lymphocyte cells in peripheral blood lymphocytes of mice vaccinated with pcDNA-PRRSV-ORF5 DNA vaccine. The results showed that the difference in ConA response to T-lymphocytes in blood was highly significant between all experimental groups and the control group (P < 0.01). The number of CD₄ ⁺ T-lymphocytes in experimental groups was significantly higher than that of the control group 7 d after vaccination. The number of CD₈ ⁺ T-lymphocytes in the experimental groups was higher than that of the control group 28 d after vaccination. Mice immunized with a higher dose (200 μg) of DNA vaccine demonstrated higher cellular immune response than those immunized with a lower dose (100 μg, 50 μg) of DNA vaccine. The results demonstrated that pcDNA-PRRSV-ORF5 DNA vaccine could induce a good cellular immune response which may be dose-dependent. __________ Translated from China J Vet Sci Mar, 2006, 26(2): 111–114 [译自: 中国兽医学报] The first three authors contribute equally to this work.     

Protective efficacy of an H5/H7 trivalent inactivated vaccine produced from Re-11, Re-12, and H7-Re2 strains against challenge with different H5 and H7 viruses in chickens

We developed an H5/H7 trivalent inactivated vaccine by using Re-11, Re-12, and H7-Re2 vaccine seed viruses, which were generated by reverse genetics and derived their HA genes from A/duck/Guizhou/S4184/2017(H5 N6)(DK/GZ/S4184/17)(a clade 2.3.4.4 d virus), A/chicken/Liaoning/SD007/2017(H5 N1)(CK/LN/SD007/17)(a clade 2.3.2.1 d virus), and A/chicken/Guangxi/SD098/2017(H7 N9)(CK/GX/SD098/17), respectively. The protective efficacy of this novel vaccine and that of the recently used H5/H7 bivalent inactivated vaccine against different H5 and H7 N9 viruses was evaluated in chickens. We found that the H5/H7 bivalent vaccine provided solid protection against the H7 N9 virus CK/GX/SD098/17, but only 50–60% protection against different H5 viruses. In contrast, the novel H5/H7 trivalent vaccine provided complete protection against the H5 and H7 viruses tested. Our study underscores the importance of timely updating of vaccines for avian influenza control.