羊驼KIT基因exon10-19 cDNA的克隆、表达及生物信息学分析

从羊驼皮肤中提取总RNA,利用RT-PCR技术,扩增了羊驼显性白毛控制基因（KIT）cDNA序列（DQ450844）,并与其它动物相应区域作了同源性比较,结果表明：羊驼KIT基因exon10-19 cDNA长1 044 bp,编码含347个氨基酸残基的蛋白;蛋白质同源性比较显示,羊驼与牛、羊、猪、人、马、猩猩等的同源性大于98%,与鼠的同源性为95%。羊驼aa2编码缬氨酸,而猪、人与牛等动物则编码异亮氨酸,均属于非极性氨基酸;羊驼aa201编码脯氨酸,是非极性氨基酸,而猪、人与牛等动物则编码丝氨酸,是不带电荷的极性氨基酸;羊驼aa344编码精氨酸,而猪、人与牛等动物则编码赖氨酸,均为带正电荷的极性氨基酸。蛋白质二级结构及功能分析结果显示：此二级结构中含有大量的α-螺旋;该蛋白编码肥大细胞/干细胞生长因子,属于酪氨酸激酶受体家族,其蛋白激酶活性位点位于248~260之间。本研究结果将为深入研究KIT基因与羊驼毛色遗传的关系奠定一定的理论基础。     

羊驼的繁殖特性

本文主要介绍了特种经济动物———羊驼的生殖生理特点、繁殖特性 ,为我国今后羊驼养殖业的发展提供理论依据     

羊驼催乳素受体基因(PRLR)exon10的克隆与序列分析

通过氰仿／异戊醇法从羊驼血液和组织中提取羊驼基因组DNA，首次扩增出羊驼PRLR基因（Prolactin Receptor，PRLR）exon10序列（GenBank登录号为DQ206831），并与其它动物相应区域作了同源性比较，结果表明：羊驼PRLR基因exon10的开放阅读框为1133bp，包括1046bp的编码区和87bp的拖尾区；同源性比较显示，羊驼PRLR基因exon10的核苷酸序列与哺乳动物（牛、绵羊、猪、狗、兔、大鼠等）的同源性较高，达80％，氨基酸序列的同源性则≥66％，而与鱼类的同源性则较低，仅为40％～45％。     

我国羊驼的管理

羊驼(Alpaca)起源于南美洲,是南美主要的毛用型经济动物,许多国家已引种成功,我国也于2001年与澳大利亚签署羊驼引种协议,于2002年5月顺利引进首批羊驼。至今,羊驼在我国生存近2年,经实践证明,羊驼引种成功。     

不同动物源性维氏气单胞菌气溶素基因的克隆及比较分析

为系统研究维氏气单胞菌毒力因子气溶素,对不同动物源性的维氏气单胞菌气溶素基因（aerA）进行了克隆,获得了1500 bp左右的目的基因,并对其序列进行了分析比较。结果显示,不同动物源性维氏气单胞菌aerA基因的核苷酸序列均具有APT Superfamily与Aerolysin Superfamily保守结构域,其核苷酸序列的同源性在95.8%以上,编码的氨基酸序列同源性在89.8%以上,说明不同源性维氏气单胞菌气溶素具有一定的保守性。该研究结果可为维氏气单胞菌的检测及疫苗制备提供一定的理论依据。     

羊驼毛鳞片超微结构的研究

羊驼(Lama pacos，Alpaca)是我国新引进的动物种，属于毛肉兼用的经济动物。羊驼属偶蹄目、骆驼科、南美洲驼属．其经济价值主要在于毛。我国对羊驼毛的研究基本处于空白阶段．国内有对小羊驼被毛物理性能的研究。我国羊驼经过一年多的饲养和繁育，已具一定的适应性，在我国气候、地理以及其他因素的影响下生产出一批被毛。羊驼90％以     

羊驼主要病毒性传染病的诊断与防治

概述了羊驼主要病毒性传染病的流行病学、临床症状、病理变化及诊断与防治。为羊驼病毒性传染病的研究与防治提供理论依据。     

microRNAs在动物皮肤及毛囊发育中的调控作用研究进展

基因表达调控是动物被毛生长和发育的决定性因素。microRNA作为一种新发现的基因调控元件,在多种哺乳动物皮肤及毛囊中均有表达,并在转录后水平调节皮肤和毛发的生长和发育过程。从microRNA水平上解析绵羊、山羊及羊驼等被毛生长特点及发生机理,为提高毛用型经济动物的毛发品质和产量提供新的思路,同时也为更深入地研究皮肤组织中microRNA的功能提供更多的理论依据。作者主要针对目前已报道的在绵羊、山羊及羊驼等哺乳动物皮肤及毛囊发育中microRNA的调控作用进行综述。     

羊驼MC1R基因克隆及其在不同毛色个体中表达水平研究

为了获取羊驼MC1R基因序列,探索MC1R基因表达水平与羊驼毛色之间的相关性,根据GenBank已发表序列(EU1358800)设计1对引物,以羊驼皮肤总RNA为模板,采用RT-PCR技术扩增并成功获得了中华白色羊驼MC1R基因cDNA序列,长度为1 081 bp,编码317个氨基酸,与已发表序列进行对比,同源性为99%,发现7处突变.根据所克隆序列设计引物,采用实时荧光定量PCR技术(QRT-PCR),分析MC1R基因在白色和棕色羊驼皮肤中的基因表达量,并对结果进行统计分析.结果表明:经过内参基因校正后,棕色羊驼皮肤中MC1R基因的相对表达量是白色羊驼相对表达量的4.32倍(P<0.01).MC1R基因表达量的差异与羊驼毛色表型之间可能存在一定的相关性.     

中国羊驼KAP1．3基因的克隆及序列分析

毛纤维中的角蛋白（keratin）和角蛋白关联蛋白（keratin-associatedproteins）的数量遗传性状位点（quantitativetraitloci）已有学者在绵羊等一些动物身上找到，其中KAP1．1、KAP1.2、KAP1．3等基因作为候选基因来间接选择羊毛细度性状。而羊驼相关候选基因的研究还未见报道，本研究提取成年羊驼的新鲜血液中基因组DNA，并以此为模板采用PCIL技术扩增KAP1．3基因进行序列分析，测序后在NCBI工作平台中进行BLASTn同源性比较，得出结论：结果表明多个物种间KAP1．3基因编码区的序列相似性在73%以上。借助DNAstar分子生物学分析软件绘制了相关动物的遗传进化图．并对羊驼的种属地位进行了进一步验证。通过羊驼与绵羊在KAP1．3的氨基酸序列上的比较只有十五处残基的差异。证实KAP1．3基因与羊驼毛细度性状相关。     

Comparison of the microbial communities of alpacas and sheep fed diets with three different ratios of corn stalk to concentrate

The objective of this study was to investigate the characteristics of ruminal microbial communities of alpacas (Lama pacos) and sheep (Ovis aries) fed three diets with varying ratios of roughage (corn stalk) to concentrate, 3:7 (LS), 5:5 (MS) and 7:3 (HS). Six alpacas (one-year-old and weighing 29.5 ± 7.1 kg) and six sheep (one-year-old and weighing 27.9 ± 2.7 kg) were used in this study, in a replicated 3 × 3 Latin square experiment. Total protozoa concentration was determined under the microscope; total fungi and methanogens were assessed using quantitative polymerase chain reaction and expressed as a percentage of total bacterial 16S rRNA gene copies; bacterial communities were investigated by targeted 16S rRNA gene (V3–V4 region) sequencing. The percentage of fungi was significantly higher in alpacas than in sheep under the LS diet, while the concentration of protozoa was significantly lower in alpacas under HS, MS and LS diets. The alpha diversity including Shannon, Chao l and ACE indices of bacterial communities was higher in alpacas than in sheep, under the LS diet. A total of 299 genera belonging to 22 phyla were observed in the forestomach of alpaca and sheep, with Bacteroidetes and Firmicutes dominating both animal species. Phyla Armatimonadetes and Fusobacteria, as well as 64 genera, were detected only in alpacas, whereas phyla Acidobacteria and Nitrospira, as well as 44 genera, were found only in sheep. The abundance of cellulolytic bacteria, including Butyrivibrio and Pseudobutyrivibrio, was higher in alpacas than in sheep under all three diets. These differences in the forestomach microbial communities partly explained why alpacas displayed a higher poor-quality roughage digestibility, and a lower methane production. Results also revealed that the adverse effects of high-concentrate diets (70%) were lesser in alpacas than in sheep.     

Targeted knockdown of canine KIT (stem cell factor receptor) using RNA interference

Canine mast cell tumours often express KIT mutations that result in constitutive activation of the c-kit receptor and which are associated with more aggressive disease. The aim of the current study was to determine whether small inhibitory RNA (SiRNA) molecules could specifically target canine KIT mRNA for knock-down. Canine beta-2 microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and KIT sequences were cloned into the psiCHECK?-2 vector. SiRNA molecules, designed to target gene-specific sequences, were co-transfected with plasmid DNA into Chinese hamster ovary (CHO) cells. Renilla and firefly luciferase activity was measured using the Dual-GLO(?) Luciferase Assay (Promega). Using this reporter system, canine housekeeping gene-specific SiRNA molecules demonstrated knockdown of their targets (72.0% knockdown for B2M and 94.5% knockdown for GAPDH). An SiRNA molecule targeting exon 2 of canine KIT successfully knocked-down reporter gene expression of a KIT(26-407) construct (90.8% knockdown). An SiRNA molecule targeting a 48 base-pair in-tandem duplication mutation in KIT exon 11 selectively knocked down expression of the KIT(1569-1966mutant) construct (93.1% knockdown) but had no effect on the KIT(1569-1918wild-type) construct. The results show that RNA interference can be used to inhibit canine KIT mRNA expression and has the potential to selectively target the mutant version of KIT that is expressed by some malignant mast cells.     

Infection with Corynebacterium pseudotuberculosis in five alpacas

Among the population of an alpaca breeding farm, 5 alpacas (22 days to 14 months old) developed focal swellings in the subcutaneous tissues of the head or neck. Infection with Corynebacterium pseudotuberculosis was confirmed on the basis of results of microbial culture of abscess material and a serum hemolysis inhibition assay to detect C. pseudotuberculosis toxin. The dams of the affected alpacas were seronegative for C. pseudotuberculosis toxin. The affected alpacas underwent surgical excision of the abscesses and were isolated from herdmates for 90 days; treatment was successful, and no other alpacas in the herd became infected. Common risk factors for sources of infection in the affected alpacas included housing in a maternity barn and a pasture. Also, the infection potentially originated from new alpacas introduced into the herd during the preceding 3 months. Infection with C. pseudotuberculosis should be considered as a differential diagnosis for camelids with peripheral lymphadenopathy or abscesses in subcutaneous tissues.     

The value of molecular expression of KIT and KIT ligand analysed using real‐time polymerase chain reaction and immunohistochemistry as a prognostic indicator for canine cutaneous mast cell tumours

This study investigated the correlation between KIT gene expression determined by immunohistochemistry and real‐time polymerase chain reaction (RT‐PCR) and the rate of tumour recurrence and tumour‐related deaths in dogs affected with mast cell tumour (MCT). Kaplan–Meier curves were constructed to compare tumour recurrence and tumour‐related death between patients. The log‐rank test was used to check for significant differences between curves. KIT‐I, KIT‐II and KIT‐III staining patterns were observed in 9 (11.11%), 50 (61.73%) and 22 (27.16%) tumours, respectively. Tumour recurrence rates and tumour‐related deaths were not associated with KIT staining patterns (P = 0278, P > 0.05), KIT (P = 0.289, P > 0.05) or KIT ligand (P = 0.106, P > 0.05) gene expression. Despite the lack of association between KIT staining pattern and patient survival time, the results suggest a correlation between aberrant KIT localization and increased proliferative activity of MCTs. RT‐PCR seems to be a sensible method for quantitative detection of KIT gene expression in canine MCT, although expressions levels are not correlated with prognosis.     

Specificity of four serologic assays for Mycobacterium avium ss paratuberculosis in llamas and alpacas: a single herd study.

An investigation was conducted for Mycobacterium avium ss paratuberculosis infections in a research herd of llamas and alpacas. Herd culture-negative status was established over a 23-month period by screening any individuals with any signs compatible with paratuberculosis (n = 1), high serology values (n = 8), or other health and research related reasons (n = 24). There were no M. avium ss paratuberculosis isolates from radiometric cultures of multiple tissue and fecal samples from these individuals and no known sources of exposure. Paratuberculosis is uncommon in North American llamas and alpacas: only 5 cases were identified after an extensive search of the Veterinary Medical Data Base, diagnostic laboratory records, publication databases, and personal communications. Therefore, serum samples from llamas (n = 84) and alpacas (n = 16) in the culture-negative herd were used to obtain preliminary estimates of test specificity for 3 enzyme-linked immunoassays (ELISAs) and an agar gel immunodiffusion (AGID) assay kit for detecting serum antibodies to M. avium ss paratuberculosis in South American camelids. The ELISAs were modifications of established bovine assays for antibody detection. With provisional cutoffs, ELISA-A had 52 false positives (specificity 48%), ELISA-B had 8 false positives (specificity 92%), ELISA-C had two false positives (specificity 98%), and the AGID had 0 false positives (specificity 100%). The range of ELISA values for culture-positive llamas and alpacas (n = 10) from other herds overlapped the range of values for culture-negative llamas and alpacas. The accuracy of the ELISAs may be improved by using age- and sex-specific cutoffs because uninfected male llamas and alpacas that were older than 1 year had higher values for some tests. These tests can be used for either llamas or alpacas; the protein-G conjugate ELISA (ELISA-B) may be useful for multispecies applications. These assays are best used for rapid presumptive diagnoses of llamas and alpacas with diarrhea and weight loss and as a screening tool for herds known to be exposed to infection. All seropositive results should be confirmed with culture.     

利用TB_PCR试剂盒对牛结核病的检测

应用聚合酶链式反应（ＰＣＲ）技术制备的ＴＢ－ＰＣＲ试剂盒对来自新疆６个牛场的２３８份血样、奶样、口腔分泌物标本中结核分枝杆菌进行检测,结果显示：ＴＢ－ＰＣＲ试剂盒对４０份奶样样本进行检测,７头为阳性,阳性检出率１７．５％。ＴＢ－ＰＣＲ试剂盒对１７８份血样标本的检测,２３头牛为阳性,阳性检出率为１２．９２％。ＴＢ－ＰＣＲ试剂盒对２０份口腔分泌物标本中结核分歧杆菌检测结果均为阴性。本试验中共计检测６个牛场的２３８份不同标本的ＰＣＲ阳性牛共计２７头,阳性检出率为３．０２％。将ＰＣＲ和传统的ＰＰＤ检测方法的结果比较显示ＰＰＤ检出阳性率高于ＰＣＲ,ＰＰＤ检出阳性牛３９头,检出阳性率６．７％。本试验对口腔分泌物标本的检测结果不理想。总之,ＴＢ－ＰＣＲ试剂盒在检测牛结核病不同标本中显示出快速、特异等优点。为今后牛结核病的检测     

应用MGB荧光探针技术确定猪KIT基因拷贝重复数

旨在建立一种简便、快速检测猪KIT基因拷贝重复数(CNVs)的方法并应用于未知样品的检测。在KIT外显子2中设计TaqMan-MGB探针和引物,建立荧光实时定量PCR检测KIT基因CNVs的方法。结果表明,TaqMan-MGB探针扩增KIT基因目的片段,不同DNA梯度浓度Ct值差异极显著(P<0.001),变异系数低(0.12%～0.26%);KIT与内标基因ESR的扩增效率相差只有2.4%;采用聚类分析推测了待测的50枚样品KIT的CNVs分别归属于2、3、4、5、6个拷贝。本研究建立的TaqMan-MGB探针实时荧光定量PCR检测KIT基因CNVs的方法,具有简便、快速、特异性和稳定性好的特点,适合于普通实验室应用。     

Progesterone and pregnanediol-glucuronid concentrations in saliva, milk and urine of female alpacas and their application in pregnancy diagnosis

The objective of the present study was the measurement of the pregnancy associated hormones progesterone (P4) and pregnanediol-glucuronide (PdG) in saliva, milk and urine of alpacas and their potential use in pregnancy diagnosis. Sample of blood, saliva, milk and urine were obtained from 36 female alpacas before mating and throughout the pregnancy. Concentrations of P4 and PdG were determined using an enzyme immunoassay (EIA). Pregnancy was checked by ultrasonography at any sampling time. The milk samples were also tested using a commercial on-farm progesterone kit which was designed for dairy cattle. EIA-Concentrations of P4 in blood, milk and urine and urine PdG concentrations were significantly higher in pregnant than in not pregnant alpacas. There was no difference in concentrations of P4 or PdG in saliva. The accuracy of the progesterone kit was 90% for diagnosis of pregnancy and 69% for non-pregnancy. However, 70% of the false positive results also showed relatively high P4 milk concentrations in the EIA. Values of P4 in blood and PdG in urine are comparable to previous reports in alpacas and therefore can be confirmed as an indicator for pregnancy. Saliva seems unsuitable in pregnancy diagnosis in alpacas, whereas milk seems to be an adequate alternative. The use of milk and urine would simplify the pregnancy diagnosis in alpacas since in contrast to the current methods (e. g. blood progesterone) the owners can take the samples. The avoidance of blood sampling results in a considerable stress reduction for the animals. P4 measurement in milk and PdG measurement in urine are good alternatives in pregnancy diagnosis during the first month of pregnancy, when a trans-abdominal ultrasonographic examination is not yet reliable. However, since high values of P4 and PdG only show the presence of active luteal tissue and therefore are indirect markers of pregnancy the diagnosis should be confirmed using ultrasound later in pregnancy.     

Wnt3a在不同毛色羊驼皮肤中的表达和定位

本研究旨在探索Wnt3a在羊驼皮肤中的表达与定位.以不同毛色的成年羊驼为研究对象,应用荧光定量PCR技术分析不同毛色羊驼皮肤Wnt3a基因的相对表达量,并运用Western blotting及免疫组织化学法对Wnt3a蛋白在不同毛色羊驼皮肤中进行表达和定位研究.结果:荧光定量PCR结果显示棕色羊驼中Wnt3a mRNA相对表达量是白色羊驼的2.9702倍;Western blotting结果表明,羊驼皮肤组织组蛋白提取物中存在相对分子质量约39 ku的产物,棕色羊驼皮肤平均蛋白表达量显著高于白色羊驼;免疫组织化学结果显示Wnt3a在羊驼皮肤毛囊的根鞘和毛球部呈阳性表达,根据光密度值分析得出Wnt3a在棕色和白色羊驼毛囊中的表达差异显著(P＜0.05).通过以上研究显示Wnt3a可能与羊驼毛色形成具有相关性.     

Assessment of the effects of exogenous long-acting insulin on glucose tolerance in alpacas

OBJECTIVE: To evaluate the effects of long-acting insulin on glucose clearance in alpacas. ANIMALS: 8 adult castrated alpacas. PROCEDURE: On 2 days, food was withheld from alpacas for 8 hours. Alpacas were randomly allocated to receive an SC injection of long-acting insulin (0.4 U/kg) or saline (0.9% NaCI) solution 1 hour before the first of 3 administrations of glucose (at 60, 480, and 1,200 minutes after treatment) on day 1 and the alternate treatment and procedure on day 2. Plasma glucose concentration was determined before and 15, 45, 120, and 240 minutes after each glucose administration, and fractional turnover rates were calculated. The data were compared between alpacas with and without insulin administration and among the 3 glucose administrations for each day. RESULTS: Compared with sham-treated alpacas, insulin-treated alpacas had significantly lower blood glucose concentrations from 180 to 600 minutes after treatment; they also had glucose concentrations significantly below baseline values from 120 to 480 minutes, at which time the mean glucose concentration was in the hypoglycemic range. Also, mean fractional turnover of glucose was significantly higher in insulin-treated alpacas from 105 through 300 minutes. CONCLUSIONS AND CLINICAL RELEVANCE: Compared with known effects of regular insulin in alpacas, the action of long-acting insulin was of slower onset but longer lasting; its administration may induce hypoglycemia, even in alpacas that receive glucose. To maintain the hypoglycemic effect, long-acting insulin may have to be administered more than once daily and blood glucose concentration should be monitored to avoid hypoglycemic complications in alpacas.