Infectious bursal disease virus (IBDV) is an important immunosuppressive virus in chickens, which caused an acute and highly contagious infectious disease and resulted in considerable economic losses in the world poultry industry. Although IBDV VP2 DNA vaccine has been extensively proved only partial protection, the efficacy of DNA vaccines against IBDV can be augmented by choosing potential molecular adjuvants. To investigate whether chicken IL-17 (chIL-17) is qualified for the molecular adjuvant, we constructed chIL-17 gene vector, co-immunized chickens with chIL-17 gene vector and IBDV VP2 DNA vector, and analyzed the effect of IL-17 gene on the immunogenicity of VP2 DNA vaccine. Results showed that co-administrated chIL-17 gene and VP2 DNA vaccine significantly increased IBDV-specific serum antibody titer, and promoted lymphocyte proliferation and interference-γ (IFN-γ) production. Mechanically, chIL-17 reduced virus titer in bursa of fabricius tissue and mortality of IBDV-infected chicken. More importantly, chIL-17 gene significantly enhanced the protection induced by the VP2 gene against virulent IBDV infection.These data show that chIL-17 is a promising immunoadjuvant to facilitate humoral as well as cellular immunity in a vaccine setting against IBDV, and suggest that it should be evaluated as a new DNA vaccine adjuvant in strategies for other poultry diseases.

Feline parvovirus (FPV) causes severe gastroenteritis and leukopenia in cats, with high morbidity and mortality, necessitating a rapid and effective antigen diagnostic test with high sensitivity and specificity. In this study, a diagnostic platform based on a combination of Recombinase-Aided Amplification (RAA) and CRISPR/Cas12a was established for detecting FPV. Cas12a recombinant protein was purified using Nickel-Nitriloacetic Acid resin after heterologous expression in Escherichia coli. The results of RAA-CRISPR/Cas12a can be detected with a fluorescence reader or lateral flow strips (LFS) for on-site detection. The RAA-CRISPR/Cas12a-LFS had a detection limit of 2.1 × 100 copies of recombinant plasmids per reaction, compared with 2.1 × 103 copies for conventional PCR analysis. Furthermore, no cross-reactivity was observed for the RAA-CRISPR/Cas12a assay with feline coronavirus, feline herpesvirus, and feline calicivirus, demonstrating reasonable specificity. Additionally, 43 cat fecal samples with suspected clinical signs were assayed with RAA-CRISPR/Cas12a-LFS and conventional PCR in parallel. The RAA-CRISPR/Cas12a-LFS showed a 100% coincident rate with PCR. In summary, a novel, visual, sensitive, and specific detection assay based on RAA and CRISPR/Cas12a was developed for FPV.

为提高《动物生物化学》课程的教学效果,培养新农科背景下创新创业型人才,该研究基于融合科研案例的线上线下混合式教学改革,开展了课前的科研文献综述泛读,课上的科研论文精读,以及问题导向的实验课设计.结果表明,基于融合科研案例的教学模式激发了学生的学习兴趣,提高了对理论知识的理解和掌握,培养了学生的科研创新思维,对教师做好科学研究具有驱动作用.

本研究旨在对鸡IL-21基因进行克隆、密码子优化、表达分析及生物活性初步研究,为进一步研究其功能奠定基础.首先利用生物信息学软件对鸡IL-21基因进行序列分析,然后,通过RT-PCR扩增鸡的IL-21基因,构建真核表达质粒pcDNA3.1A-IL-21-wt/MH,根据人偏爱密码子对鸡IL-21基因进行密码子优化,构建真核表达质粒pcDNA3.1A-IL-21-opt/MH.测序正确后转染HEK293T细胞进行表达,经His标签纯化后,通过脾脏淋巴细胞增殖实验鉴定纯化后的IL-21蛋白生物活性.结果显示,扩增的鸡IL-21序列与Genbank中的IL-21序列相对比有2个碱基发生突变(141C→141T,312C→312T),但不影响氨基酸序列;生物信息学分析表明,鸡IL-21基因含有信号肽序列可介导其分泌表达,同时含有1个潜在的N-糖基化位点;Western blot在培养基和细胞内均检测到鸡IL-21蛋白的表达;密码子优化后1L-21表达水平与野生型相比显著升高(P＜0.000 1);MTT法检测发现,纯化后的IL-21具有增强淋巴细胞增殖的功能.综上所述,本试验成功构建了鸡IL-21的真核表达载体,通过密码子优化提高了 IL-21在真核细胞中的表达且表达的蛋白具有良好的生物活性,为进一步探索IL-21蛋白功能奠定一定的基础.

白细胞介素-17(interleukin-17,IL-17)是T细胞诱导炎症的早期启动因子,可促进促炎性细胞因子的释放和刺激炎症反应,同时IL-17也能够抵抗某些病原,因此具有生物佐剂作用.鸡(Gallus gallus)的IL-17是否具有生物佐剂作用尚不清楚.为探讨鸡IL-17的生物佐剂作用,需要制备鸡IL-17的重组蛋白.为此本研究通过RT-PCR扩增了鸡的IL-17cDNA序列,利用生物学软件对其基因序列及氨基酸序列进行比较分析.为提高鸡IL-17基因在真核细胞中的表达水平,依据人(Homo sapiens)和鸡偏爱的密码子对IL-17基因进行密码子优化,分别构建了野生型和密码子优化的真核表达载体,并在人胚胎肾(human embryonic kidney,HEK)293T细胞进行了表达.实验结果显示,扩增的鸡IL-17基因序列与GenBank的相比有2个碱基的差异和1个存在于信号肽序列中氨基酸的突变,所以未改变成熟鸡IL-17蛋白质氨基酸的序列,表明鸡的IL-17基因存在着多态性.生物信息学分析表明,鸡IL-17基因含有1个典型的信号肽序列以介导鸡IL-17的分泌表达,同时蛋白中含有1个N-糖基化位点.通过在HEK 293T细胞的瞬时表达,证明密码子优化可明显提高鸡IL-17的表达水平,表达水平由野生型IL-17基因的13μg/T75细胞培养瓶提高到密码子优化的IL-17基因的45μg/T75细胞培养瓶,提高了3.5倍,同时优化后的重组蛋白可以刺激淋巴细胞的增殖和诱导鸡成纤维细胞产生IL-6.本研究表明密码子优化可明显提高IL-17基因在真核细胞中的表达并且优化表达的IL-17蛋白仍具有生物活性.这为进一步研究鸡IL-17的生物佐剂作用提供了有利条件.

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.

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.

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.

Porcine circovirus 3 (PCV3) is a recently identified virus that is associated with reproductive failure, porcine dermatitis and nephropathy syndrome, and multi-systemic inflammation. To investigate the molecular epidemic characteristics and genetic evolution of PCV3 in northern China, a commercial TaqMan-based real-time quantitative PCR kit was used to detect PCV3 in 435 tissue specimens collected from pigs with various clinical signs from 105 different swine farms in northern China. The results showed that 48 out of 105 (45.7%) farms and 97 out of 435 (22.3%) samples tested positive for PCV3. Of the 97 PCV3-positive samples, 80 (82.5%) tested positive for other pathogens. PCV3 was found more frequently in pigs with reproductive failure than in those with other clinical signs. This study is the first to detect PCV3 in Tianjin. The complete genome sequences of six PCV3 isolates and the capsid (Cap) protein gene sequences of 11 isolates were determined. Based on the predicted amino acids at positions 24 and 27 of the Cap protein and their evolutionary relationships, the 17 PCV3 strains obtained from northern China and 49 reference strains downloaded from the GenBank database were divided into four major groups (3a-3d). An analysis of selection pressure and polymorphism indicated that the PCV3 Cap protein seems to be evolving under balancing selection, that the population is in dynamic equilibrium, and that no population expansion occurred during the study period. Our results provide new information about the molecular epidemiology and evolution of PCV3.

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.

猪细小病毒(PPV)是导致母猪出现生育障碍的重要病因之一,研究表明猪细小病毒的进化比较保守,对于宿主的要求比较专一.然而,近年发现的PPV2和PHoV(香港株)与原先的PPV具有非常大的差异.因此,该文对猪细小病毒的形态结构、基因结构、蛋白的表达、复制培养、结构蛋白和非结构蛋白的研究展开了叙述.

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.

Porcine bocavirus (PBoV), which belongs the genus Bocaparvovirus, has been identified throughout the world. However, serological methods for detecting anti-PBoV antibodies are presently limited. In the present study, an indirect enzyme-linked immunosorbent assay (PBoV-rNP1 ELISA) based on a recombinant form of nucleoprotein 1 (NP1) of PBoV was established for investigating the seroprevalence of PBoV in 2025 serum specimens collected in north-central China from 2016 to 2018, and 42.3% of the samples tested positive for anti-PBoV IgG antibodies, indicating that the seroprevalence of PBoV is high in pig populations in China.

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.