龙眼蜂蜜抗体鉴定技术的开发

龙眼蜜为台湾特色蜂蜜,备受消费者喜爱。然而,市面上充斥以糖浆调制的人造龙眼蜜,不仅侵害了消费者的权益,也影响正规龙眼蜜的产销,损害了台湾蜂农的利益。本研究针对龙眼蜜含有花粉粒的特性,利用酵素免疫分析的技术,开发龙眼蜂蜜的鉴定方法。采取确定来源的龙眼蜜稀释后离心取沉淀,以Na—bicarbonate冰温萃取花粉成分,经透析浓缩后作为抗原,用Freund完全佐剂乳化,免疫BALB／c雌性小鼠,每隔3-4周再补强注射。补强免疫后10-14天采血分离血清,以ELISA测抗体力价。结果显示,龙眼蜜的花粉萃取液能有效诱发小鼠免疫反应,产生的多源抗体能区别真蜜与合成蜜。     

Segmentation of the Pathophysiological Stages of Diabetic Changes in the db/db Mouse

The db/db mouse is one of the diabetes mellitus animal models and if the pathophysiological stages of diabetic changes in the mouse model could simulate the stages in human diabetes, the db/db mouse could be used to better evaluate drug candidates. Blood insulin, HbA1c levels and morphological features of pancreatic islets in db/db mice were evaluated to determine the pathophysiological stage. At 6 weeks of age, db/db mice showed the highest level of plasma insulin and lowest level of HbA1c, and histopathological examination revealed enlarged islets with a circular shape and hypertrophic islet cells. By 9 and 12 weeks of age, the plasma insulin levels had decreased to mid levels and HbA1c had increased to mid to high levels; histopathological examination at this time revealed two types of islets coexisting, enlarged circular islets and small irregular-shaped islets. By 15 and 22 weeks of age, plasma insulin had decreased further to low levels and HbA1c was at its highest level; the histopathological examination at this time revealed an increase in irregular-shaped and small islets. Based on blood insulin levels, HbA1c levels and histopathology findings in the db/db mice in this study, the clinical staging of diabetic changes were recognized. The pathophysiological stages of diabetes mellitus in this animal model were similar to the stages in humans.     

Functional and antigenic properties of GlpO from Mycoplasma mycoides subsp. mycoides SC: characterization of a flavin adenine dinucleotide-binding site deletion mutant

L-α-glycerophosphate oxidase (GlpO) plays a central role in virulence of Mycoplasma mycoides subsp. mycoides SC, a severe bacterial pathogen causing contagious bovine pleuropneumonia (CBPP). It is involved in production and translocation of toxic H₂O₂ into the host cell, causing inflammation and cell death. The binding site on GlpO for the cofactor flavin adenine dinucleotide (FAD) has been identified as Gly ₁₂−Gly₁₃−Gly ₁₄−Ile₁₅−Ile₁₆−Gly ₁₇. Recombinant GlpO lacking these six amino acids (GlpOΔFAD) was unable to bind FAD and was also devoid of glycerophosphate oxidase activity, in contrast to non-modified recombinant GlpO that binds FAD and is enzymatically active. Polyclonal monospecific antibodies directed against GlpOΔFAD, similarly to anti-GlpO antibodies, neutralised H₂O₂ production of M. mycoides subsp. mycoides SC grown in the presence of glycerol, as well as cytotoxicity towards embryonic calf nasal epithelial (ECaNEp) cells. The FAD-binding site of GlpO is therefore suggested as a valuable target site for the future construction of deletion mutants to yield attenuated live vaccines of M. mycoides subsp. mycoides SC necessary to efficiently combat CBPP.     

猪瘟抗体检测技术研究现状及发展趋势

简要叙述了FAVN、NPLA、ELISA、兔体中和等猪瘟抗体检测技术的原理及其应用现状,并通过对猪瘟抗体检测技术发展趋势的介绍,显示了猪瘟参考血清等相关标准物质在猪瘟抗体检测技术应用与发展中的价值及其今后的发展前景。     

小鼠输卵管上皮细胞的原代培养及纯化方法研究

建立小鼠输卵管上皮细胞原代培养及纯化方法.小鼠输卵管上皮细胞原代培养采用组织块培养法和酶消化法.酶消化法于37℃分为4个处理组进行.处理1以2.5 g/L胰酶+0.4 g/L EDTA消化5、10、20 min;处理2以0.5 g/L胰酶+0.08 g/L EDTA消化35、50、75 min;处理3以0.3 g/L的Ⅰ型胶原酶消化60、90、240 min;处理4以2.5 g/L胰酶+0.4 g/L EDTA和0.3 g/L Ⅰ型胶原酶(1∶2)消化60 min,150 min.原代细胞纯化采用差速贴壁和反复差速贴壁法.传代采用胰酶两步消化法进一步纯化输卵管上皮细胞.组织块法原代培养成纤维细胞生长优势明显,传代纯化细胞效果不佳.用酶消化法原代培养时,处理1效果不理想;处理2采用3个消化时间时均取得比较理想的结果;处理3消化240 min时结果较好;处理4消化150 min效果较好.原代细胞纯化,反复差速贴壁得到的上皮细胞较纯,进一步传代纯化细胞取得了理想的结果.小鼠输卵管上皮细胞原代培养采用酶消化法结合反复差速贴壁分离纯化细胞,传代采用两步消化法,可以成功地进行小鼠输卵管上皮细胞的分离纯化培养.     

地顶孢霉培养物对保育仔猪生产性能及免疫水平的影响

试验选择28日龄杜×长×大三元杂交仔猪32头,随机分为2组,试验组在基础日粮中添加0.25%地顶孢霉培养物,对照组饲喂基础日粮,试验期30 d。结果表明,试验组仔猪日增重比对照组提高10.4%,差异显著（P<0.05）,饲料转化率提高2.6%,腹泻率降低57.7%;试验组平均猪瘟抗体效价极显著高于对照组（P<0.01）,PRRS免疫抗体阳性率也明显高于对照组。因此,地顶孢霉培养物具有促进仔猪生长和提高免疫力的功能。     

培养基质中添加酯化葡甘露聚糖和DON对小鼠脾细胞生长和转化的影响

将体外培养的脾细胞分为5组,以RPMI-1640为基础培养基,第1组为空白对照组;第2组为阳性对照组,添加500 μg/L DON;第3组、第4组和第5组在添加500 μg/L DON的基础上分别添加0.05%、0.10%和0.15%的EGM。在培养72 h后,测定各组培养基质中MDA含量、SOD活性、脾细胞存活率和淋巴细胞转化率。结果表明,第1组、第4组和第5组的脾细胞存活率、SOD活性极显著高于第2组(P<0.01);第1组、第4组和第5组的淋巴细胞转化率显著高于第2组(P>0.05);第2组培养液中MDA含量显著高于第1组、第4组和第5组(P<0.05);第1组、第4组和第5组各项指标差异不显著(P>0.05)。由此表明,EGM能很好地吸附DON,减轻DON对小鼠脾细胞的毒性作用;EGM在饲料中的适宜添加剂量为0.10%。     

鸡堆型艾美耳球虫特异性单抗轻链可变区基因的克隆与序列测定

将已扩增出的鸡堆型艾美耳球虫特异性单抗轻链可变区基因进行纯化,并用纯化的基因片段与pMD-18T载体连接,将重组载体转化于感受态细胞JM109,筛选出阳性重组子,提取阳性重组子质粒并进行测序,得到特异性单抗轻链可变区的基因序列。为鸡堆型艾美耳球虫特异性单链抗体基因的构建奠定了基础。     

猪血凝性脑脊髓炎病毒的血清学调查

应用血凝和血凝抑制试验检测了从辽宁省部分地区采集的猪血清中血凝性脑脊髓炎病毒（HEV）的抗体滴度。结果表明,368份血清样品中,有162份呈抗体阳性反应,阳性率高达44.0%。被采集血清的猪未表现出临床症状,说明该地区存在血凝性脑脊髓炎病毒的隐性感染。     

抗麦草畏多克隆抗体的制备及其间接竞争ELISA检测方法的建立

为建立麦草畏的免疫分析方法,采用碳化二亚胺法将麦草畏(DIC)与卵清蛋白(OVA)和牛血清蛋白(BSA)偶联,分别合成免疫原与包被原;用合成的免疫原免疫新西兰大白兔获得抗麦草畏多克隆抗体,该抗体效价为1∶1.28×105,且该抗体与麦草畏的结构类似物2,3,5-三氯苯甲酸、2,3,6-三氯苯甲酸、2-氨基-3,5-二氯苯甲酸的交叉反应率均小于0.1%。以包被原1∶8 000和多抗溶液1∶80 000的稀释倍数为工作浓度,建立麦草畏间接竞争ELISA(ciELISA)检测方法,此方法检测麦草畏的线性范围为1～100ng.mL-1,线性回归方程为y=8.684 4ln x+15.001,R2=0.994 4,最低检测限为IC20=1.779ng.mL-1;玉米粉和面粉空白样品的麦草畏添加回收实验表明,麦草畏添加值为5～30μg.kg-1时,玉米粉中的麦草畏回收率为53.08%～92.37%,面粉的回收率则分别为66.5%～94.63%。