利用非转座子载体介导的转基因家蚕表达人胰岛素样生长因子Ⅰ

为了实现利用非转座子载体介导的转基因家蚕整体表达人胰岛素样生长因子Ⅰ(hIGF-Ⅰ),将hIGF-Ⅰ基因克隆进非转座子类型的昆虫细胞表达载体pIZT/V5-His,构建转基因载体pIZT/V5-His-hIGF-Ⅰ。利用精子介导法将该转基因载体导入家蚕卵,通过绿色荧光筛选并结合PCR和Dot blotting检测鉴定,表明已成功获得hIGF-Ⅰ转基因家蚕。对培育至G2代的转基因家蚕5龄幼虫蛋白质样品进行Western blotting分析,结果显示hIGF-Ⅰ在转基因家蚕中获得表达,重组hIGF-Ⅰ的分子质量约12.5 kD;ELISA检测hIGF-Ⅰ在G2代转基因家蚕5龄幼虫全蚕以及后部丝腺、脂肪体冻干粉中的质量比分别为65、411、469 ng/g。试验结果再次证实通过非转座子载体pIZT/V5-His介导可以将外源基因导入家蚕基因组,并实现外源基因在转基因家蚕中整体表达。     

ZJFC-Ⅰ防霉剂对竹重组材的防霉效果

以黑曲霉,绿色木霉、桔青霉为试验霉菌,以竹重组材为试验对象,使用6个浓度梯度的ZJFC-Ⅰ防霉剂进行处理。经过1个月的防霉试验后,分析各浓度对3种霉菌的防治效力。结果表明:ZJFC-Ⅰ在0.7%浓度时为极限浓度,试件经过ZJFC-Ⅰ处理后,再在表面涂布一层保护油,防治效力进一步提升。     

鸭Ⅰ型肝炎病毒RT-LAMP可视化检测方法的建立

为建立一种快速检测鸭Ⅰ型肝炎病毒(DHV I)的方法,本研究根据基因库中DHV Ⅰ基因的保守序列,设计特异性环介导等温扩增(LAMP)引物,建立了DHV I的RT-LAMP可视化检测方法。该方法的敏感性可达10fg,高于常规PCR方法 100倍;全部反应可在1 h内完成;可以通过肉眼观察颜色直接判定结果;对其它鸭常见病原体的检测结果均为阴性。实验结果表明建立的RT-LAMP方法简便、快速、灵敏、特异,可用于DHV Ⅰ感染的快速检测。     

马媾疫锥虫SYBR GreenⅠ荧光定量PCR检测方法的建立

为建立一种特异、快速的媾疫锥虫检测方法,本研究通过PCR方法从马媾疫锥虫动基体基因组中扩增得到395 bp的特异的保守序列,将其克隆到pMD-18T载体中构建重组质粒标准品,以10倍倍比稀释的质粒标准品为模板,进行SYBR GreenⅠ荧光定量PCR扩增并制作标准曲线,建立马媾疫锥虫荧光定量PCR的检测方法。结果表明:该方法的检测灵敏度可达到1拷贝/μL,并且与马属动物其他传染病无交叉反应。其组间及组内变异系数分别小于3.183%和3.842%。该方法的建立为快速及特异性检测马媾疫锥虫提供了有效的方法。     

J亚群禽白血病SYBR Green Ⅰ荧光定量PCR检测方法的建立

为建立一种能够快速、灵敏和特异地检测J亚群禽白血病病毒(ALV-J)的SYBR Green Ⅰ荧光定量PCR方法,本研究以ALV-J原型病毒株HPRS-103的pol基因3′端与gp85编码基因之间的保守区域(5 258 bp～5 802 bp)为检测目的片段,构建重组质粒并作为靶基因,通过对其浓度、引物浓度和退火温度的优化,建立了ALV-J SYBR Green Ⅰ荧光定量PCR方法.结果显示该方法的最低检测限度为2.36×102拷贝/μL,比普通PCR高100倍;与其他禽源病毒无交叉反应;批内和批间变异系数均小于5％.表明本研究建立的方法具有良好的特异性、稳定性和灵敏性.采用该方法对100只临床病鸡进行检测,ALV的检出率为44％.随机选择10只感染阳性鸡,检测病毒基因在心脏、肝脏、脾脏、肺脏和肾脏中的分布与表达水平,结果表明ALV-J在各主要脏器中均有分布,但以肾脏中的病毒表达量最高.     

表达Ⅰ型鸭病毒性肝炎病毒VP1蛋白重组腺病毒的构建

为构建表达Ⅰ型鸭病毒性肝炎病毒(DHV-I)VP1蛋白的重组腺病毒,本实验采用RT-PCR方法扩增DHV-Ⅰ HP-1株VP1基因,克隆于腺病毒穿梭质粒pShuttle-CMV中,并将其电转化于含腺病毒pAdeasy骨架质粒的大肠杆菌BJ5183中,通过同源重组制备含有重组腺病毒的质粒.重组质粒经PacⅠ酶切线性化后转染AD293细胞,获得重组腺病毒(rAd-VP1).间接免疫荧光和western blot鉴定结果显示,DHV-Ⅰ的VP1蛋白在重组腺病毒感染的AD293中获得表达.rAd-VP1的制备为鸭免疫实验效果评价奠定了基础.     

胰岛素样生长因子Ⅰ的研究进展

胰岛素样生长因子Ⅰ（IGF-Ⅰ）是胰岛素类激素家族中的成员之一,是一种在分子结构上与胰岛素类似的多肽蛋白物质.IGF-I在人体及动物体内具有极其重要并且丰富的生物学功能,如促生长、促分化、参与糖代谢、蛋白质代谢和脂肪代谢等,并对消化系统、泌乳及生殖有一定的影响.文章就IGF-I的来源,分子结构及生物学功能作一简单概述.     

Reversibility of the effects of GnRH‐vaccination used to suppress reproductive function in mares

Reasons for performing study: Active immunisation against gonadotrophin‐releasing hormone (GnRH) provides a reversible method for control of oestrous behaviour and fertility in mares. Previous reports failed to demonstrate the interval to resumption of cyclic ovarian activity after GnRH‐vaccination. Hypothesis: Administration of the GnRH‐vaccine Improvac in a large group of mares of various ages will result in effective, reliably reversible suppression of ovarian activity within a 2 year period. Methods: The mares, subdivided into 3 age categories, were vaccinated twice (with a 35 day interval) using 400 µg Improvac and monitored via blood samples until Day 720 after initial vaccination for serum progesterone concentration determination by radioimmune assay and anti‐GnRH antibody titre by enzyme immunoassay. Samples were collected until individuals resumed cyclic ovarian activity. Results: All mares showed suppression of cyclic ovarian activity by clinical examination and serum progesterone concentration (SPC) ≤1 nmol/l by Day 70 and 92.2% resumed cyclic activity by SPC at Day 720 with a mean interval = 417.8 days (s.d. ± 23.9; range 232–488 days, median 344 days). A significant age effect (P = 0.028) on the interval, but not on GnRH‐antibody titre response, was observed between the youngest (≤4 years) and oldest (≥11 years) categories. Conclusions: Immunising adult mares of all ages with Improvac resulted in a reversible suppression of cyclic ovarian activity in most mares. An age effect, with the youngest mares showing a longer interval to reversibility, was observed.     

Chlamydia infection in cats in New Zealand

Conjunctival swabs collected in 1991-92 from 333 pedigree and non-pedigree cats were tested for the presence of Chlamydia spp. antigen using an ELISA antigen kit. Forty (18.4%) of the 217 samples from cats with conjunctivitis were positive. Seven (6%) of 116 samples from cats which were in contact with cats with conjunctivitis but which showed no clinical signs at the time of sample collection were positive. Positive-testing cats were frequently from multi-cat households. Chlamydia spp. is present and associated with conjunctivitis in cats in New Zealand. Infection may occur concurrently with viral diseases. Feline calicivirus was recovered from 27 (21 with conjunctivitis) of 37 cats tested in five catteries. Four cats (with conjunctivitis) were FIV-positive.