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1.
《上海畜牧兽医通讯》2016,(1)
抗精子抗体11被认为在雄性生殖道的先天免疫及生殖方面起着重要的作用,牛抗精子抗体11E(SPAG11E)主要存在睾丸、附睾等生殖道中。本实验根据GenBank中牛SPAG11E基因序列(登录号:DQ838983.1)设计特异性引物,提取牛睾丸组织总RNA,经RT-PCR获得SPAG11E基因序列,构建原核表达载体,并转入BL21(DE3),利用IPTG诱导表达,同时检测了重组蛋白体外抑菌活性。结果显示,成功扩增了牛SPAG11E基因的CDS序列,其与GenBank公布序列相一致;正确构建了原核表达载体pET-32a(+)-SPAG11E,最佳表达条件为IPTG终浓度1.0mmol/L、诱导时间4h;重组蛋白对检测的6种菌没有明显的抑菌作用。 相似文献
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试验旨在了解在鸡睾丸中高表达的1个长链非编码RNA （long non-coding RNA,lncRNA）及其预测靶基因的时空表达规律,研究二者在鸡弱精子症中的调控作用。根据弱精子症和正常北京油鸡公鸡睾丸转录组测序筛选到的1个高表达的lncRNA （MSTRG.15568.9）,采用顺式（cis）作用模式预测其潜在靶基因SPAG4（sperm-associated antigen 4）,进一步采用实时荧光定量PCR方法进行表达量分析。分别选择3只0、5、20、30、45、60周龄正常北京油鸡公鸡,检测MSTRG.15568.9与SPAG4基因在不同周龄公鸡睾丸中的表达量差异;选择30周龄3只正常公鸡,采集睾丸、肝脏和脾脏等8个部位组织样品,检测MSTRG.15568.9与SPAG4基因在不同组织间的表达规律;选择45周龄弱精子症公鸡和正常公鸡各3只,对比MSTRG.15568.9与SPAG4基因在睾丸的表达量差异。结果显示,MSTRG.15568.9与SPAG4存在明显的时空表达差异,且二者表达趋势基本一致。在不同周龄的鸡睾丸组织中,MSTRG.15568.9和SPAG4的表达趋势相近,MSTRG.15568.9在20周龄的表达量显著高于0、5、30、45、60周龄（P<0.05）,0和5周龄表达量显著低于20、30、45和60周龄（P<0.05）;SPAG4在45周龄表达量最高,其次是20周龄（P<0.05）。MSTRG.15568.9和SPAG4在睾丸和肝脏中的表达量均显著高于脾脏、肾脏等组织（P<0.05）;在正常睾丸组织中的表达量均显著高于弱精子症睾丸组织（P<0.05）。综上所述,MSTRG.15568.9与SPAG4基因具有较明显的组织表达特异性,且MSTRG.15568.9可能调控SPAG4基因的表达,参与精子发生与精子活力调控;但其具体作用机制需要进一步探索。本研究可为鉴定与鸡弱精子症调节机制相关的功能基因提供参考。 相似文献
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通过对１５头西门塔尔种公牛冷冻精液精子畸形率的测定，认为南昌地区气温（季节）变化对精子畸形率会产生很大影响。经统计分析并得出结论，高温（３２℃以上）对种公牛冷冻精子畸形率影响较大，即气温愈高，精子畸形率愈高。冬、夏季西门塔尔牛精子畸形率差异极显著（Ｐ＜０．０１）。 相似文献
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本文旨在研究猪睾丸ACE启动子驱动的SPAG6 shRNA在猪睾丸支持细胞（ST）中的干扰作用。利用PCR法从ST细胞基因组中扩增猪睾丸ACE启动子,构建重组质粒ACE-pIRES2-DsRed、ACE-pGL3-basic,通过红色荧光蛋白表达和双荧光素酶报告基因检测试验检测猪睾丸ACE启动子在ST细胞中的转录活性;根据SPAG6 mRNA靶序列设计合成3条siRNA序列,通过脂质体转染至ST细胞,利用实时荧光定量PCR及Western Blot检测siRNA的干扰效率;基于人miR-30a结构设计SPAG6 shRNA,构建重组载体ACE-sh-SPAG6-pcDNA3.1,Western Blot检测其对SPAG6的干扰效果。结果表明,成功构建重组质粒ACEpIRES2-DsRed、ACE-pGL3-basic,并证明猪睾丸ACE启动子在ST细胞中具有较强的转录活性;各试验组均能对靶基因SPAG6 mRNA的表达产生抑制作用,以siRNA-3的作用最佳。siRNA-3转染48 h后,SPAG6蛋白表达量显著下降（P<0.001）,成功筛选出SPAG6基因的最佳干扰序列;成功... 相似文献
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SOX5蛋白对公鸡精子发生和精子活力的作用及其定位 总被引：1，自引：0，他引：1
本研究旨在探明SOX5蛋白在家禽睾丸发育中的表达规律,分析其在睾丸发育和精子活力调控中的潜在作用。采用RT-qPCR和Western blot技术检测0、5、15、40和60周龄北京油鸡公鸡睾丸中SOX5mRNA和蛋白的表达,并比较其在40周龄正常公鸡与弱精子症公鸡睾丸中的表达差异,通过免疫组化技术对SOX5蛋白在睾丸组织中的表达进行定位。结果表明,SOX5 mRNA和蛋白在不同周龄公鸡的睾丸中均有表达,且差异显著(P0.05)。SOX5mRNA和蛋白表达量趋势一致;随着周龄的增加,其表达呈现先上升后降低的趋势:0和5周龄SOX5蛋白主要在精原细胞和支持细胞中表达;15周龄SOX5蛋白在支持细胞、各级精母细胞、圆形精子细胞和成熟精子中均高表达;40周龄表达量有所降低。SOX5基因和蛋白的表达趋势与公鸡性成熟和繁殖机能衰退的趋势一致,即性成熟前后和繁殖高峰期表达量较高,在性发育前和生精机能减退时表达量较低。此外,40周龄弱精子症公鸡睾丸中,SOX5的表达显著低于正常公鸡(P0.05)。综上表明,SOX5的表达量与公鸡睾丸发育水平和精子活力存在一定的相关,可能对其具有重要调控作用。 相似文献
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旨在探究肌球蛋白结合蛋白C1（myosin binding protein C1,MyBPC1）对牛骨骼肌卫星细胞增殖与成肌分化的影响,为进一步研究MyBPC1在细胞分化和肌肉发育过程中的调控作用提供依据。本研究利用西门塔尔胎牛原代牛骨骼肌卫星细胞体外诱导成肌分化模型模拟牛骨骼肌的生长发育过程。采用qRT-PCR和Western blot检测MyBPC1的细胞时序表达谱。试验分为两组。在RNA水平每组4个重复,每个重复20 μL;在蛋白水平每组3个重复,每个重复15 μg。采用qRT-PCR和Western blot检测牛骨骼肌卫星细胞转染MyBPC1的过表达效果,并进一步检测细胞增殖期标志因子Pax7、Ki67以及细胞分化期标志因子MyHC、MyOG的表达变化情况,观察牛骨骼肌卫星细胞肌管形成状态。结果,MyBPC1在牛骨骼肌卫星细胞分化前后表达水平存在极显著差异,牛骨骼肌卫星细胞诱导分化后MyBPC1的mRNA和蛋白表达量均极显著高于增殖期（P<0.01）。过表达MyBPC1后,细胞分化形成的肌管数量明显多于对照组,增殖标志因子Pax7的mRNA水平和蛋白表达水平无显著差异,分化标志因子MyHC的mRNA水平和蛋白表达水平极显著高于对照组（P<0.01）。过表达MyBPC1可以促进牛骨骼肌卫星细胞体外成肌分化,为进一步开展MyBPC1对牛骨骼肌卫星细胞的调控机制奠定基础。 相似文献
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《中国兽医杂志》2017,(1)
为了探讨温和热应激、脂多糖(LPS)和谷氨酰胺(Gln)诱导牛睾丸支持细胞(b SCs)HSP72的时效表达规律,本试验将传至第3代b SCs分成热应激、LPS和Gln处理组,用CCK-8试剂盒检测细胞活性,用RT-PCR和Western-Blot方法分别检测处理后0、2、4、6、8、12、14h和16 h时细胞内HSP72 mRNA和蛋白表达。结果显示,3种方式均能诱导HSP72表达,且HSP72蛋白分别在热应激、Gln和LPS预处理后2、4h和12 h时表达量最高。结果表明,热应激或Gln均比LPS诱导牛睾丸支持细胞HSP72表达的时间早,提示可考虑用Gln减少由LPS诱导的细胞损伤。 相似文献
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旨在分析Toll样受体7/8(TLR7/8)在公猪精子及生殖器官中的表达情况，并探讨是否能通过其配体处理的方式分离猪X精子和Y精子。本研究采用qRT-PCR分析3头健康成年公猪睾丸、附睾头、附睾体、附睾尾以及精子中TLR7和TLR8基因的mRNA表达水平，利用免疫组化检测公猪睾丸和附睾中TLR7和TLR8蛋白的表达，并通过细胞免疫荧光分析其在不同物种(健康成年小鼠、公牛和公猪)精子中的表达情况，将其配体R848与猪精子共孵育，研究其对精子活力以及X/Y精子分离的影响。结果表明，TLR7和TLR8 mRNA在公猪睾丸、附睾头、附睾体、附睾尾组织以及精子中均表达；免疫组化结果显示，TLR7/8蛋白在睾丸中主要表达于生殖细胞，在附睾中主要表达于柱状细胞微绒毛中；细胞免疫荧光结果表明，TLR7和TLR8蛋白只表达于小鼠和牛X精子尾部，Y精子中不表达，但TLR7和TLR8蛋白在公猪X和Y精子中都表达且表达模式无显著差异，TLR7蛋白主要表达于猪精子头部顶体区域，TLR8蛋白主要表达于猪精子尾部；与对照组相比，用TLR7和TLR8配体R848孵育猪精子后，精子活力降低，但上层精子的性别比例无显著差... 相似文献
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HU Xiu-zhong XIANG Min CHENG Lei ZHOU Yuan YU Jie LIU Xiao-hua WANG Ding-fa XIA Yu ZHANG Li-qing 《中国畜牧兽医》2017,44(5):1446-1451
To explore the influence of heat stress on the expression level of bovine sperm associated antigen 11D (SPAG11D),which was sperm membrane protein,the mRNA and protein expression levels of SPAG11D were tested in primary bovine testicular cells and sperm. The normal sperm and low activity sperm were collected from Jersey cattle, Bos bubalus and Simmental cattle. The results showed that mRNA and protein expression levels of SPAG11D were highest at 36℃ than others (32, 34, 38 and 40℃) after primary bovine testicular cells cultured for 24 h. And the mRNA and protein expression levels were higher in high temperature groups than low temperature groups. The mRNA expression of SPAG11D in 48 h was consistent with 24 h, but the protein expression levels decreased at high temperature (38 and 40℃). In addition, the protein expression of SPAG11D was lower in low activity sperm than in normal sperm (Jersey cattle and Bos bubalus), and there was no significant change in Simmental cattle sperm. 相似文献
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Garrett AJ Rincon G Medrano JF Elzo MA Silver GA Thomas MG 《Journal of animal science》2008,86(12):3315-3323
Expression of the GH receptor (GHR) gene and its binding with GH is essential for growth and fat metabolism. A GT microsatellite exists in the promoter of bovine GHR segregating short (11 bp) and long (16 to 20 bp) allele sequences. To detect SNP and complete an association study of genotype to phenotype, we resequenced a 1,195-bp fragment of DNA including the GT microsatellite and exon 1A. Resequencing was completed in 48 familialy unrelated Holstein, Jersey, Brown Swiss, Simmental, Angus, Brahman, and Brangus cattle. Nine SNP were identified. Phylogeny analyses revealed minor distance (i.e., <5%) in DNA sequence among the 5 Bos taurus breeds; however, sequence from Brahman cattle averaged 27.4 +/- 0.07% divergence from the Bos taurus breeds, whereas divergence of Brangus was intermediate. An association study of genotype to phenotype was completed with data from growing Brangus bulls (n = 553 from 96 sires) and data from 4 of the SNP flanking the GT microsatellite. These SNP were found to be in Hardy-Weinberg equilibrium and in phase based on linkage disequilibrium analyses (r(2) = 0.84 and D'= 0.92). An A/G tag SNP was identified (ss86273136) and was located in exon 1A, which began 88 bp downstream from the GT microsatellite. Minor allele frequency of the tag SNP was greater than 10%, and Mendelian segregation was verified in 3 generation pedigrees. The A allele was derived from Brahman, and the G allele was derived from Angus. This tag SNP genotype was a significant effect in analyses of rib fat data collected with ultrasound when bulls were ~365 d of age. Specifically, bulls of the GG genotype had 6.1% more (P = 0.0204) rib fat than bulls of the AA and AG genotypes, respectively. Tag SNP (ss86273136), located in the promoter of GHR, appears to be associated with a measure of corporal fat in Bos taurus x Bos indicus composite cattle. 相似文献
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为研究长途运输应激对西门塔尔牛体重、血液生化指标和细胞因子含量的影响,随机选取12头、平均体重（292.1±38.2）kg的西门塔尔牛,在最高气温30℃下,经过36 h、1 450 km的敞篷卡车运输,运输前后称重并采集颈静脉血样,测定其体重及血液生化指标的变化情况。结果表明：①与运输前体重相比,经36 h、1 450 km运输后,试验牛平均体重极显著降低（P<0.01）,由292.1 kg降低到262.8 kg,体重损失10.03%。②与运输前相比,运输36 h时西门塔尔牛血清皮质醇（COR）含量显著提高（P<0.05）、血糖（GLU）水平极显著增高（P<0.01）,运输后7~14 d下降到运输前水平（P>0.05）;运输应激对血清胆固醇水平无显著影响（P>0.05）。③运输应激极显著提高了西门塔尔牛血清C-反应蛋白（CRP）含量（P<0.01）,显著提高血清白细胞介素-6（IL-6）、γ-干扰素（IFN-γ）和肿瘤坏死因子-α（TNF-α）的含量（P<0.05）,运输后7~14 d下降到运输前或更低水平（P>0.05）;运输应激对血清白细胞介素-1β（IL-1β）含量无显著影响（P>0.05）。综上所述,长途运输可显著降低肉牛的体重和抑制机体免疫机能,经长途运输后的西门塔尔牛需要约14 d的恢复期。 相似文献
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为研究营养水平对杂交肉牛心脏型脂肪酸结合蛋白（FABP3）基因表达及肌内脂肪含量（IMF）的影响,以西门塔尔、利木赞和夏洛莱杂交肉牛为研究对象,利用实时荧光定量PCR技术,分别测定高、中、低营养水平的3种杂交肉牛背部脂肪中FABP3mRNA表达水平,并测定其背最长肌IMF。结果：（1）高营养组中夏洛莱牛IMF高于同营养组其他2种肉牛,其背部脂肪中FABP3mRNA水平也显著高于另外2种杂交肉牛（P〈0.01）;中营养组中西门塔尔和利木赞牛IMF均高于同营养组的夏洛莱牛,但FABP3mRNA的水平无显著差异（P〉0.05）;低营养组中夏洛莱牛IMF和FABP3mRNA水平均显著低于另外2种肉牛（P〈0.01）;（2）夏洛莱杂交肉牛背部脂肪中FABP3mRNA水平与IMF具有显著的相关性（P〈0.05）,而西门塔尔和利木赞牛中FABP3mRNA水平与IMF无显著相关性（P〉0.05）。该结果表明提高营养水平影响FABP3mRNA表达并在一定程度上影响IMF,但影响显著与否存在品种差异,在肉牛育种和牛肉生产中应当同时考虑营养因素和遗传因素。 相似文献
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Montgomery JL Blanton JR Horst RL Galyean ML Morrow KJ Wester DB Miller MF 《Journal of animal science》2004,82(7):2043-2049
Feedlot steers (n = 36) from three biological types (Bos indicus, Bos taurus-Continental, and Bos taurus-English) were used to determine the Ca, P, and vitamin D3 status of feedlot cattle. The USDA yield and quality grade traits were measured at slaughter, and the concentrations of vitamin D3 (VITD) and the metabolites 25-hydroxyvitamin D3 (25-OH D) and 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D) were determined in LM, liver, kidney, and plasma. Plasma and muscle Ca and P concentrations also were determined. Biological type of cattle affected a number of carcass traits. Carcasses from Bos taurus-English cattle had more marbling, resulting in higher quality grades (P < 0.05). Carcasses from Bos taurus-Continental cattle had lower calculated yield grades (P < 0.05) than did carcasses from cattle in the other biological types. In general, differences in carcass traits resulting from biological type were consistent with other reports. Plasma and LM Ca and P concentrations were not affected (P = 0.06) by biological type of cattle, indicating that Ca and P homeostasis is a conserved trait across the different types of cattle. Plasma VITD and 25-OH D concentrations were not affected (P = 0.41) by biological type, whereas plasma 1,25-(OH)2 D concentration was lower (P < 0.05) in Bos taurus-English cattle than in Bos taurus-Continental and Bos indicus cattle. Liver VITD and 25-OH D were not affected by biological type (P = 0.76), but liver 1,25-(OH)2 D concentration was greater (P < 0.05) in Bos indicus cattle than in Bos taurus-Continental cattle. Kidney vitamin D metabolite concentrations were not affected by biological type of cattle (P = 0.21). Muscle VITD concentration was greater (P < 0.05) in Bos taurus-English cattle than in the other two biological types, and muscle 25-OH D concentrations were greater (P < 0.05) in Bos taurus-English cattle than in Bos indicus cattle. Muscle 1,25-(OH)2 D concentration was less (P < 0.05) in the Bos taurus-Continental cattle than in the other two biological types. Cooking eliminated vitamin D metabolite differences among the biological types. Our results suggest that Bos indicus cattle had greater 1,25-(OH)2 D (the biologically active form) in tissues, and greater 1,25-(OH)2 D plasma concentrations than Bos taurus cattle. Thus, the need for VITD supplementation and optimal levels of Ca and P in feedlot diets might differ between Bos indicus and Bos taurus cattle. 相似文献
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McGuire K Manuja A Russell GC Springbett A Craigmile SC Nichani AK Malhotra DV Glass EJ 《Veterinary immunology and immunopathology》2004,99(1-2):87-98
The pathogenic mechanisms involved in tropical theileriosis, caused by the tick-borne protozoan parasite Theileria annulata, are unclear. Pathology is associated with the schizont stage of the parasite, which resides within bovine macrophages. Breed-specific differences in pathology have been observed in cattle, several Bos indicus breeds are relatively resistant to tropical theileriosis whilst Bos taurus cattle are highly susceptible. Infected cells express pro-inflammatory cytokines and it has been hypothesized that these cytokines play a major role in the pathology of the disease. Therefore, using quantitative RT-PCR we investigated the expression of the key candidates, interleukin 1 beta (IL-1beta), IL-6 and tumour necrosis factor alpha (TNF-alpha), in T. annulata low passage infected cell lines derived ex vivo from experimental infection of resistant and susceptible cattle. mRNA for each cytokine was detected in all cell lines investigated at levels higher than those observed in resting monocytes. However, the analyses did not identify any breed-specific differences. Therefore, these results are not consistent with the hypothesis that differential regulation of infected cell derived pro-inflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) accounts for the breed-related differences in resistance and susceptibility to T. annulata infection. Other, currently unknown mechanisms may be of greater importance. 相似文献
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草原红牛ACSL3基因CDS区克隆、生物信息学分析及组织表达研究 总被引：1，自引：1，他引：0
试验旨在克隆草原红牛长链酰基辅酶A合成酶3(long-chain acyl-CoA synthetase 3,ACSL3)基因编码区并对其进行生物信息学分析,同时在mRNA和蛋白质水平上分析ACSL3基因在草原红牛不同组织中的表达差异。利用RT-PCR技术和TA克隆的方法获得草原红牛ACSL3基因CDS序列;利用在线软件对ACSL3基因进行生物信息学分析,分析ACSL3基因与其他物种的同源性并构建系统进化树,分析ACSL3基因编码蛋白质的基本理化性质、潜在磷酸化位点、O-糖基化位点、N-糖基化位点、信号肽、二硫键、跨膜区结构、亚细胞定位及该基因编码蛋白的二级结构、三级结构;通过实时荧光定量PCR和Western blotting技术检测ACSL3基因在草原红牛各组织间的mRNA和蛋白表达水平。结果显示,试验成功克隆了草原红牛ACSL3基因CDS区,全长2 163 bp,编码720个氨基酸,蛋白分子质量为80.28 ku,理论等电点为8.74,属于亲水性蛋白。通过NCBI中BLAST比对发现,草原红牛与牛、绵羊、猪、人、大鼠、小鼠、鸡的ACSL3基因核苷酸序列同源性分别为99%、97%、93%、91%、88%、88%和78%;系统进化树结果表明,草原红牛与牛、绵羊的亲缘关系最近,与鸡的亲缘关系最远。该蛋白序列有7个二硫键,66个磷酸化位点,9个O-糖基化位点,3个N-糖基化位点,不存在信号肽,但存在1个跨膜区。二级结构和三级结构分析结果表明,ACSL3蛋白通过无规则卷曲连接,蛋白质结构以α-螺旋和β-转角为主,为混合型蛋白。mRNA和蛋白表达量检测结果显示,ACSL3基因在肾脏和肌肉组织中表达量较高,显著高于其他组织(P<0.05);在胃、肝脏和心脏中中度表达,显著高于脾脏、肺脏、肠和脂肪(P<0.05);在脾脏、肺脏、肠和脂肪中相对低表达,说明草原红牛ACSL3基因可能与体内脂肪沉积和脂质代谢等调控功能有关。本试验结果为进一步研究ACSL3基因在草原红牛中脂质代谢及脂肪沉积等方面的调控作用提供了基础材料。 相似文献
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试验旨在评价细胞因子IL-6和IL-17 mRNA转录水平与牛分枝杆菌感染之间的关系,及其在牛结核病诊断中的应用潜力。通过皮内变态反应试验和IFN-γ释放试验临床筛选结核病阳性牛和结核病阴性牛,采集试验动物抗凝全血,分离、收集外周血淋巴细胞,分别用牛结核菌素（PPD-B）、禽结核菌素（PPD-A）、重组蛋白CFP-10-ESAT-6（CE）、pET-32a载体标签蛋白（PET）或PBS 37℃培养6 h,用实时荧光定量PCR检测细胞因子IL-6、IL-17和IFN-γ的mRNA相对转录水平。结果显示,PET和空白对照PBS类似,不能刺激细胞因子mRNA转录水平的提高,表明CE中包含的PET对试验的影响可忽略不计;牛外周血淋巴细胞经PPD-B、PPD-A或CE刺激后,结核病阳性牛样品中IL-17和IFN-γ的mRNA转录水平均显著高于结核病阴性牛（P<0.05）,其中PPD-B刺激效果强于CE和PPD-A,而CE刺激的特异性更好;选取CE作为最佳刺激源,结果显示,IL-17和IFN-γ的mRNA转录水平之间相关性良好（spearman r=0.79）,并初步建立了基于IL-17和IFN-γ转录水平的实时荧光定量PCR检测方法;以此方法对14头结核病阳性牛进行临床检验,IL-17实时荧光定量PCR法的阳性样本检出率为85.7%,高于IFN-γ（71.4%）。本研究结果初步表明,牛分枝杆菌特异性抗原（PPD-B、CE）诱导的IL-17 mRNA转录水平与牛结核病相关,以CE为刺激源建立的IL-17实时荧光定量PCR检测方法具有用于牛结核病诊断的潜力。 相似文献
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