山羊ACSS2基因的克隆、序列分析及组织表达研究

本研究旨在对山羊乙酰辅酶A合成酶2（acetyl-CoA synthetase 2,ACSS2）基因进行克隆和生物信息学分析,并检测其在山羊不同泌乳时期乳腺组织中的表达量变化。以山羊乳腺组织RNA为模板,采用RT-PCR方法扩增并克隆山羊ACSS2基因完整CDS区序列,对测序结果进行生物信息学分析,并对ACSS2基因在山羊不同泌乳时期乳腺组织中的表达量进行分析。结果显示,山羊ACSS2基因CDS区序列长2 106 bp,编码701个氨基酸;山羊ACSS2基因与牛、马、人、犬、猪、小鼠和鸡的同源性分别为97.8%、92.0%、91.3%、91.3%、91.1%、88.1%和73.3%。蛋白理化性质分析结果表明,ACSS2蛋白分子质量为78.72 ku,理论等电点为6.03,属于酸性蛋白;跨膜结构和信号肽分析表明,ACSS2蛋白不含跨膜结构和信号肽;结构域分析表明,该蛋白含有1个乙酰辅酶A合成酶N端结构域。亚细胞定位分析结果表明,该蛋白主要分布在内质网（44.4%）、线粒体（33.3%）、细胞质（11.1%）和细胞核（11.1%）中。蛋白质结构预测发现ACSS2蛋白含有α-螺旋（29.10%）、延伸链（21.54%）、β-转角（9.84%）及无规则卷曲（39.52%）。实时荧光定量PCR分析结果表明,ACSS2基因在不同泌乳时期均有表达,其中在泌乳中期表达量最高,在干奶期表达量最低。本试验结果为进一步研究山羊ACSS2基因在脂质代谢过程中的功能及转录调控机制提供了参考。     

The use of quantitative PCR to detect Felis catus papillomavirus type 2 DNA from a high proportion of queens and their kittens

Squamous cell carcinomas are common feline skin cancers that have been associated with infection with Felis catus papillomavirus type 2 (FcaPV-2). Currently, little is known about the epidemiology of FcaPV-2 infection. The aim of this study was to develop a real-time PCR assay to quantify FcaPV-2 DNA in plucked hairs and skin swabs from 11 healthy breeding queens and their kittens. Samples were taken prior to kittening and then 2, 7 and 28 days after kittening to determine the age at which the kittens were first exposed to the virus. FcaPV-2 DNA was amplified from all of the queens and from 91% of the kittens at 2 days of age. There was a wide range in the quantity of FcaPV-2 DNA detected, from 1 to 92,520 copies per swab, and from 0.01 to 234 copies per copy of reference gene DNA in the hair plucks. The quantity of FcaPV-2 DNA detected in samples collected from the kittens was strongly correlated to that of their respective queens and the mean viral DNA load was similar for cats within a household but varied significantly between households. This is the first time that quantitative PCR has been used to detect FcaPV-2 DNA and the results suggest that the virus is ubiquitous but there is a wide variation of viral DNA loads. Kittens appear to be exposed to FcaPV-2 early in life, presumably from direct contact with their queen. These results are important when determining if FcaPV-2 infection of cats is preventable.     

BRCA1基因多态性及其与奶牛乳房炎抗性的相关性研究

为了研究BRCA1基因突变与荷斯坦奶牛体细胞数和体细胞评分的关系,试验通过对BRCA1基因外显子13、14进行克隆、序列比对和挖掘已有突变的方法确定该基因的多态位点,采用SNaPshot技术检测了BRCA1基因25025 T>A和46126 G>T突变位点在北京郊区荷斯坦奶牛群体中的分布,并对突变位点与体细胞数和体细胞评分进行了关联分析。结果表明,荷斯坦奶牛BRCA1基因2个位点均检测到3种基因型,其中25025 bp位点TT基因型为优势基因型,46126 bp位点GT基因型为优势基因型。25025 bp位点AA基因型个体体细胞数(P<0.05)和体细胞评分(P<0.01)都显著低于TT和TA基因型;46126 bp位点TT基因型个体体细胞数显著低于GG和GT基因型个体(P<0.05),但3种基因型个体体细胞评分无显著差异(P>0.05)。本研究结果初步表明,BRCA1基因25025和46126 bp位点可作为中国荷斯坦牛乳房炎抗性的标记辅助选择。     

Cloning of Buffalo AQP9 Gene and Analysing its Expression in the Buffalo Tissues

Cloning buffalo AQP9 gene and analyzing its expression in buffalo tissues.A pair of primers was designed according to the released bovine AQP9 sequences in GenBank,which was used to clone buffalo AQP9 gene.The AQP9 gene was amplified by RT-PCR,whose nucleotide sequence and protein structure were analyzed by bioinformatics methods.The expression of AQP9 in buffalo tissues was assayed by Real-time quantitative PCR.The expression of AQP9 gene in buffalo ovary and testis tissue was detected by immunohistochemical staining method.The results showed that the cloned ORF length of buffalo AQP9 gene was 888 bp,which coded 295 amino acids.The results of multiple sequence comparison showed that the nucleotide sequence of buffalo AQP9 shared 99%,90%,97% and 88% homologeous compared with that of Bos taurus,Sus scrofa,Ovis ariessis and Homo sapiens,respectively,while shared 99%,86%,97%,83% homologeous for amino acids,respectively.Phylogenetic tree analysis indicated that AQP9 gene was highly conservative in the evolutionary process.Real-time quantitative PCR results showed that AQP9 gene expressed in buffalo liver,lung,brain,skin,testis and ovary tissues with different levels,had the most abundant expression in liver,followed by in skin and testis,less observed in lung and ovary.The results of immunohistochemical staining showed that the expression of AQP9 protein varied with the development of buffalo ovarian tissue,and gradually enhanced with follicle development.In testicular tissue,AQP9 protein expressed in spermatocyte and leydig cells of developmental stage testis.These results indicated that we had successfully cloned buffalo AQP9 gene sequences.The expression and its function of AQP9 in buffalo ovaries and testes might play an important role in follicle development and spermatogenesis.     

Ascorbic acid ameliorates dysregulated folliculogenesis induced by mono-(2-ethylhexyl)phthalate in neonatal mouse ovaries via reducing ovarian oxidative stress

Phthalates, including di-(2-ethylhexyl)phthalate (DEHP), are common industrial chemicals in the environment. Recent evidence indicates that DEHP and its active metabolite mono-(2-ethylhexyl)phthalate (MEHP) negatively modulate reproductive functions and induce reactive oxygen species. Ascorbic acid (AA) is a dietary requirement for primates, and it acts as a potent free radical scavenger to protect tissues against oxidative stress. In this study, to investigate the toxic effects of MEHP on the follicle development and the beneficial role of AA, neonatal mouse ovaries were treated with different concentrations of MEHP with or without AA for 6 days. Then, the follicle constitution and oxidative status were compared in different groups. Results showed MEHP accelerated primordial follicle recruitment by increasing the percentage of primary and secondary follicles and decreasing the percentage of primordial follicles in the ovaries. Moreover, MEHP-induced ovarian oxidative stress by significantly increasing malondialdehyde (MDA) concentration and the expression of GSS and SOD1. When ovaries were co-administrated with MEHP and AA, follicle constitution was normalized, and the oxidative status was significantly decreased. These results suggested that AA ameliorated MEHP-induced ovarian oxidative stress and follicular dysregulation, which attested the clinical significance of AA for ovary protection in the case of MEHP exposure.     

氨基色谱柱测定蜂蜜中4种糖方法研究

《中国药典》(2015年)蜂蜜中规定:用Prevail Carbohyrate ES色谱柱,以乙腈-水(75:25)为流动相,示差折光检测器测定蜂蜜中的果糖、葡萄糖、蔗糖及麦芽糖。此次试验证明用普通氨基色谱柱替代Prevail Carbohyrate ES色谱柱,待测组分与其他组分分离良好,精密度试验满足要求,回收率试验结果大于93%,结果准确、可靠,普通氨基柱可替代Prevail Carbohyrate ES色谱柱,用于蜂蜜中糖类物质的测定。     

SNAT2介导蛋氨酸对牛乳腺上皮细胞增殖与自噬的调节作用

钠离子依赖性中性氨基酸转运体2（SNAT2）是一种氨基酸转运蛋白,可转运中性氨基酸,广泛分布于多种细胞中。氨基酸既可作为蛋白质合成的底物,也是调节细胞新陈代谢的关键信号分子,但SNAT2是否介导氨基酸调节BMECs增殖和自噬尚未见报道。本研究利用CASY细胞计数和Western blotting技术检测SNAT2过表达和siRNA干扰后牛乳腺上皮细胞（BMECs）增殖情况以及SNAT2对自噬标志蛋白LC3-Ⅰ/Ⅱ表达量的影响,并利用免疫荧光检测细胞自噬斑点（LC3-Ⅱ）变化。结果显示,SNAT2过表达时,p-PI3K、p-mTOR和Cyclin D1表达量增加,反之,p-PI3K、p-mTOR和Cyclin D1表达量下降。SNAT2抑制时,LC3-Ⅱ表达量增加,免疫荧光检测自噬斑点增多。添加自噬增强剂海藻糖（trehalose,Tre）和蛋氨酸（methionine,Met）后,与单一添加Tre组相比,Met+Tre组p-mTOR表达量增加,LC3-Ⅱ表达量降低,胞浆内绿色自噬斑点减少;添加Tre和Met并抑制SNAT2时,p-mTOR表达量下降,LC3-Ⅱ表达量增多,胞浆内绿色自噬斑点增加。以上结果表明,SNAT2可介导Met通过调控PI3K-mTOR/Cyclin D1信号通路调节BMECs的增殖与自噬。     

Effect of the β‐carotene oxygenase 2 genotype on the content of carotenoids,retinol and α‐tocopherol in the liver,fat and milk of rabbit does,reproduction parameters and kitten growth

Mutations in the β‐carotene oxygenase 2 (BCO2) gene can impair the function of the enzyme that breaks down carotenoids. As a result, gradual accumulation of unoxidized carotenoids in animal tissues gives them a yellow colour. The aim of the study was to determine the content of carotenoids, retinol and α‐tocopherol in the liver, fat and milk of rabbit does with three different genotypes determined by AAT‐deletion mutation at codon 248 of the BCO2 gene and to find out whether differences in the concentrations of the above compounds in the tissues and milk of the does affect reproduction parameters and the rearing rate of kittens. The experimental materials comprised 36 does, 12 of each genotype of the BCO2 gene, with their litters. Females with their litters were placed in individual cages, on deep litter. Between days 7 and 13 of lactation, samples of milk were collected from the does. The kittens stayed with their mothers until 35 days of age. After weaning, the does were sacrificed. Tissue samples of liver and perirenal fat were collected for chemical analyses. Additionally, based on samples taken from one female, RNA expression levels were determined from the mammary gland and liver, adipose tissue and skin. It was found that homozygous does with deletion at codon 248 of the BCO2 gene were characterized by considerably higher concentrations of xanthophylls and beta‐carotene in the liver, adipose tissue and milk than does with the remaining genotypes. However, the differences in the content of the above compounds in milk had no influence on litter weight or the number and rearing rate of kittens. Additionally, RNA expression of the BCO2 gene was found in the mammary tissue of lactating doe and its level was similar to those noted in the liver and adipose tissue.     

REGIONAL BINDING INDEX OF THE RADIOLABELED SELECTIVE 5-HT2A ANTAGONIST 123I-5-I-R91150 IN THE NORMAL CANINE BRAIN IMAGED WITH SINGLE PHOTON EMISSION COMPUTED TOMOGRAPHY

The pattern of the specific 5-HT2A (5-hydroxytryptamine 2A receptor) antagonist 123I-5-I-R91150 was measured in 10 healthy dogs without neurologic and behavior abnormalities. Eight cortical regions (left and right fronto-, temporo-, parieto-, and occipitocortical area), one global subcortical region (including the thalamic system) were compared with a reference region lacking receptors; that is, the cerebellum. The 123I labeled radioligand was injected intravenously 100-200 minutes before acquisition. Both transmission and emission data were obtained with a triple head gamma camera equipped with high-resolution fanbeam collimators. The emission data were corrected for scatter and attenuation. To delineate different cerebral regions more accurately, the regions of interest (ROI) defined in a former study on brain perfusion measured with 99mTc-ethyl cysteinate dimer (ECD) in the same dogs were used. The co-registration of the 99mTc-ECD and the 123I-5-I-R91150, obtained from each dog, was realized with the help of corresponding transmission maps. By normalizing each regional cerebral activity to the activity observed in the cerebellum, the regional radioactivity (binding index) could be relatively quantified. Highest brain uptake was noted in the frontocortical brain areas (right: 1.85, left: 1.89), followed by the temporocortical region (right: 1.58, left: 1.56). Least uptake was noted in the more caudal and middle brain regions [occipito- (right: 1.46, left: 1.41), parietocortical (right: 1.30, left: 1.26), and striatal region (1.19)]. No gender nor age influence was noted in this series. The 123I labeled serotonin-2A receptor ligand seems to have similar cortical binding in the normal canine brain, as shown in humans and other animal species. A frontocortical to occipitocortical (rostrocaudal) binding index gradient was identified within the dog, which has not been seen in imaging studies from humans and other animal species. The significance of these results will need further investigation. This normative data can be used to compare regional brain uptake of the 123I-radioligand to dogs with behavioral disorders related to the serotonergic system, in future studies.     

用ELISA方法进行猪圆环病毒病血清学调查

本研究用猪2型圆环病毒ORF2基因的表达产物包被酶标板，建立ELISA诊断方法，用此方法对临床1257份送检血清进行了检测，其总阳性率为57．28%(720／1257)，仔猪阳性率为30．83％(119／386)，肥猪阳性率为64．93％(137／211)，母猪阳性率为71．50％(419／586)，公猪阳性率为70．27％(52／74)。这一结果表明猪圆环病毒在我国流行较普遍，且抗体阳性率随猪体年龄的增长而升高。