贵州从江香猪PDK4、FGF10基因的克隆及组织表达分析

试验旨在克隆获得PDK4、FGF10基因,并研究大白猪与从江香猪不同组织中PDK4、FGF10基因mRNA的表达差异。采用RT-PCR分别克隆从江香猪PDK4、FGF10基因并进行生物信息学分析,利用实时荧光定量PCR技术检测PDK4、FGF10基因在大白猪和从江香猪不同组织中mRNA的相对表达量。结果显示,从江香猪PDK4基因的编码区全长1 224 bp,编码407个氨基酸;FGF10基因的编码区全长636 bp,编码211个氨基酸。经BLAST软件进行同源性比对,发现从江香猪PDK4基因与羊、马、人的核苷酸序列同源性分别为93%、92%和91%;FGF10基因与羊、牛、人、鼠的核苷酸序列同源性分别为94%、93%、93%和90%。由PDK4基因系统进化树可知,从江香猪与牛、绵羊亲缘关系较近;由FGF10基因系统进化树可知,从江香猪与绵羊、牛、人、猕猴亲缘关系较近,与小鼠和鸡亲缘关系较远。实时荧光定量PCR结果显示,在从江香猪不同组织中,PDK4基因在肾脏中的表达量最高,在胃和脂肪中表达量较高,FGF10基因在胃中表达量最高,在肾脏和脂肪中表达量较高,两个基因在背最长肌中的表达量均最低;在大白猪的不同组织中,PDK4、FGF10基因在脂肪中的表达量均最高,PDK4基因在背最长肌中的表达量最低,而FGF10基因在心脏中表达量最低。本试验成功克隆了从江香猪PDK4、FGF10基因,并检测了其在大白猪与从江香猪不同组织中的表达,为进一步研究PDK4、FGF10基因在脂质代谢及脂肪沉积等方面的调控作用提供科学依据。     

Characterization of CTLA-4, PD-1 and PDL-1 of swamp and riverine type water buffaloes

Characterization of CTLA-4, PD-1 and PDL-1 genes from swamp and riverine type water buffaloes was done by molecular cloning, sequencing and phylogenetic analysis. The cloned cDNA of CTLA-4, PD-1 and PDL-1 contained an open reading frame of 666, 849 and 870 nucleotides, encoding a polypeptide of 221, 282 and 298 amino acids, respectively. Nucleotide sequence homology of both CTLA-4 and PDL-1 had 99.8% in swamp and riverine type, which gives the identical polypeptide. Meanwhile, PD-1 genes of swamp and riverine type water buffaloes had 99.2% of homology in nucleotide sequence, which has substitution of two amino acid residues. The hexapeptide motif, phosphatidylinositol 3′-kinase and potential glycosylation sites were conserved within the tribe Bovinae. Phylogenetic analysis confirmed the degree of relationship between the bubaline species and justify the distinctness of each breeds by the bootstrap value generated.     

人血小板因子Ⅳ在家蚕杆状病毒表达载体系统中的表达

将人血小板因子Ⅳ (HumanPlateletFactorⅣ ,简称hPF4)基因重组于家蚕杆状病毒转移载体pBacPAK8中 ,获得重组转移载体pBacPAK PF4,并与线性化病毒Bm BacPAK6DNA共转染家蚕培养细胞 ,在细胞内发生同源重组 ,获得重组病毒BacPAK PF4。Southern杂交结果表明重组病毒基因组中含有hPF4基因。重组病毒以MOI=10感染家蚕培养细胞 (2× 10 6个细胞 )和家蚕 5龄幼虫 ,表达产物用体外培养的血管内皮细胞测定其生物活性 ,测得表达量在家蚕培养细胞中第 3天达到最高值为 6 0 88μg/ 2× 10 6个细胞 ;在蚕体内表达第 5天达到最高值 ,表达量明显高于家蚕培养细胞     

白羽瘤头鸭不同生长阶段的甲状腺活动

本文报道了用放射免疫分析法测定５７例不同日龄的白羽瘤头鸭（Ｃａｉｒｉｎａｍｏｓｈａｔａ）外用血清中Ｔ４的含量和用组织学方法检查甲状腺滤泡的发育作为甲状腺活动的指标，研究不同日龄的生长速度与甲状腺活动的关系。５７例白羽瘤头鸭的血清Ｔ４含量平均为１．３６±０．３７μｇ／１００ｍｌ，其中１—４０日龄甲状腺的分泌活动除２０日龄外，其他的均随着日龄增长而升高，分泌的高峰出现在４０日龄，以后随日龄增长而下降。４０日龄组血清Ｔ４的含量与１、１０、２０、６０、和９０日龄组比较，差异极显著（Ｐ＜０．０１），血清Ｔ４的波动与相对生长率之间有统计学强的正相关，差异极显著（Ｐ＜０．０１）。组织切片检查，刚出壳的雏鸭甲状腺滤泡小。１０—４０日龄甲状腺滤泡随日龄的增长而增大，滤泡腔被甲状腺球蛋白所组成的蛋白质凝胶的均质液所充满、着色深而均匀。６０—９０日龄鸭甲状腺滤泡虽然增大，但其内部凝胶的均质液充满不完全或空虚，着色浅而不均匀。各日龄组甲状腺滤泡的变化和甲状腺分泌的含量变化与Ｔ４的波动相一致。     

A pig–human comparative RH map comprising 20 genes on pig chromosome 13q41 that harbours the ETEC F4ac receptor locus

The enterotoxigenic Escherichia coli (ETEC) F4ac is a major cause of diarrhoea in newborn and young pigs. The locus for the intestinal ETEC F4ac receptor (F4acR) has been mapped to pig chromosome (SSC) 13q41 with known homology to human chromosome (HSA) 3q21 and q29. However, the causative gene and mutation(s) remain unknown. The aim of this study was to characterize gene-derived markers on SSC13q41 for fine mapping of the F4acR locus, and construct a high-resolution pig–human comparative map to select positional candidate genes for F4acR. Pig-specific sequence-tagged site markers were developed for 20 genes that are located in a 6.8-Mb region on HSA3q21 and q29, and a total of 34 single-nucleotide polymorphisms (SNPs) were identified in 14 of 20 markers developed. Eighteen markers were mapped to SSC13q41, while the other two markers ( PLXNA1 and KLF15 ) were assigned to SSC13q32 and SSC7q13, respectively, by radiation hybrid mapping. This result showed that there was a small conserved segment on SSC7 corresponding to HSA3q21. A framework map comprising 18 markers on SSC13q41 was established, refining the synteny breakpoint on SSC13q41 to a region of 12.3 centiRay. The comparative radiation hybrid (RH) map revealed three interesting candidate genes for F4acR from the human genome, viz. MUC4 , MUC13 and MUC20 . Linkage analysis with six marker polymorphisms revealed that MUC4 had the most significant linkage with the F4acR locus.     

金霉素和代谢物差向金霉素在肉鸡肌肉和肝脏中的残留分布

为了研究金霉素(CTC)、差向金霉素(ECTC)在肉鸡肌肉、肝脏中的残留分布情况,样品经提取液提取后过Oasis HLB SPE小柱净化,采用电喷雾正离子模式(ESI+)和多反应监测模式(MRM),用高效液相色谱-串联质谱仪检测;试验组鸡按体重以50 mg/(kg·d)剂量内服CTC,连续5 d,每天给药1次。结果表明:该方法测定肉鸡肌肉和肝脏中CTC检测限和定量限分别为5.0、10.0μg/kg。ECTC检测限和定量限分别为20.0、30.0μg/kg。CTC和ECTC在肉鸡肌肉中平均回收率分别在73.0%~95.20%和81.80%~101.60%,相对标准偏差(RSD)分别在8.85%~12.18%和2.05%~9.29%;在肉鸡肝脏中平均回收率分别在67.06%~90.80%和78.64%~106.98%,RSD分别在2.90%~8.88%和4.72%~9.48%;肉鸡肌肉和肝脏中CTC、代谢物ECTC的残留量分别在给药后第1、5 d时达到最高。肉鸡肝脏中ECTC的残留量约占总残留量的1/2。停药后CTC及其代谢物ECTC代谢缓慢,在停药第6 d时,仍能检测到CTC及其代谢物ECTC,但其残留量均低于最高残留限量(MRLs)。从保障食品安全方面考虑,建议我国将CTC和ECTC均作为金霉素的残留标示物进行监测。     

猪圆环病毒Ⅱ型体外诱导3D4/2细胞炎症模型的建立

【目的】通过对猪圆环病毒Ⅱ型（PCV2）体外感染3D4/2细胞浓度、时间与细胞炎症水平进行探讨,建立PCV2体外感染3D4/2细胞炎症反应模型,以期为后期药物调控PCV2诱发3D4/2细胞炎症反应的研究奠定基础。【方法】将3D4/2细胞分为对照组及10⁰、10^-1、10^-2和10^-3 PCV2感染组,每组3个重复。对照组用DMEM培养,各PCV2感染组用不同稀释倍数PCV2液培养,2 h后均更换为含5%胎牛血清（FBS）的DMEM维持液进行培养,培养4、8、12和24 h后分别收集细胞及细胞上清液。采用Griess法检测一氧化氮（NO）水平,DCFH-DA荧光探针法检测活性氧（ROS）水平,酶标法检测还原型谷胱甘肽（GSH）水平,分光光度法检测黄嘌呤氧化酶（XOD）和髓过氧化物酶（MPO）活性,ELISA法测定白细胞介素-1β（IL-1β）、IL-6、肿瘤坏死因子-α（TNF-α）、IL-10、γ干扰素（IFN-γ）、IL-8、单核细胞趋化蛋白1（MCP-1）以及环氧合酶1（COX-1）和COX-2的分泌水平。【结果】10⁰至10^-3 PCV2作用4、8、12和24 h均能够成功感染3D4/2细胞。与对照组相比,10⁰ PCV2在感染3D4/2细胞4、8、12、24 h后ROS水平均极显著升高（P<0.01）,10^-1至10^-3 PCV2感染3D4/2细胞8、12、24 h后ROS水平显著或极显著升高（P<0.05;P<0.01）;10⁰至10^-3 PCV2感染3D4/2细胞8、12、24 h后,细胞内NO浓度及MPO活性显著提高（P<0.05）,细胞上清液中的IL-1β、IL-6、TNF-α、IL-10、IFN-γ、IL-8和MCP-1水平及COX-1活性均显著或极显著升高（P<0.05;P<0.01）,其中10⁰ PCV2感染3D4/2细胞后,各炎症因子水平上升最显著,且随着时间的延长,NO浓度逐渐升高,XOD活性逐渐降低。【结论】PCV2可诱导3D4/2细胞炎症反应,且10⁰ PCV2体外感染3D4/2细胞4~12 h是建立炎症模型的最佳条件。     

LPS-TLR4/MD-2–TNF-α signaling mediates alcohol-induced liver fibrosis in rats

Liver fibrosis results from liver inflammation and progresses to liver cirrhosis or liver cancer. It is known that nonalcoholic liver disease is mediated by the Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2)–tumor necrosis factor-alpha (TNF-α) signaling pathway. This study aimed to investigate whether alcoholic liver disease is also mediated by this pathway. To this end, we first established rat models of liver fibrosis by administering alcohol. Next, the rats were injected with anti-TLR4 and anti-MD-2 antibodies. Real Time Quantitative PCR (RT-qPCR) and Western blotting were used to detect the activation of the TLR4/MD-2–TNF-α signaling pathway and hepatic stellate cells (HSCs). Moreover, the expression of molecules related to liver fibrosis was estimated. The morphology of rat liver tissue was observed through hematoxylin–eosin staining and Masson staining. For in vitro studies, Kupffer cells (KCs) isolated from the liver were transfected with si-TLR4 and si-MD-2 and co-cultured with HSCs to determine the activity of HSCs. It was found that alcohol treatment activated the TLR4/MD-2–TNF-α signaling pathway and upregulated the molecules associated with liver fibrosis. However, inhibition of TLR4 and MD-2 partially reversed this trend. Notably, in vitro studies indicated that knockdown of TLR4 and MD-2 in KCs partially inhibited LPS-induced activation of KCs and HSCs. Overall, this study showed that alcohol induces liver fibrosis via the LPS-TLR4/MD-2–TNF-α signaling pathway.     

7种长蠹科昆虫的线粒体DNA ND4基因序列比较分析

长蠹科昆虫严重危害林木和仓贮物品。该文应用非损伤性DNA测序技术测定了来自不同国家的长蠹科害虫的线粒体DNA ND4基因的部分序列。在获得的204bp的序列中，7种昆虫的序列变异丰富，多数变异发生在密码子的第3位点上。将实验结果与形态学特征比较分析，探讨7个种的形态差异与基因序列的差异；结果表明，7种昆虫不仅在外形特征上存在差异，而且在ND4基因序列上的差异程度也很显著，平均为21．94％。这为分类鉴定提供了充分的证据。