抗猪流行性腹泻病毒单克隆抗体的研制及其特性鉴定

以新生乳猪增殖猪流行性腹泻病毒（ＰＥＤＶ）,通过蔗糖密度梯度离心将其纯化,获得较为完整的病毒颗粒。病毒ＥＬＩＳＡ效价为１∶３×１０５,蛋白含量为３．９６ｇ／Ｌ。将提纯的ＰＥＤＶ免疫ＢＡＬＢ／ｃ小鼠,取免疫鼠脾细胞与ＳＰ２／０骨髓瘤细胞进行融合,融合率为９５．６％。用间接ＥＬＩＳＡ进行筛选,共检出３８个阳性孔,阳性率为２８．８％。对其中１５个阳性值较高的孔进行３次再克隆,阳性率达１００％,最后获得１５株稳定分泌特异性抗ＰＥＤＶ单抗（ＭｃＡｂ）的杂交瘤细胞。ＥＬＩＳＡ阻断试验与交叉试验证明,其分泌的抗体具有高度特异性。腹水和细胞培养上清的ＥＬＩＳＡ效价分别为１∶１０３～１∶１０６和１∶１２８～１∶５１２。经鉴定,１５株ＭｃＡｂ的分子类型均为ＩｇＧ,均无免疫沉淀特性。杂交瘤细胞的染色体数目为９０～１１０,平均９５。尤为重要的是,这些杂交瘤细胞所分泌的抗体,有的对ＰＥＤＶ具有中和作用,能够阻断病毒对猪只的侵害作用。     

黄芪多糖对雏鸡外周血T淋巴细胞转化功能的影响

将１０８只１日龄伊莎系蛋用公雏均分为３组：一组为对照,其余２组在３日龄时,于背侧颈部皮下分别注射０．２、０．４ｍＬ黄芪多糖注射液（０．０１ｇ／ｍＬ）１次,再分别于７、２１、３５、４９日龄时采用ＭＴＴ比色法及微量全血培养３Ｈ－ＴｄＲ掺入法检测外周血Ｔ淋巴细胞转化率的动态变化。结果表明,黄芪多糖对２１、３５日龄雏鸡Ｔ淋巴细胞转化功能有增强作用,且与剂量有相关性,而对７、４９日龄雏鸡的作用不明显。ＭＴＴ比色法与３Ｈ－ＴｄＲ掺入法的检测结果无显著差异（Ｐ＜０．０５）。     

Effect of ABA,arginine and sucrose on protein content of date palm somatic embryos

Abscisic acid (ABA), arginine and sucrose were evaluated for their effects on the morphology, germination rates and protein content of date palm somatic embryos (SE). Different concentrations of these supplements in the culture medium were used. The comparative study of SE length and thickness between treated and untreated SE revealed no differences, except for ABA (20 μM), which increased thickness. A decrease of water content (WC) in favor of an increase in dry weight (DW) was observed in all treated SE, especially with sucrose (90 g l⁻¹) and ABA (20 μM). Only ABA (20 and 4 μM) caused a proliferation rate of the cultures higher than those in the control. Although all the tested compounds increased protein content, ABA (20 μM) was more effective in protein enrichment than arginine and sucrose treatments. The SDS-PAGE protein profiles showed a significant difference between treated and untreated SE. A protein band of 22 kDa, identified as glutelin in a previous work, was accumulated after treatment with 20 μM ABA or 3 mM arginine. These findings may contribute to further understanding of the mechanisms involved in the accumulation of specific storage proteins in several plants.     

基于纳米材料的免疫层析试纸在兽药残留检测中的应用

兽药残留是食品安全的重要影响因素,开发兽药残留检测方法是保证食品安全的重要手段。免疫层析试纸是一种操作简单、生产成本低且具有良好灵敏度的检测方法,适用于现场检测、大规模食品筛选,在兽药残留检测中具有巨大的应用价值。纳米材料可提供特殊的检测信号,是影响免疫层析试纸检测灵敏度和定量分析的关键因素。文章主要对胶体金、磁性纳米材料、量子点以及时间分辨荧光微球这几种纳米标记材料进行介绍,指出各自的优缺点及应用情况,并对该技术在兽药残留检测中的应用趋势进行展望。     

抗丝状支原体丝状亚种单克隆抗体的制备及初步应用

旨在制备抗丝状支原体丝状亚种(Mmm)的特异性单克隆抗体(MAb),为牛传染性胸膜肺炎(CBPP)病原诊断的免疫学方法提供特异性抗体。本研究利用生物信息学技术分析了Mmm国内分离株Ben-1不同传代株的全基因组序列,选取M0071蛋白作为研究对象。将原核表达的可溶性重组蛋白M0071(rM0071)作为免疫原免疫BALB/c小鼠,通过有限稀释法和间接ELISA方法筛选得到能稳定分泌抗rM0071蛋白的单克隆抗体的杂交瘤细胞株。进一步制备单抗腹水并纯化,利用Western blot方法对该单抗进行特异性鉴定,同时测定其抗体效价和抗体亚类。随后利用间接免疫荧光试验(IFA)评价该单抗对细胞感染Mmm的检测能力。结果表明成功获得1株单克隆细胞株3C4A1,将其分泌抗体命名为MAb 3C4A1。特异性结果表明,MAb 3C4A1能与Mmm的分离株和标准株发生特异性反应,而不与山羊支原体山羊肺炎亚种、丝状支原体山羊亚种、牛鼻支原体、无乳支原体、牛支原体、leachii支原体和牛A型巴氏杆菌等发生反应。抗体亚类鉴定MAb 3C4A1属于IgG1亚类、轻链为κ链。经间接ELISA测定其抗体效价为1∶256 000。IFA试验结果表明,MAb 3C4A1仅与感染EBL细胞的Mmm发生绿色荧光反应,而与牛鼻支原体、无乳支原体、牛支原体感染的细胞不发生荧光反应,特异性良好。本研究制备的MAb 3C4A1具有良好的特异性和免疫反应性,可作为CBPP病原免疫学诊断的工具,为进一步研制CBPP病原鉴别诊断试剂盒提供了基础材料。     

头孢氨苄免疫层析表面增强拉曼光谱检测方法研究

【目的】 建立一种简便、快速、灵敏度高、特异性强的头孢氨苄免疫层析表面增强拉曼光谱(SERS)检测新方法。【方法】 制备金银复合核壳结构的纳米贵金属,用来标记头孢氨苄抗体和拉曼信号分子5,5’-二硫代双2-硝基苯甲酸 (DTNB),合成拉曼免疫探针,用透射电子显微镜和双光束紫外分光光度计对纳米金和修饰拉曼信号分子的金银复合核壳结构进行表征。将合成的拉曼免疫探针固定在微孔板上,待测样品中的头孢氨苄与包被在硝酸纤维素膜上的头孢氨苄抗原竞争结合拉曼免疫探针,通过免疫层析试纸条结合共聚焦拉曼光谱仪读取DTNB的信号值,建立头孢氨苄定量检测技术,并应用于实际牛奶样品的检测。【结果】 透射电子显微镜和双光束紫外分光光度计表征结果显示,纳米银成功包裹在金颗粒表面。用共聚焦拉曼光谱仪检测免疫层析试纸条上拉曼信号分子的特征峰,拉曼信号分子特征峰为1 062、1 154、1 334和1 558 cm^-1,其中1 334 cm^-1处特征峰信号最强,选取此处峰高定量测定头孢氨苄的含量。头孢氨苄免疫层析表面增强拉曼光谱检测的线性范围为0~1 ng/mL,检测限为0.001 ng/mL,头孢氨苄的半数抑制质量浓度为0.05 ng/mL。与头孢克洛交叉反应率为111.1%,与头孢曲松钠、头孢夫辛酯、头孢噻吩钠交叉反应率均低于0.1%。实际样品加标浓度为1、4和8 ng /mL,回收率分别为94.4%、103.3%和97.5%。【结论】 本研究建立的头孢氨苄免疫层析表面增强拉曼光谱检测方法操作简便、快速、灵敏,耗材成本低廉,可满足奶牛养殖农场和乳品加工企业进行大批量样品初筛检测需求。     

提高根癌农杆菌介导的香石竹遗传转化效率的研究

以无菌苗叶片为外植体,在根癌农杆菌介导下建立并优化了香石竹5个品种的遗传转化体系。预培养2d可明显提高转化率;香石竹品种间在转化上存在差异;培养基中添加20μmol的AgNO3抑制不定芽的分化。转化植株在含25mg·L-1卡那霉素的生根培养基上培养,生根率为72 1%,GUS检测结果55%的转化植株呈蓝色,PCR扩增表明阳性率为32 2%,Southern杂交证实外源基因已整合到植物基因组中。     

Liquid culture and transformation of hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.)

Hinoki cypress (Chamaecyparis obtusa) is one of the most important timber resource forest trees in Japan. Because seed production from a seed orchard of hinoki cypress is not constant every year, micropropagation from a limited amount of material is useful. Up to now, the conventional tissue culture method using solid medium has been used. Here a new method using liquid culture in tubes rotated vertically is described. Shoot primordium of hinoki cypress was inoculated in Campbell and Durzan’s (CD) liquid medium containing different cytokinins (6-benzylaminopurine (BAP), Zeatin, thidiazurone (TDZ)), and the container tubes were rotated vertically around the axis at 2 times / min. Culture room temperature was 25°C and light condition was 16 h photoperiod per day of fluorescent lamps. Zeatin at 1μM concentration was the best for maintaining the shoot primordium production and TDZ induced callus on the surface of the shoot primordia. After shoot primordium multiplication in the liquid culture, they were transplanted to agar medium for shoot elongation. A high concentration of agar (up to 16 g/L) or AVF (anti vitrification factor from Dr. Nairn, 1995) was effective to prevent vitrification of the shoots. Transformation of shoot primordium was done using particle bombardment with vectors containingβ-glucuronidase (GUS) gene or herbicide resistance gene (bar). Positive result for transient transformation was observed with the histo-chemical study for transformation with GUS. Integration of a useful herbicidebar gene into the shoot primordium culture system was also tried and stably transformed plants were obtained. This is the first report of stable transformation of Japanese conifer using practically useful gene. The generous supply of AVF-B from Dr. B.J. Nairn, Tasman Forestry, NZ is also appreciated.     

Suppression of influenza virus infection by the orf virus isolated in Taiwan

Orf virus (ORFV), a member of parapoxvirus, is an enveloped virus with genome of double-stranded DNA. ORFV causes contagious pustular dermatitis or contagious ecthyma in sheep and goats worldwide. In general, detection of viral DNA and observing ORFV virion in tissues of afflicted animals are two methods commonly used for diagnosis of orf infection; however, isolation of the ORFV in cell culture using virus-containing tissue as inoculum is known to be difficult. In this work, the ORFV (Hoping strain) isolated in central Taiwan was successfully grown in cell culture. We further examined the biochemical characteristic of our isolate, including viral genotyping, viral mRNA and protein expression. By electron microscopy, one unique form of viral particle from ORFV infected cellular lysate was demonstrated in the negative-stained field. Moreover, immunomodulating and anti-influenza virus properties of this ORFV were investigated. ORFV stimulated human monocytes (THP-1) secreting proinflammatory cytokines IL-8 and TNF-α. And, pre-treatment of ORFV-infected cell medium prevents A549 cells from subsequent type A influenza virus (IAV) infection. Similarly, mice infected with ORFV via both intramuscular and subcutaneous routes at two days prior to IAV infection significantly decreased the replication of IAV. In summary, the results of a current study indicated our Hoping strain harbors the immune modulator property; with such a bio-adjuvanticity, we further proved that pre-exposure of ORFV protects animals from subsequent IAV infection.     

非洲猪瘟病毒p30蛋白单克隆抗体制备及线性抗原表位定位

【目的】制备非洲猪瘟病毒（African swine fever virus,ASFV）p30蛋白的单克隆抗体（monoclonal antibodies,MAbs）并初步分析其所识别的线性抗原表位,为ASFV及其抗体检测方法的建立及p30蛋白结构和功能的研究奠定基础。【方法】将原核表达并纯化的p30重组蛋白作为免疫原,免疫6—8周龄BALB/c雌鼠,每两周免疫1次,共免疫3次,首次免疫是抗原与等体积的弗氏完全佐剂乳化后免疫,第二次和第三次免疫与等体积的弗氏不完全佐剂乳化,3次免疫后1 w断尾采血,间接酶联免疫吸附试验（ELISA）检测血清抗体效价,选择血清效价最高的小鼠进行加强免疫,3 d后取小鼠脾淋巴细胞与SP2/0骨髓瘤细胞按照4﹕1的比例使用PEG进行常规细胞融合。利用重组p30蛋白作为包被抗原,间接ELISA筛选阳性杂交瘤细胞,有限稀释法进行克隆纯化,直至筛出能够稳定分泌抗体的MAbs。将ASFV接种于猪肺泡巨噬细胞,以筛选的MAbs为一抗、兔抗鼠HRP-IgG为二抗,进行间接免疫荧光试验（IFA）。将感染和未感染ASFV的细胞沉淀处理后进行 SDS-PAGE并转印至硝酸纤维素膜,分别以IFA鉴定为阳性的MAbs上清为一抗、兔抗鼠HRP-IgG为二抗,进行Western blotting分析,筛选获得p30 MAbs。根据已知序列设计引物扩增p30ab与p30bc两段截短基因,其中p30ab代表由第86—153位氨基酸残基的截短体,p30bc代表由第120—187位氨基酸残基的截短体,原核表达部分重叠的截短p30蛋白,最终获得重组蛋白GST-p30ab与重组蛋白GST-p30bc。分别以GST-p30ab和GST-p30bc融合蛋白为包被抗原,以5株MAbs为一抗,以兔抗鼠HRP-IgG为二抗, 通过间接ELISA方法初步定位p30蛋白的抗原表位。【结果】以纯化的重组蛋白为包被抗原,经间接ELISA试验筛选出25株可分泌抗重组 p30蛋白的杂交瘤细胞株。IFA结果显示,5株MAbs（8F4、1D3、1H2、6C3和8E11）与ASFV感染的猪肺泡巨噬细胞IFA 试验呈阳性;Western blotting结果显示,5株MAbs均能够与ASFV感染的细胞呈阳性反应,与未感染病毒的细胞呈阴性反应。试验构建的p30截短体重组蛋白GST-p30ab以可溶和包涵体两种形式表达,而GST-p30bc仅以包涵体形式表达,以两组截短体融合蛋白为包被抗原,通过间接ELISA检测出MAbs 8F4、1H2和6C3与两个重组蛋白均能有效结合,证明MAbs 8F4、1H2和6C3抗原识别区域为两组截短蛋白重叠区域,即第120—153位氨基酸;MAbs 8E11与1D3则只能与GST-p30ab蛋白结合, 证明MAbs 8E11与1D3抗原识别区域为两个重组蛋白的非重叠区域,即第86—119位氨基酸。【结论】本研究可溶性地表达了p30蛋白的第86—153位氨基酸截短体重组蛋白,制备了5株p30 MAbs,定位到2个p30蛋白抗原表位。结合ELISA和IFA,可建立十分可靠的ASFV及其抗体的检测手段。