牛FABP3转基因小鼠的遗传及表达特性研究

旨在研究和分析牛FABP3基因在小鼠体内遗传和表达特性,对培育优质高产转基因肉牛新品种具有重要意义。本研究利用前期获得的4只FABP3转基因小鼠,通过制定的4组交配方案获得子代小鼠,并利用PCR、实时荧光定量PCR和组织原位表达技术对转基因牛FABP3基因小鼠的子代遗传特征,以及FABP3基因在G0代和G1代小鼠心、肝、肾、骨骼肌组织的表达情况进行了检测。RT-PCR检测结果表明,在G0代转基因小鼠中,牛FABP3基因能够表达;牛FABP3基因在转基因小鼠中可以遗传,G1代转基因小鼠平均阳性率达68.4%;相对定量PCR结果表明,转基因小鼠总FABP3基因在G1代小鼠心肌和骨骼肌中表达量相对于阴性个体有不同程度的增加;组织原位检测表明,在G0代个体中表达量较高,G1代个体中虽能看到绿色荧光,但略有降低。本研究表明,牛FABP3基因存在于小鼠基因组中,并能够被表达,且可以遗传给后代,这为后期进一步研究牛FABP3基因对小鼠骨骼肌脂肪含量的影响奠定坚实基础。     

FABP5和CRABP2基因在绵羊不同组织中的mRNA表达研究

FABP5和CRABP2同属于脂肪酸结合蛋白,在动物体内均受视黄酸调节,调控脂质氧化和能量利用。本研究利用实时定量荧光PCR技术,对山西肉用绵羊母本品系10月龄去势公羊的皮下脂肪、心脏、肾、肝、肺、肌肉6种组织中FABP5和CRABP2基因的mRNA表达进行检测,同时利用GEO DataSets数据比较2个基因在人和小鼠各组织的mRNA表达,以探讨FABP5和CRABP2的组织表达规律。结果表明:FABP5和CRABP2在人、小鼠和绵羊的脂肪、心脏、肾、肝、肺和肌肉组织中均有表达,且有组织表达差异和种属表达差异;在绵羊中,FABP5和CRABP2均在脂肪组织中的mRNA表达量最高,且与其他组织差异显著(P<0.05);FABP5和CRABP2mRNA均在肝脏中表达最低。本实验结果为进一步研究FABP5和CRABP2基因在绵羊中的功能奠定基础。     

FABPs基因表达量与高黎贡山猪脂肪酸含量的相关分析

为探究FABP1、FABP3基因在高黎贡山猪肝脏、背脂和背最长肌3个组织中的相对表达量和脂肪酸含量的关系,本研究以高黎贡山猪为研究对象,采用气相色谱法测定其背最长肌脂肪酸含量,利用RT-qPCR技术检测FABP1、FABP3基因在高黎贡山猪背脂、背最长肌和肝脏中的相对表达量,并与脂肪酸含量进行相关分析。高黎贡山猪FABP1基因的表达趋势为：肝脏>背脂>背最长肌,FABP3基因的表达趋势为：背最长肌>肝脏>背脂;经与脂肪酸含量进行相关性分析,发现在肝脏组织中FABP1基因与十九酸的含量呈显著负相关;在背最长肌组织中FABP3基因与二十二碳一烯酸含量呈显著正相关,与二十碳五烯酸呈极显著正相关。结果表明,FABP3基因与高黎贡山猪多不饱和脂肪酸中的二十二碳一烯酸和二十碳五烯酸含量有相关性,但FABP1、FABP3能否作为影响高黎贡山猪脂肪酸含量的关键基因还需进一步探究。     

北川白山羊FABP3基因cDNA的克隆及表达研究

脂肪酸结合蛋白(Fatty aicd Binding Protein 3,FABP3)是细胞内脂类伴侣。本研究采用RT-PCR和RACE技术,分离并克隆山羊FABP3基因的cDNA序列,结合实时定量分析山羊FABP3在10个不同组织和5个不同泌乳时期的相对表达。结果表明:FABP3基因的m RNA序列全长为714 bp,包括5'非翻译区64 bp,CDS区402 bp和3'非翻译区248 bp,共计翻译为133个氨基酸,山羊FABP3与Gen Bank中牛(Bos taurus)、小鼠(Mus musculus)和人(Homo sapiens)的核苷酸同源性较高,分别为96%、89%、87%;蛋白质结构分析显示,FABP3蛋白分子量为14.76 ku,等电点为6.11,不具有信号肽序列,没有跨膜结构;组织表达分析表明,FABP3基因在山羊的心脏组织中表达量最高,小肠次之,表达量最少的为肌肉组织;山羊5个不同泌乳时期乳腺组织FABP3的表达量分析表明,泌乳盛期的表达量最高,约为干奶期的130倍。     

脂肪酸结合蛋白7的研究进展

脂肪酸结合蛋白7(FABP7)是脂肪酸结合蛋白(FABPs)家族成员之一,FABP7基因对大脑发育及中枢神经系统的调节至关重要。同时,FABP7位于脂质代谢的经典通路PPAR通路中,在脂质代谢、基因调控、细胞生长和分化过程中发挥重要的作用。目前,关于FABP7基因的研究主要集中于大脑发育及中枢神经系统的调节,对于家畜脂质代谢分子机制方面的研究相对较少。本文对FABP7的结构、分布及其在脂质代谢、神经系统和癌细胞增殖相关研究进展进行阐述,为今后利用FABP7基因相关育种研究提供参考。     

猪心脏和脂肪型脂肪酸结合蛋白基因表达与肌内脂肪含量的关系

肌内脂肪含量是猪肉食用品质的 1个主要决定因素 ,是高度遗传的 ( Hovenier等 ,1 993)。在纯种的杜洛克猪群中已经证实 ,A- FABP( FABP4)和 H- FABP( FABP3)基因位点的遗传变异与肌内脂肪含量显著相关 ( Gerbens等 ,1 998,1 999)。对梅山×荷兰大白猪杂交群的分析进一步支持H- FABP(而非 A- FABP)参与肌内脂肪沉积的观点 ( Gerbens等 ,2 0 0 0 )。然而 ,尽管有这样一些的联系 ,在对 2个猪群的分析中都不能排除与 A-FABP、H- FABP基因密切相连的基因的影响。为了调控肌内脂肪含量 ,A- FABP、H- FABP基因的遗传学变异最…     

转基因小鼠多重PCR快速检测方法的建立

以转赖氨酸基因小鼠为研究对象,根据转入基因的载体结构分别设计赖氨酸基因、新霉素抗性基因、转基因启动子及小鼠基因组内参基因特异性引物,优化反应条件,建立转基因小鼠多重PCR检测方法。结果表明,建立的方法可以用于转赖氨酸基因小鼠的检测,同时为转基因动物检测标准的建立提供试验依据。     

延边黄牛FABP4基因组织表达及基因多态性分析

为了解FABP4基因在延边黄牛各组织中的表达特征,深入探讨FABP4基因在肌内脂肪沉积中的作用以及该基因是否可以作为延边黄牛肉质性状的候选基因。本研究运用实时荧光定量PCR方法检测延边黄牛不同组织FABP4基因的表达水平及其与肌内脂肪沉积的相关性;同时运用直接测序和PCR-RFLP技术对FABP4基因的SNPs位点进行检测,并与延边黄牛肉质性状进行相关性分析。结果显示FABP4基因在延边黄牛肾脏、肺脏、心脏、脾脏、肝脏、后腿肌及背最长肌中均有表达,其中心脏中的表达量最高,肾脏中的表达量最低。在心脏中的表达量极显著高于脾脏、后腿肌、肺脏、肝脏、肾脏组织(P0.01)。背最长肌中FABP4基因表达量与肌内脂肪(IMF)含量呈极显著正相关(P0.01),相关系数为0.994。后腿肌组织中FABP4基因表达量与IMF含量呈显著正相关(P0.05),相关系数为0.952。在FABP4基因中存在呈现中度多态的g.3691 GA位点,该位点GG基因型的水分含量显著高于GA和AA基因型,GA基因型的脂肪含量显著高于GG和AA基因型,GG和AA基因型的蛋白含量显著高于GA基因型。研究结果提示,FABP4基因参与延边黄牛肌内脂肪沉积调控,可以作为延边黄牛肉质性状的候选基因。     

S100a10基因对小鼠前体脂肪细胞白色和棕色成脂分化的影响

旨在用小鼠模型探究全基因组关联分析(GWAS)筛选到的西门塔尔牛大理石花纹评分性状正相关基因S100钙结合蛋白A10( S100A10 )对小鼠前体脂肪细胞成脂分化的影响。本研究以来源于C57BL/6品系小鼠腹股沟两侧白色脂肪组织的前体脂肪细胞为材料,体外进行白色/棕色成脂分化。通过siRNA干扰 S100a10 基因表达,通过油红O染色检测前体脂肪细胞分化效果,通过荧光定量PCR(qRT-PCR)、蛋白免疫印迹和细胞免疫荧光检测基因和蛋白表达变化。结果显示,敲低 S100a10 后,通过油红O染色发现脂滴明显变少;通过qRT-PCR检测白色成脂分化标志基因 Pparg 、 C/ebpa 、 Fabp4 、 Glut4 、 Adiponectin 、 Leptin 表达量显著下降( P <0.01);棕色成脂分化基因 Ucp1、Pgc1a、Fabp4、Elovl3、Cox7a 表达量显著下降( P <0.01)。敲低 S100a10 后,通过蛋白免疫印迹检测发现,FABP4和PPARG在白色成脂分化中表达量显著下降( P <0.01),通过细胞免疫荧光检测发现FABP4在白色成脂分化中表达量下降;敲低 S100a10 后通过蛋白免疫印迹和细胞免疫荧光检测发现,UCP1和FABP4在棕色成脂分化中表达量显著下降( P <0.01)。综上表明,敲低 S100a10 基因后抑制小鼠前体脂肪细胞的白色/棕色成脂分化,本研究将为探究 S100A10 基因对于肉牛肌内脂肪沉积的影响提供科学依据。     

MyoG转基因小鼠模型制作、鉴定及组织学分析

为了研究MyoG基因对小鼠肌肉形成的影响,研究构建了MogG转基因小鼠并采用荧光定量RT-PCR方法检测转基因小鼠中外源基因拷贝数;H.E.染色后统计了转MyoG基因小鼠肌纤维数目以及直径。结果表明:转MyoG基因小鼠比目鱼肌的肌纤维直径相对于对照组明显增大(P0.05)。说明MyoG基因对慢肌纤维数目无影响而对直径有影响。     

IL-4对肝片吸虫感染诱导的巨噬细胞中脂肪酸结合蛋白2分布的影响

为阐明小鼠感染肝片吸虫后巨噬细胞中脂肪酸结合蛋白2(FABP2)的分布,采用肝片吸虫囊蚴为感染源,经口分别感染雌性BALB/c野生型(WT)及其IL-4单抗处置小鼠,在运用特异性PCR鉴定成功感染小鼠后获取巨噬细胞,并设立人工巯基醋酸盐诱导巨噬细胞对照组进行体外培养。用荧光定量PCR检测选择性激活巨噬细胞的标记蛋白Relmα、Ym1和 Arginase1以确定其激活状态。采用特异性抗体对所获取的巨噬细胞细胞质染色后用共聚焦显微镜摄取不同时间点从不同小鼠体内获取的巨噬细胞染色后的图片。野生型BALB/c巨噬细胞中Relmα、Ym1和Arginase1均大量表达;IL-4单抗处置BALB/c小鼠和巯基醋酸盐诱导小鼠中巨噬细胞中的Relmα、Ym1和Arginase1的表达量较野生型小鼠中的极显著或显著下降。FABP2在FeMΦ中并不分布于细胞质膜下,而是呈点状广泛分布于细胞质中,其荧光强度在12~24 h间呈上升趋势,在24~48 h间却呈下降趋势。     

Progress in the development of Fasciola hepatica vaccine using recombinant fatty acid binding protein with the adjuvant adaptation system ADAD

Fatty acid binding proteins (FABP) have been designed as a potential vaccine against fasciolosis. In this work, the immunoprophylaxis of the recombinant Fh15 FABP from F. hepatica (Fh15) in adjuvant/immunomodulator ADAD system was evaluated using mice and sheep challenged with F. hepatica. The ADAD system combines the Fh15 antigen with an immunomodulator (hydroalcoholic extract of Polypodium leucotomos; PAL) and/or an adjuvant (saponins of Quillaja saponaria; Qs) in a water/oil emulsion (30/70) with a non-mineral oil (Montanide). All the infected control mice died by 41-48 days post-infection. The mice vaccinated with ADAD only with PAL+Fh15 present a survival rate of 40-50% and those vaccinated with ADAD containing PAL+Qs+Fh15 had a survival rate of 50-62.5%. IgG1 antibodies were lower in surviving mice in comparison with non-surviving mice. The sheep vaccinated with ADAD PAL+Qs+Fh15 showed lower fluke recovery (43%), less hepatic lesions and higher post-infection daily weight gain than F. hepatica infected control animals. Thus, the ADAD system using recombinant fatty acid binding proteins from F. hepatica could be a good option to develop vaccines against F. hepatica.     

Fatty acid‐binding protein expression in the gastrointestinal tract of calves and cows

Fatty acid‐binding protein (FABP) has high affinity for long‐chain fatty acids and appears to participate in the metabolism and intracellular transport of lipids. Liver‐ and intestinal‐type FABP (L‐FABP and I‐FABP, respectively) are expressed in the small intestine. However, in the gastrointestinal tract of ruminants, expression and localization of FABPs are unknown. In this study, we investigated the expression of I‐FABP and L‐FABP in the gastrointestinal tract of cattle. I‐ and L‐FABP had higher messenger RNA (mRNA) and protein expression levels in the duodenum and jejunum relatively to other gastrointestinal regions in both calves and cows. Furthermore, L‐FABP mRNA and protein expression were high in the colon. Both these protein types were confirmed to be in the cytosol of jejunal epithelial cells, where they were found in the villi rather than in the crypts. We concluded that duodenal and jejunal FABPs might be involved in the metabolism of fatty acids mainly in epithelial cells in cattle.     

脂肪酸结合蛋白4对BCG诱导巨噬细胞自噬的调控作用

旨在探讨脂肪酸结合蛋白4（fatty acid binding protein,FABP4）在牛分枝杆菌卡介苗（BCG）感染的巨噬细胞中对脂肪酸代谢与自噬的调控作用。本研究利用小干扰RNA技术敲减小鼠巨噬细胞系RAW264.7中FABP4的表达,并结合BCG感染,采用免疫印记和免疫荧光等技术,检测了FABP4蛋白表达、脂肪酸累积、脂肪酸β氧化和自噬相关蛋白表达等指标。结果表明,BCG感染极显著上调了小鼠巨噬细胞RAW264.7中FABP4的表达（P<0.001）并促进了脂肪酸的累积。FABP4小干扰RNA（siRNA-FABP4）能显著降低BCG感染后巨噬细胞中脂肪酸含量,伴随着肉毒碱棕榈酰移位酶1A（CPT1A）的表达上调（P<0.05）,与ATP产量的提升（P<0.05）。同时,siRNA-FABP4极显著下调了自噬调控关键因子AMPK及自噬相关蛋白p-ULK1、ATG5、ATG7、ATG12和LC3B的表达（P<0.01）。以上研究结果表明,在BCG感染RAW264.7细胞过程中,下调了FABP4的表达,促进了脂肪酸的氧化,减少了巨噬细胞内脂肪酸的含量,并通过AMPK信号通路抑制了细胞自噬的发生。     

Role of Fatty Acid Binding Protein 4 in Regulating Macrophage Autophagy Induced by BCG Infection

To investigate the role of FABP4 on regulating fatty acid metabolism and autophagy in macrophage infected with BCG, small interfering RNA for FABP4 were transfected into RAW264.7 cells alone or combined with BCG infection. The immunoblot and immunofluorescence were used to detect the expression of FABP4, the accumulation of lipid droplets, the expression of fatty acid β-oxidation and autophagy related factors. The results showed that the infection of BCG increased the expression of FABP4 and accompanied with the accumulation of lipid droplets in RAW264.7. Moreover, the knockdown of FABP4 decreased lipid droplets but promoted the expression of carnitine palmitoyltransferase 1A (CPT1A) (P <0.05) and the production of ATP (P <0.05). Meanwhile, the knockdown of FABP4 suppressed the expression of AMPK, p-ULK1, ATG5, ATG7, ATG12 and LC3B which are autophagy related factors (P<0.001). Our results indicated that the knockdown of FABP4 promoted fatty acid oxidation, decreased fatty acid level and suppressed autophagy via AMPK signal pathway in BCG infected RAW264.7 cells.     

Vaccination of mice against schistosoma bovis with a recombinant fatty acid binding protein from Fasciola hepatica

Two strains of mice (NMRI and C57/BL) were each immunized with a 15kDa recombinant Fasciola hepatica fatty acid binding protein (FABP) (Fh15) and challenged percutaneously with Schistosoma bovis cercariae. C57/BL mice immunized with Fh15 had significant reductions in S. bovis worm burden recoveries (72% reductions over controls). When using NMRI mice, Fh15 in Freund's adjuvant failed to induce significant protection against S. bovis. In C57/BL mice, only antibodies to the IgG2a isotype increased after the second immunization and remained high through 8 weeks of S. bovis infection. This is the first time that a heterologous recombinant molecule from F. hepatica has been used in vaccination against S. bovis, obtaining a significant reduction in the number of worms in C57/BL mice.     

草原红牛FABP7基因克隆、生物信息学及组织表达差异分析

本研究旨在克隆草原红牛脂肪酸结合蛋白7基因(FABP7)的CDS区序列并对其进行生物信息学分析,同时检测其在牛各个组织中的mRNA表达水平。利用RT-PCR技术克隆草原红牛FABP7基因CDS区序列,并使用多种生物软件和在线工具进行生物信息学分析,通过qPCR技术检测FABP7基因在各组织间mRNA的表达水平。结果表明:草原红牛FABP7基因CDS区全长399 bp,编码132个氨基酸,蛋白分子量为14.96 ku,理论等电点为5.38,属于亲水性蛋白;通过NCBI-BLAST对比发现,草原红牛与普通牛、羊、猪、人、鼠、鸡的核苷酸序列同源性分别为99%、98%、94%、92%、85%、85%,系统进化树结果发现,草原红牛与普通牛亲缘关系最近,与鸡的亲缘关系最远;FABP7蛋白序列有1个二硫键,14个磷酸化位点,1个N-糖基化位点,不存在信号肽和跨膜区;在蛋白的二级结构和三级结构中发现,FABP7蛋白主要存在2个α-螺旋结构和10条β-折叠,为混合型蛋白。FABP7基因在小肠组织表达量最高,在心脏、脂肪和胃中中度表达。     

Allotransplantation of Transgenic Mouse Ovaries Expressing Enhanced Green Fluorescent Protein under the Control of the Murine Phosphoglycerate Kinase 1 Promoter

The aim of this study was to generate a transgenic mouse that ubiquitously expressed enhanced green fluorescent protein (EGFP) under the control of the murine phosphoglycerate kinase 1 promoter by allotransplantation of transgenic mouse ovaries. The EGFP transgenic mice expressed green fluorescence in many organs, and the fluorescence was detected as early as the embryonic stage. Ovaries from the EGFP transgenic mice were allotransplanted into recipients and these mice were mated with normal male mice. Histological sections of EGFP‐allotransplanted ovaries from the recipient mice showed the well development and formation at follicles and corpora lutea. The green fluorescence was clearly detectable at the allotransplanted section of the ovaries, which had fused with the normal ovary. The average size of the first litter from these mice was 6.8 ± 1.2 pups per recipient, and 17.8% of the pups expressed EGFP. These results demonstrated that allotransplantation of transgenic ovaries can restore a normal reproductive lifespan and can be used to generate a ubiquitously expressing EGFP animal model.     

Effect of Fatty Acid Binding Protein 5 on Milk Fat Synthesis of Bovine Mammary Epithelial Cells

The fatty acid binding protein 5 (FABP5) is an intracellular lipid carrier. The TG GPO-POD assay kit and BODIPY staining methods were used to detect lipid secretion and triglyceride content,and the effect of FABP5 on SREBP-1c expression were detected by Real-time PCR and Western blotting methods. The results showed that high purity bovine mammary epithelial cells (BMECs) were successfully isolated and purified,and the eukaryotic expression vector pGCMV-IRES-EGFP-FABP5 was constructed in this experiment. Compared with the blank control group and empty vector group,the lipid secretion and triglyceride content, and the expression of SREBP-1c and FAS,ACC were extremely significantly increased when the FABP5 was overexpressed (P<0.01). FABP5 siRNA1 was selected as the optimal interference fragment, and when FABP5 was inhibited,the lipid secretion and triglyceride content,the expression of SREBP-1c,FAS and ACC were extremely significantly decreased (P<0.01).The results indicated that FABP5 could promote the synthesis of milk fat in BMEC by up-regulating the expression of SREBP-1c.