北京地区规模化奶牛场牛病毒性腹泻病血清学调查

对北京郊区6个未进行牛病毒性腹泻病（BVD）免疫牛场的546份奶牛血清样品,使用牛病毒性腹泻抗体ELISA试剂盒进行检测,并对其中3个牛群进行牛病毒性腹泻病毒（BVDV）血清抗原筛查。共检出阳性血清514份,总阳性率94.1%,其中5个牛场的场内血清抗体阳性率在95%以上。牛病毒性腹泻持续性感染牛（PI牛）筛查的3个牛群均有阳性牛检出。结果表明,北京地区规模化奶牛场存在牛病毒性腹泻感染和接触史,应采取净化措施进行控制。     

转基因抗虫玉米Bt蛋白表达量的研究

应用酶联免疫吸附测定法(ELISA)定量检测转基因抗虫玉米不同生长期不同部位的Bt蛋白表达量。结果显示:Bt毒蛋白在玉米乳熟末期的第15叶中表达量(0.225%)最高,在花粉中表达量(0.011%)最低。叶、茎、髓、花丝、穗轴及粒的Bt蛋白含量及比例随着作物的生长呈上升的趋势。Bt毒蛋白最低检出量为0.5ng/mL。     

Antibody-ELISA for Trypanosoma evansi: Application in a serological survey of dairy cattle, Thailand, and validation of a locally produced antigen

Trypanosoma evansi is generally considered a mild pathogen in bovines. However, in Asia, acute and chronic signs have been observed in cattle, with high levels of parasitaemia, abortion and death. Investigations in Asian cattle are needed to better understand this epidemiological situation. To generate comparable data at a regional level, development and standardization of an antibody-enzyme linked immunosorbent assay for T. evansi (ELISA/T. evansi) was initiated and applied in an epidemiological survey carried out in dairy cattle in Thailand. A batch of 1979 samples was collected from dairy farms located throughout the country's four regions. Soluble T. evansi antigens initially produced in France were also produced in Thailand for comparison and technology transfer. Screening of 500 samples allowed us to identify reference samples and to determine the cut-off value of the ELISA. Seropositive animals – some of them confirmed by PCR – were found in the four regions, in 12 out of 13 provinces, in 22 out of 31 districts, in 56 farms out of 222 (25%, 95%CI ± 6%) and in 163 animals out of 1979 (8.2, 95%CI ± 1.2%). Estimated seroprevalence in 35 farms ranged between 1% and 30%, and in 21 farms it was >30%. Approximately 25% of survey cattle were exposed to the infection, in various situations. A sub-sample of 160 sera was tested on both antigens. Wilcoxon's (Z = 1.24; p = 0.22) and McNemars's tests (CHI2 = 3.55; p = 0.09) did not show any significant differences, showing that the locally produced antigen is suitable for further evaluation in the surrounding countries. Use of this standardized serological method will broaden knowledge of the prevalence and impact of the disease at the regional level in South-East Asia. Further validation of this ELISA will be necessary in other host species such as buffalo, horse and pig.     

猪圆环病毒2型灭活疫苗中病毒抗原含量实时荧光定量PCR检测方法的建立

依据GenBank公布的猪圆环病毒2型Cap基因序列,在保守区域设计特异性引物和TaqMan探针,优化反应体系,建立评价猪圆环病毒2型灭活疫苗中病毒含量的实时荧光定量PCR检测方法,对方法的特异性、敏感性和重复性进行试验,并验证灭活剂用量和灭活时间对检测结果的影响。结果显示：该方法只对猪圆环病毒2型基因有特异性扩增,其他3种对照病毒基因的扩增结果均为阴性;检测灵敏度达到10^2.0 TCID₅₀/mL,比普通PCR方法高100倍;方法的重复性好,对同一样品进行10次检测,变异系数为2.28%;不同灭活剂用量和灭活时间对结果的影响较小,不会因各厂家使用的灭活剂用量和灭活时间不同,影响疫苗对比实验的公平性。该研究成功建立了一种评价猪圆环病毒2型灭活疫苗中病毒含量的实时荧光定量PCR检测方法,用于猪圆环病毒2型灭活疫苗样品中病毒抗原含量的定量,其结果可以反映不同猪圆环病毒2型灭活疫苗样品中抗原含量差异,为研究猪圆环病毒2型灭活疫苗病毒抗原含量评估方法提供了新的思路。     

巴西橡胶树类甜味蛋白基因HbTLP1的克隆及表达分析

根据模式植物拟南芥中同类蛋白的氨基酸序列在橡胶树转录组数据库中进行同源搜索并设计引物,通过RT-PCR方法扩增巴西橡胶树TLP同源基因的cDNA片段,命名为Hb TLP1.该片段包含一个984 bp的开放阅读框,编码327个氨基酸的多肽.生物信息学分析结果表明,该氨基酸序列含有一个超级THAUMATIN-Like结构域,与杨树、大豆、葡萄和拟南芥的TPL蛋白有较高的同源性.半定量分析结果表明,该基因的表达受水杨酸(SA)和鞭毛蛋白(flg22)的诱导,但不受茉莉酸(JA)的影响.以上结果揭示该基因为橡胶树的TPL同源基因,参与SA介导的抗病信号途径和植物的先天免疫反应.     

Milk progesterone enzyme-linked immunosorbent assay as a tool to investigate ovarian cyclicity of water buffaloes in relation to body condition score and milk production

Background

Application of assisted reproductive technologies in buffaloes is limited to some extent by farmers’ inability to detect oestrus because of its poor expression. The present study aimed at investigating reliability of a milk progesterone enzyme-linked immunosorbent assay (ELISA) to assess the ovarian cyclicity during post partum, oestrus and post-breeding periods in water buffaloes.

Methods

Progesterone concentrations were measured by an ELISA in milk of 23 postpartum buffaloes in relation to oestrus, pregnancy, body condition score (BCS) and milk production. Two milk samples were taken at 10 days intervals, every month starting from day 30 and continued to day 150 post partum. BCS and milk production were recorded during sample collection. Milk samples from bred buffaloes were collected at Day 0 (day of breeding), Days 10–12 and Days 22–24. Defatted milk was preserved at −80°C until analysis. Pregnancy was confirmed by palpation per rectum on Days 70–90.

Results

Seventeen buffaloes had 47 ovulatory cycles, one to four in each, 13 were detected in oestrus once (28 % oestrus detection rate). Progesterone concentration ≥1 ng/ml in one of the two 10-day-interval milk samples reflected ovulation and corpus luteum formation. The intervals between calving to first luteal activity and to first detected oestrus varied from 41 to 123 days (n = 17) and 83 to 135 (n = 13) days, respectively. Eight buffaloes were bred in the course of the study and seven were found pregnant. These buffaloes had a progesterone profile of low (<1 ng/ml), high (≥ 1 ng/ml) and high (≥ 1 ng/ml) on Day 0, Days 10–12 and Days 22–24, respectively. Buffaloes cycling later in the postpartum period had fewer missed oestruses (P < 0.05). Buffaloes with a superior BCS had a shorter calving to oestrus interval and produced more milk (P < 0.05).

Conclusions

Milk progesterone ELISA is a reliable tool for monitoring ovarian cyclicity and good BCS may be an indicator of resuming cyclicity in water buffalo.     

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。     

Application of time-resolved fluorometry to immunoassays for bovine reproductive hormones

The principle of time‐resolved fluorometry with lanthanide chelates was established in the 1980s, but in the field of animal sciences it has not been widely applied to immunoassays. However, immunoassays that utilize time‐resolved fluorometry are possible alternatives to radioimmunoassays, since they can attain high sensitivity without safety risks. In this short review, we introduce the development of time‐resolved immunoassays for inhibin A, inhibin B and follicle‐stimulating hormone (FSH), and describe their application to the investigation of FSH regulation in male and female cattle. The results obtained using these newly developed immunoassays indicate that inhibin A acts as a feedback regulator for FSH secretion in female cattle, whereas inhibin A, and probably inhibin B, do so in male cattle.