Background Humoral immunity plays an important role in the prevention of canine distemper. Anti-CD virus (CDV) antibody has strong antiviral activity and is widely used in the treatment of CD. However, with the increase of CD cases, the availability of therapeutic CD antibody fell short of the clinical needs. Results The high-titer antiserum with the high-titer neutralizing activity against CDV was obtained from the donkeys (Dezhou Donkey) immunized with the inactivated CDV vaccine. The donkey anti-CDV IgG was purified from the donkey serum, which was identified to significantly inhibit the CDV replication in the cultured Vero cells and effectively reduce the clinical symptoms and increase the survival rates (75%) of CDV-infected dogs (Shih-tzu Dog), similar to that treated with the dog-derived anti-CDV IgG. These results indicate that donkey-derived IgG is a potential substitute for dog-derived IgG to treat the CD in clinic. Conclusions Administration of donkey-derived anti-CDV IgG can ameliorate clinical symptoms and inhibit virus replication, thereby increasing the survival of CDV-infected dogs. This study opens up a new source of therapeutic antibody for CD treatment.

采用ELISA检测方法对2018年1月至2019年5月从河北省11个地市195个不同规模猪场采集的10 479份血清进行gE抗体的检测,采用PCR方法对流产胎儿进行猪伪狂犬病病毒(pseudorabies virus,PRV)的抗原检測,并将部分阳性样本进行gE全基因的扩增和遗传变异分析.血清学检测结果显示,所检测的河北省11个地市均存在不同程度的PRV感染,血清样本gE抗体平均阳性率为50.05％;检测的195个猪场中,161个gE抗体为阳性,场群阳性率为82.56％,且存栏50～300头母猪的规模化猪场阳性率最高;不同阶段的猪群中,种猪群空怀母猪的阳性率最高,为73.75％.病原检测结果显示,204个流产胎儿中有54个为PRV阳性,阳性率为26.47％;对扩增的8株PRV进行遗传进化分析,结果显示河北省流行的毒株与2011年后的流行毒株亲缘关系较近,且均在第48位和第492位各存在1个天冬氨酸的插入,为变异毒株.以上结果提示,河北省猪场PRV的感染较为普遍.

Tanshinone IIA (Tan IIA) possesses potent anti-atherogenic function, however, the underlying pharmacological mechanism remains incompletely understood.Previous studies suggest that oxidized LDL (oxLDL)-induced NLRP3 (NOD-like receptor (NLR) family, pyrin domain-containing protein 3) inflammasome activation in macrophages plays a vital role in atherogenesis.Whether the anti-atherogenic effect of Tan IIA relies on the inhibition of the NLRP3 inflammasome has not been investigated before.In this study, we found that Tan IIA treatment of high-fat diet fed ApoE-/-mice significantly attenuated NLRP3 inflammasome activation in vivo.Consistently, Tan IIA also potently inhibited oxLDL-induced NLRP3 inflammasome activation in mouse macrophages.Mechanically, Tan IIA inhibited NF-κB activation to downregulate pro-interleukin (IL) -1β and NLRP3 expression, and decreased oxLDLinduced expression of lectin-like oxidized LDL receptor-1 (LOX-1) and cluster of differentiation 36 (CD36), thereby attenuating oxLDL cellular uptake and subsequent induction of mitochondrial and lysosomal damage -events that promote the NLRP3 inflammasome assembly.Through regulating both the inflammasome 'priming' and 'activation' steps, Tan IIA potently inhibited oxLDL-induced NLRP3 inflammasome activation, thereby ameliorating atherogenesis.

旨在分离纯化牛天然肺泡表面活性蛋白A(SP-A)和分析其抗菌活性,首先通过共价交联法制备出可特异吸附SP-A的麦芽糖-Sepharose(MS)胶粒,然后用MS胶粒从牛肺泡洗出液中吸附牛SP-A,从而得到牛SP-A.SDS-PAGE和Western blot检测表明,牛SP-A单体的相对分子质量为30 ku,二聚体为60 ku.分离纯化的牛SP-A能够凝聚大肠杆菌,表明SP-A具有生物活性.抗菌活性检测结果表明,牛SP-A在体外可明显抑制金黄色葡萄球菌、无乳链球菌和致病性大肠杆菌的增殖,尤其是对革兰阴性菌的抑制作用更为明显,表明本试验制备的牛天然SP-A具有抗菌活性,这为进一步研究其生物学特性及抗菌和抗病毒活性提供了必要的条件,也为SP-A作为抗菌制剂的研发提供了新的思路.

旨在探明1,25(OH)2D3对猪伪狂犬病病毒(pseudorabies virus,PRV)诱导小鼠流产的影响.采用颈背部皮下注射不同剂量PRV建立PRV诱导小鼠流产模型;通过实时荧光定量PCR检测不同剂量1,25(OH)2D3预处理组和生理盐水预处理组小鼠卵巢和子宫内VDR mRNA的水平及PRV量;利用组织学病理切片技术观察PRV引起的1,25(OH)2D3预处理组和生理盐水处理组小鼠子宫和卵巢的病理变化.结果 显示,成功建立了小鼠流产模型,确定了引起小鼠流产的PRV最佳接种剂量为0.35 mL 1 000 TCID50 PRV;用300 μg/kg 1,25(OH)2D3预处理,小鼠VDR表达水平最高,能有效抑制病毒的增殖,对PRV感染小鼠流产的抑制作用最强.组织病理学检查发现,相较于生理盐水预处理的PRV感染组,1,25(OH)2D3预处理组小鼠卵泡腔内颗粒细胞增多、红细胞量减少;子宫腔内红细胞数量减少或消失,子宫内膜皱褶增多.结果 表明,1,25(OH)2D3能有效抑制PRV增殖,降低PRV诱导的小鼠流产.

CD83, either in its membrance-bound form (mCD83) or soluble form (sCD83), is an important immunomodulatory molecule in humans and mice. While mCD83 is immunostimulatory, sCD83 exhibits striking immunosuppressive activities, suggesting that sCD83 may be used to combat inflammatory diseases, such as rheumatoid arthritis, graft-versus-host disease and habitual abortion. Although many studies had shed lights on the role of CD83 in humans and mice, little is known about CD83 in other animals. Recently, we showed that porcine CD83 had similar biochemical characteristics and immunoregulatory functions as its human counterpart. However, whether porcine sCD83 (psCD83) is involved in maintaining the immunological tolerance at the maternal-fetal interface and thereby prevents embryo loss and abortion during pregnancy is unclear. In this study, we used LPS-induced animal model to analyze the effect of porcine sCD83 on the mouse abortion. Results showed that psCD83 could significantly alleviate LPS-induced abortion in mice, indicating that the psCD83 had the function of fetal protection. Mechanically, psCD83-mediated fetal protection was related to the promotion on Th2 cytokine production, Treg cell differentiation and trophoblast invasion. This study provides a molecular basis for the fetal protection of psCD83, as well as a potential target for the regulation of maternal-fetal interfacial immune tolerance.

Canine parvovirus (CPV) non-structural protein-1 (NS1) plays crucial roles in CPV replication and transcription, as well as pathogenic effects to the host. However, the mechanism was not fully understood. Lack of NS1 antibody is one of the restricting factors for NS1 function investigation. To prepare NS1 monoclonal antibody (mAb), the NS1 epitope (AA461 ~ AA650) gene was amplified by PCR, and inserted into pGEX-4T-1vector to construct the prokaryotic expression vector of GST-tag-fused NS1 epitope gene. The NS1 fusion protein was expressed in E. coli, and purified with GSH-magnetic beads, and then used to immunize BALB/c mice. The mouse splenic lymphocytes were isolated and fused with myeloma cells (SP 2/0) to generate hybridoma cells. After several rounds of screening by ELISA, a hybridoma cell clone (1B8) stably expressing NS1 mAb was developed. A large amount of NS1 mAb was prepared from mouse ascites fluid. The isotype of NS1 mAb was identified as IgG1, which can specifically bind NS1 protein in either CPV-infected cells or NS1 vector-transfected cells, indicating the NS1 mAb is effective in detecting NS1 protein. Meanwhile, we used the NS1 mAb to investigate NS1 dynamic changes by qRT-PCR and location by confocal imaging in CPV-infected host cells and showed that NS1 began to appear in the cells at 12 h after CPV infection and reached the highest level at 42 h, NS1 protein was mainly located in nucleus of the cells. This study provided a necessary condition for further investigation on molecular mechanism of NS1 function and pathogenicity.

为探讨Th1和Th2细胞因子在单增李斯特菌(Listeria monocytogenes,LM)致小鼠流产中的作用,用LM感染妊娠小鼠和小鼠巨噬细胞,统计胚胎存活率,酶联免疫测定(enzyme 1inked lmmuno sorbent assay,ELISA)小鼠子宫组织中和巨噬细胞培养基中主要的Th1和Th2细胞因子水平.结果显示,LM诱导的流产小鼠子宫中Th1细胞因子水平显著增加,溶血素O(listeriolysin O,LLO)基因缺陷型(△hly)LM感染组胚胎存活率和子宫中Th1、Th2细胞因子水平无显著变化;LM感染的巨噬细胞分泌的Th1细胞因子和白细胞介素-4(interleukin,IL-4)水平显著增加,IL-10水平显著降低,△hly LM感染组Th1和Th2细胞因子水平无显著变化.因此,LM感染后子宫中Th1/Th2细胞因子失衡诱导了小鼠流产,这可为临床防治LM诱导的流产提供科学依据.

为通过E.coli表达系统制备猪细小病毒NS1抗原表位区蛋白,本试验利用DNAstar软件确定含有抗原表位的NS1蛋白片段,然后运用PCR方法从含有NS1基因的载体中扩增NS1抗原表位区基因片段,并将该基因片段插入到原核表达载体pGEX-4T-1中,构建成与GST标签融合的NS1抗原表位基因的表达载体pGEX-GST-NS1,将表达载体转化BL21大肠杆菌筛选出工程菌.经IPTG诱导表达NS1融合蛋白,经GST-琼脂糖凝胶磁珠分离纯化,制备出与GST融合的NS1抗原表位蛋白.经SDS-PAGE以及Western blot鉴定,制备的NS1融合蛋白分子量和预期一致,并且可以和抗GST标签抗体进行特异反应,表明NS1抗原蛋白已成功制备,为下一步NS1抗体的研制提供了必要的条件.

为探讨犬细小病毒(CPV)引起宿主细胞凋亡的分子机制,用CPV感染MDCK细胞,感染后通过台盼蓝染色法检测不同时间的细胞存活率,用Annexin V-FITC/PI法检测磷脂酰丝氨酸外翻情况,用试剂盒检测Caspase-3活性,分析CPV诱导细胞的凋亡.并通过流式细胞术检测细胞线粒体的损伤和ROS的产生探讨其分子机制.结果 显示,随着病毒感染时间的不断延长,CPV可明显降低MDCK细胞存活率,促进磷脂酰丝氨酸外翻,增强Caspase-3活性,表明CPV可引起细胞凋亡.同时还发现CPV可明显提高细胞内ROS的水平,引起线粒体的损伤,这可能是CPV诱导细胞凋亡的重要分子基础.由此可见,CPV诱导MDCK细胞凋亡与CPV增加细胞ROS的产生和线粒体的损伤有关.

Emerging evidence suggests that CD83, a dendritic cells (DCs) maturation marker in humans and mice, may prossess immunomodulatory capacities. Although porcine CD83 shares ∼75% sequence homology with its human counterpart, whether it functions as an immunoregulatory molecule remains unknown. To investigate porcine CD83 function, we deleted it in porcine DCs by RNA intereference. Results show that membrane-bound CD83 (mCD83) promotes DC-mediated T cell proliferation and cytokine production, thus confirming its immunoregulatory capacity. Intriguingly, porcine soluble CD83 (sCD83) treatment instead led to inhibition of DC-mediated T cell activation. Moreover, porcine sCD83 also inhibited differentiation of prepheral blood mononuclear cells (PBMCs) into DCs. These results collectively indicate that in addition to being a DC maturation maker, both membrane bound and souble porcine CD83 serve as immunoregulatory molecules with opposite effects on DC-mediated T cell activation and DC differentiation.

Chicken infectious bursal disease (IBD) is still incompletely controlled worldwide. Although IBD virus (IBDV) VP2 DNA vaccine was considered a safe vaccine for IBD prevention, the immunogenicity by itself remains poor, resulting in the failure of effectively protecting chickens from infection. We and others demonstrated that chicken IL-2 (chIL-2) and chIL-7 have the capacity to enhance the immunogenicity of the VP2 DNA vaccine. However, whether chIL-2 and chIL-7 can mutually enhance the immunogenicity of VP2 DNA vaccine and thereby augment the latter's protection efficacy remains unknown. By using chIL-2/chIL-7 bicistronic gene vector to co-immunize the chickens together with the VP2 DNA vaccine, we now show that chIL-2 and chIL-7 significantly increased IBDV VP2-specific antibody titers, T cell proliferation, and IFN-γ production, resulting in the ultimate enhancement of vaccine-induced protection efficacy relative to that of chIL-2 or chIL-7 gene vectors alone. These results suggest that chIL-2 and chIL-7 can mutually enhance VP2 DNA vaccine's efficacy, thereby establishing a concrete foundation for future optimization of IBDV VP2 DNA vaccine to prevent/treat chicken IBD.

Inflammation benefits the host by promoting the elimination of invading pathogens and clearance of cellular debris after tissue injury. Inflammation also stimulates tissue repair and regeneration to restore homeostasis and organismal health. Emerging evidence suggests that mitochondrial DNA (mtDNA), the only form of non-nuclear DNA in eukaryotic cells, is a major activator of inflammation when leaked out from stressed mitochondria. Here, we review the current understanding on the role of mtDNA in innate immunity, discussing how dysregulated mtDNA metabolism can promote chronic inflammation and disease progression.

犬细小病毒(Canine parvovirus,CPV)非结构蛋白1(nonstructural protein 1,NS1)是CPV的重要功能蛋白,参与病毒的复制和转录,同时也是宿主细胞的毒性蛋白.为探索其生物学功能,需要制备重组NS1蛋白.本研究采用人(Homo sapiens)CD5信号肽序列介导NS1蛋白在真核细胞中进行分泌表达,以制备高纯度的NS1重组蛋白.首先,根据人CD5信号肽序列(GenBank No.NM_014207.4),设计合成1对两端带有限制性内切酶位点的互补全长CD5信号肽序列(2条寡核苷酸链);然后将2个寡核苷酸链进行退火处理,生成CD5信号肽基因,进而通过酶切位点插入真核表达载体pcDNA3.1-NS1/H的NS1基因5'端,与NS1基因融合,构建形成与His标签融合的NS1分泌型真核表达载体pcDNA3.1-CD5-NS1/H.将该表达载体转染人胚胎肾(human embryonic kidney,HEK)293T细胞使其表达,用Ni-NTA琼脂糖胶粒通过亲和层析法从细胞培养基中纯化重组NS1蛋白.SDS-PAGE和Western blot检测结果表明,纯化的NS1蛋白纯度达95％以上,并能与抗His标签抗体进行特异结合,表明所构建的表达载体可以介导NS1在真核细胞中进行分泌型表达.为提高表达水平,进一步分析载体用量、转染时间和表达时间对NS1表达的影响,初步确定NS1表达的最适条件为在T75细胞培养瓶中载体用量30μg、转染时间5 h、表达时间48 h.本研究可为进一步探讨NS1的生物学功能提供基础资料.

Autophagy plays a crucial role in eliminating protein aggregates, damaged organelles and invading pathogens. Genetically engineered cell line stably expressing green fluorescent protein (GFP)-tagged microtubule-associated protein light chain 3 (LC3) is extensively used to test autophagy through observing GFP puncta formation in the cells by fluorescence imaging. However, canine LC3 (cLC3) gene has not been cloned, therefore, GFP-tagged canine LC3 (GFP-cLC3) detection system has not been established. To generate GFP-cLC3 stably expressing canine-derived macrophages, the cLC3 cDNA was first amplified by RT-PCR and inserted into pEGFP-C1 plasmid to create GFP-cLC3 gene fusion. This genetic element was then transducted into canine macrophages mediated by lentivirus vector to generate the canine macrophages stably expressing fusion protein. Results showed that the sequence of cLC3 cloned in this study is highly homologous with other animals (80-95% homology). Phenotypic and functional analysis of these engineered cells revealed that GFP-cLC3 was indeed stably expressed and rapamycin or starvation can effectively induce GFP puncta formation in the cells, indicative of autophagosome formation. These GFP-cLC3-expressing cells may thus be useful to study autophagy in canine.

Porcine parvovirus (PPV) is an important pathogen causing reproductive failure in pigs. PPV-induced cell apoptosis has been recently identified as being involved in PPV-induced placental tissue damages resulting in reproductive failure. However, the molecular mechanism was not fully elucidated. Here we demonstrate that PPV nonstructural protein 1 (NS1) can induce host cell apoptosis and death, thereby indicating the NS1 may play a crucial role in PPV-induced placental tissue damages and reproductive failure. We have found that NS1-induced apoptosis was significantly inhibited by caspase 9 inhibitor, but not caspase 8 inhibitor, and transfection of NS1 gene into PK-15 cells significantly inhibited mitochondria-associated antiapoptotic molecules Bcl-2 and Mcl-1 expressions and enhanced proapoptotic molecules Bax, P21, and P53 expressions, suggesting that NS1-induced apoptosis is mainly through the mitochondria-mediated intrinsic apoptosis pathway. We also found that both PPV infection and NS1 vector transfection could cause host DNA damage resulting in cell cycle arrest at the G1 and G2 phases, trigger mitochondrial ROS accumulation resulting in mitochondria damage, and therefore, induce the host cell apoptosis. This study provides a molecular basis for elucidating PPV-induced cell apoptosis and reproductive failure.

通过PCR方法从猪细小病毒(PPV)基因组中扩增NS1基因,将其克隆到pcDNA3.1A质粒中,构建成与Myc/His标签融合的NS1真核表达载体.将表达载体转染PK-15细胞使其表达.利用猪偏爱的密码子对NS1全基因进行了密码子优化,通过构建表达载体和转染细胞,检测并比较了密码子优化前后的表达水平.结果显示:扩增的NS1基因与GenBank序列一致,构建的表达载体结构正确;Western blot检测表明,NS1基因能够在PK-15细胞中进行表达,但表达水平很低,有时检测不到,从而严重影响对其的进一步研究;密码子优化后NS1基因的表达水平由优化前的7μg/T25培养瓶增加至32μg/T25培养瓶,是原来的4倍.可见密码子优化可明显提高NS1蛋白在PK-15细胞中的表达,这为下一步研究NS1蛋白的功能提供了有利条件.

为了制备大量廉价的可溶性犬细小病毒(CPV) VP2蛋白抗原,试验截选了VP2抗原表位集中的基因片段VP2-70进行密码子优化及合成,将合成基因克隆到pET-32a(+)载体中,构建原核表达载体pET-32-VP2-70,转化BL21(DE3)工程菌,IPTG诱导表达后进行Ni-NTA亲和层析纯化,Western-blot检测验证其生物学活性.结果表明:试验构建的VP2-70表达载体能够介导VP2-70融合蛋白在大肠杆菌中高效表达,表达量高出野生型VP2-70融合蛋白16 mg/L;Westem-blot检测显示,该蛋白具有很好的免疫反应原性,可为CPV亚单位疫苗和免疫学诊断方法的研究提供候选抗原.

为制备驴源抗犬细小病毒(canine parvovirus,CPV)抗体,首先利用F81猫肾细胞扩增CPV-2a病毒株,经甲醛灭活和氢氧化铝胶乳化,制成CPV灭活疫苗,病毒滴度为10-8.5 TICD50,然后通过肌肉分点注射免疫驴,连续免疫3次(第1次注射3 mL,后2次各注射6 mL),每次免疫前采血,分析驴血清的抗体滴度和中和抗体效价;利用盐析法和离子交换层析法分离和纯化驴血清IgG.用IgG处理幼犬(50 mg/kg),用CPV-2a毒株对IgG处理的犬进行攻毒,分析IgG抗CPV的活性.结果显示,免疫3次后驴血清的CPV抗体滴度为1∶8 192,中和抗体效价为28.5.用制备的IgG处理幼犬未发现不良反应,经CPV-2a攻毒实验证实IgG处理犬的发病率和死亡率分别为33％,0％,均明显低于非处理组(分别为100％,83％),说明制备的驴源IgG具有明显的抗CPV活性.

随着高校教育改革的不断深入和课程授课时数的减少,以学生为学习主体的教育模式成为高等教育的主导模式,而如何帮助学生明确学习目的和重点,提高学生学习的能动性和效率,成为亟待解决的问题.高等院校课程改革的核心是课堂教学改革,而课堂教学模式改革则是课堂教学改革的突破口.