Studies on Mucosal Immunity Induced by Transmissible Gastroenteritis Virus Nucleocapsid Protein Recombinant Lactobacillus casei in Mice and Sow     

WANG Gui-hua  HOU Xi-lin  YU Li-yun  LIU Jian-kui  WEI Chun-hua 《中国农业科学(英文版)》2009,8(2):231-237

Mucosal immunity plays an important role in protecting pigs against transmissible gastroenteritis virus （TGEV） infection. To elicit mucosal immune response against TGEV, we developed a surface antigen display system using the poly-γ- glutamate synthetase A （pgsA） protein of Bacillus subtilis as an anchoring matrix to express recombinant fusion proteins of pgsA and nucleocapsid protein of TGEV in Lactobacillus casei. Surface location of fusion protein was verified by ELISA and indirect immunofluorescence test. Oral and intranasal inoculations of pregnant sow and mice with recombinant L. casei resulted in high levels of serum immunoglobulin G （IgG） and secretory immunoglobulin A （slgA） against recombinant N protein as demonstrated by ELISA. More importantly, the level of specific slgA in colostrum significantly increased compared with that of IgG. The serum IgG levels of the piglets increased after suckling colostrum produced by sows was previously inoculated with recombinant L. casei. These results indicate that immunization with recombinant L. casei expressing TGEV N protein on its surface elicited high levels of specific slgA and circulating IgG against TGEV N protein.  相似文献   

猪圆环病毒Ⅱ型单克隆抗体的制备及应用     

车勇良  周伦江  王隆柏  魏宏  陈少莺  陈如敬  庄向生 《西北农林科技大学学报(自然科学版)》2010,(3):12-16,22

【目的】制备猪圆环病毒Ⅱ型(PCV2)Cap蛋白单克隆抗体,并对其进行鉴定和应用。【方法】以超离纯化的猪圆环病毒Ⅱ型作为免疫原,按常规方法免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞(SP2/0)融合,经间接ELISA筛选获得了2株分泌PCV2-Cap蛋白单克隆抗体的杂交瘤细胞,分别命名为8-60和10-48,对其生物学特性进行鉴定,并将其作为一抗,建立了间接免疫荧光诊断方法。【结果】8-60和10-48的染色体数平均为98～105对,间接ELISA检测其细胞培养上清液效价分别为1∶16和1∶32,小鼠腹水效价分别为1∶3×212和1∶3×213,抗体的免疫球蛋白亚型均为IgM。间接免疫荧光结果显示,8-60和10-48能与PCV2发生反应;利用这种特性,建立了能诊断PCV2病原的间接免疫荧光诊断方法,实际应用结果表明,其诊断结果与PCR方法诊断结果基本一致,阳性和阴性的符合率分别为93.75%和100%。【结论】以制备的猪圆环病毒Ⅱ型(PCV2)Cap蛋白单克隆抗体为一抗,建立了间接免疫荧光诊断方法。  相似文献   

鹅α干扰素基因在COS-7细胞中的瞬时表达及抗病毒功能初探     

刘菲  唐卫杰  程安春  汪铭书  赵婷婷  高丽芹 《浙江大学学报(农业与生命科学版)》2011,37(4)

为了研究鹅α干扰素( goIFN-α)基因的特性和功能,将天府肉鹅的α干扰素基因克隆到pcDNA3.1(+)真核表达载体上,构建重组质粒pcDNA3.1-goIFN-α,脂质体介导将其转入COS-7细胞中;应用间接免疫荧光、酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)和免疫印迹(Western-blot)法检测goIFN-α基因在COS-7细胞中的转录和表达情况,并对COS-7细胞表达上清液进行抗病毒活性检测.结果表明:目的蛋白在转染12 h后就可以被检测出,36 h前目的蛋白在上清液中快速增加,之后增加速度变慢;重组质粒能够在COS-7细胞内得到高效表达,表达的蛋白分子质量为38ku,比预测的分子质量(约21 ku)大;间接免疫荧光显示,细胞内出现黄绿色荧光,胞核附近最亮;表达产物经细胞病变抑制法测定,显示出具有明显的抗猪水泡性口炎病毒(VSV)及小鹅瘟病毒(GPV)的活性.  相似文献   

Characterization and Differentiation into Adipocytes and Myocytes of Porcine Bone Marrow Mesenchymal Stem Cells     

DU  Min-qing HUANG  Yue-qin LU  Nai-Sheng SHU  Gang ZHU  Xiao-tong WANG  Li-na GAO  Ping XI  Qian-yun ZHANG  Yong-liang WANG  Song-bo JIANG  Qing-yan 《农业科学学报》2014,13(4):837-848

Bone marrow mesenchymal stem cells （BMSCs） could differentiate into various cell types including adipocytes and myocytes, which had important scientific significance not only in the field of tissue regeneration, but also in the field of agricultural science. In an attempt to exhibit the characterization and differentiation into adipocytes and myocytes of porcine BMSCs, we isolated and purified porcine BMSCs by red blood cell lysis method and percoll gradient centrifugation. The purified cells presented a stretched fibroblast-like phenotype when adhered to the culture plate. The results of flow cytometry analysis and immunofluorescence staining demonstrated that the isolated cells were positive for mesenchymal surface markers CD29, CD44 and negative for hematopoietic markers CD45 and the adhesion molecules CD31. Cells were induced to differentiate into adipocytes with adipogenic medium containing insulin, dexamethasone, oleate and octanoate. Oil Red O staining demonstrated that the porcine BMSCs successfully differentiated to adipocytes. Moreover, the findings of real-time PCR and Western blotting indicated that the induced cells expressed adipogenic marker genes （PPAR-y, C/EBP-c~, perilipin, aP2） mRNA or proteins （PPAR-3,, perilipin, aP2）. On the other hand, porcine BMSCs were induced into myoctyes with myogenic medium supplemented with 5-azacytidine, basic fibroblast growth factor, chick embryo extract and horse serum. Morphological observation by hochest 33342 staining showed that the induced cells presented as multi-nucleus muscular tube structure. And myogenic marker genes （Myf5, desmin） mRNA or proteins （MyfS, MyoD, myogenin, desmin） were found in the induced cells. In addition, the results of immunofluorescence staining revealed that myogenic marker （Myf5, MyoD, myogenin, desmin, S-MyHC） proteins was positive in the induced cells. Above all, these results suggested that the isolated porcine BMSCs were not only consistent with the characterization of mesenchymal stem cells, but also exhibited the multipotential capacity to form adipocytes and myocytes, which provided the basis to investigate the regulation mechanism involved in the selective differentiation of porcine BMSCs.  相似文献   

集约化猪场沼液生态处理工艺优化试验效果评价          下载免费PDF全文

李文英 《农学学报》2014,4(8):85-87

沼液的深度净化处理是集约化畜禽养殖废水无害化处理的必由之路,本研究旨在筛选处理猪场厌氧消化液的最佳工艺条件组合,为养殖废水资源化循环利用提供科学依据和技术支撑。采用水质相关国家标准方法,选取2个复合菌剂（A、B）,3个接种量（0.5、1.0、5.0%）,2个通气量（2mg/L、4mg/L）,比较研究不同处理条件组合对猪场沼液的净化效果。实验表明：12个组合处理均对猪场沼液具有不同程度的净化效果,能够有效去除猪场沼液生化需氧量（BOD5）、总磷（TP）、粪大肠菌群（FC, Fecal Coliform）,各自去除率最高分别达到90.0%、82.0%、99.9%。综合各项评估指标,工艺1、4、6优于其它组合,其中工艺4为最优组合。  相似文献   

传染性法氏囊病病毒上海超强毒株VP2-4-3基因真核表达质粒的构建与表达   总被引：3，自引：0，他引：3  

蒋静  孙建和陆苹 《华中农业大学学报》2004,23(2):179-182

应用Long and Accurate—PPCR(LA—PCR)技术。扩增克隆了鸡传染性法氏囊病病毒上海超强毒(vvIB—DV)的VP2—4—3基因。应用粘性末端连接策略,将VP2—4—3基因克隆人真核表达载体pALTER—MAX。经酶切鉴定,表明VP2—4—3基因为正向插入,位于CMV启动子下游。该真核表达质粒在脂质体的介导下转染Vero细胞,经特异性抗IBDV抗体的免疫荧光检测,发现在细胞内有特异蛋白表达。  相似文献   

小麦叶片原生质体微管骨架的免疫荧光标记及其影响因素   总被引：10，自引：0，他引：10  

党磊  王冬梅  刘娟  王智炘 《河北农业大学学报》2002,25(3):23-27

以小麦叶肉细胞原生质体为材料，利用免疫荧光标记并辅以共聚焦激光扫描显微镜检术（Confocal la-ser scanning microscopy,CLSM)观察，对含有叶绿体较多的小麦叶肉细胞微管骨架的标记方法及其影响因素进行了探索。结果表明，来自未完全展开叶片的原生质体中，微管的数量多且骨架的排列方式较复杂，而从完全展开的叶片获得的原生质体中，微管数量很少，实验中发现，外加Ca^2 离子浓度的增加，使微管的稳定性降低，并确定了导致微管解聚的Ca^2 浓度。  相似文献   

应用间接免疫荧光法检测禽网状内皮组织增生病病毒抗体的研究   总被引：5，自引：0，他引：5  

荣骏弓  尹训南 《中国畜禽传染病》1998,20(5):284-286

用禽网状内皮组织增生病病毒（ＲＥＶ）感染ＳＰＦ鸡胚次代成纤维细胞涂片为抗原，建立了检测ＲＥＶ抗体的间接免疫荧光法（ＩＦＡ）。确定了待检血清稀释度１：６４和兔抗鸡ＩｇＧ荧光抗体的稀释度１：３２的工作浓度。应用该ＩＦＡ方法对人工感染ＲＥＶ的２０只３０日龄ＳＰＦ鸡进行了检测，接种后４天时均呈阴性反应，７天时８只鸡呈阳性反应，１４天２０只全部呈阳性的反应。通过对ＮＤＶ、ＩＢＤＶ、ＡＬＶ、ＭＤＶ、ＣＩＡＶ、  相似文献   

用异源抗体免疫荧光技术检测禽脑脊髓炎病毒和抗体     

杜元钊  李增光 《动物检疫》1992,9(3):9-11

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免疫荧光法快速监测SPF鸡群的新城疫病毒抗体     

赵智博  李军 《中国兽医杂志》1991,17(7):8-9

血凝抑制试验(HI)仍是目前国内最常用的检查鸡新城疫病毒(NDV)抗体的血清学方法。HI试验具有简单,快速等优点。当血清1:10以上稀释时,一般不发生非特异性抑制。HI的缺点在于检查低水平抗体时不够灵敏,感染NDV的不同毒株后产生的  相似文献