壳聚糖对肉仔鸡血清花生四烯酸含量、磷脂酶A2活性及小肠胞内磷脂酶A2 mRNA表达的影响

本试验旨在研究日粮中添加不同水平壳聚糖对肉仔鸡血清花生四烯酸含量、磷脂酶A2活性及小肠胞内磷脂酶A2mRNA表达的影响.试验选择1日龄健康爱拔益加肉仔鸡240只(公母各占1/2),随机分为6个处理,每个处理5个重复,每个重复8只鸡.对照组饲喂基础日粮,其他5种试验日粮分别在基础日粮中添加50、200、500、1000和2 000 mg/kg的壳聚糖配制而成.试验期42 d.结果表明,肉仔鸡血清花生四烯酸含量、磷脂酶A2活性及十二指肠、空肠、回肠的胞内磷脂酶A2mRNA表达均随日粮壳聚糖添加量的增加呈显著二次曲线增加(P<0.05);其中以500和1 000 mg/kg壳聚糖组较高,但当日粮壳聚糖添加量为2 000 mg/kg时则有不同程度的降低.由此可知,壳聚糖对肉仔鸡免疫功能的影响可能与磷脂酶A2的活性及小肠胞内磷脂酶A2mRNA的表达发生改变有关.     

三穗鸭肌细胞增强因子基因多态性与屠宰性状的相关性分析

为了解三穗鸭肌细胞增强因子(myocyte enhancer factor 2A,MEF2A)SNPs与屠宰性状的相关性,本研究以60只三穗鸭为研究对象,采用PCR-SSCP方法结合PCR产物直接测序技术对三穗鸭MEF2A基因多态性进行检测,并进行SNPs与屠宰性状各指标的相关分析。结果表明,在MEF2A基因中共发现了2个SNPs:第11外显子的g.47915G>A位点和g.47918G>A位点,g.47915G>A位点发生的G/A突变使密码子由GAA变为AAA,翻译出的氨基酸由谷氨酸变成赖氨酸,g.47918G>A位点的G/A突变引起的密码子由GAT变成AAT,翻译出的氨基酸由天冬氨酸变成天冬酰胺。SNPs与屠宰性状的关联性分析表明,g.47915G>A和g.47918G>A位点影响全净膛率。这一结果揭示了MEF2A基因的多态性对三穗鸭屠体性状具有重要的影响。     

Interaction of bovine immunoglobulin gamma 2 (IgG2) with staphylococcal protein A: evidence for IgG2a and IgG2b sub-subclasses in the bovine

The reactivity of bovine IgG with protein A is confusing with respect to which of the bovine IgG class and subclasses are reactive. We have, therefore, re-examined the interaction of bovine immunoglobulins with protein A. The results presented in this paper indicated that at pH 8.0 protein A binds only immunoglobulin of the IgG2 subclass. The bound IgG2 can be readily recovered from an immobilized protein A column at pH 5.0. Furthermore, the antigenic IgG2 eluted demonstrated two charged species which could readily be separated by ion-exchange chromatography. These results indicate that IgG2 in the bovine exists in two sub-subclasses, IgG2a and IgG2b. The two sub-subclasses of IgG2 could be rapidly isolated with a good yield in two-steps namely protein A affinity chromatography followed by ion exchange chromatography.     

Polymorphisms of MEF2A Gene and its Relationship with Slaughter Traits in Sansui Duck

To understand the relationship between single nucleotide polymorphism sites (SNPs) of myocyte enhancer factor 2A (MEF2A) gene and slaughter traits in Sansui duck, a total of 60 individuals of Sansui ducks were selected to investigate in this study, direct sequencing of PCR and PCR-SSCP methods were used on single nucleotide polymorphisms of MEF2A gene, and genetic effects of its on slaughter traits were analyzed.The results showed that two SNPs which include g.47915G>A and g.47918G>A of exon 11 were found in MEF2A gene, and the G/A mutation in the g.47915G>A SNP resulted in the change of codon from GAA to AAA, and the coding amino acid from Glu to Lys, and the G/A mutation in the g.47918G>A SNP resulted in the change of codon from GAT to AAT, and the coding amino acid from Asp to Asn.The result of association of SNPs with slaughter traits showed various results were as follows:g.47915G>A and g.47918G>A had affected the eviscerated percentage.This result revealed that the polymorphism of MEF2A gene had basilic influence on slaughter traits.     

MAT2A/2B promote porcine intramuscular preadipocyte proliferation through ERK signaling pathway

Intramuscular fat (IMF) content has been identified as a crucial factor of porcine meat quality. MAT2A and MAT2B coordinately catalyzes the synthesis of the major biological methyl donor S‐adenosylmethionine (SAMe). However, the regulatory effect of MAT2A and MAT2B on porcine intramuscular preadipocyte proliferation has not been clarified. In this study, we investigated the effect of MAT2A and MAT2B and its potential mechanism during porcine intramuscular proliferation. We demonstrated that overexpression of MAT2A and MAT2B promoted the cell cycle progression of porcine preadipocyte by flow cytometry and EdU‐labeling assay, as well as promoted the expression of cell cycle marker genes including Cyclin B, Cyclin D, and Cyclin‐dependent kinase 4, but reduced the expression of cell cycle inhibitor P27. Consistently, knockdown of MAT2A and MAT2B inhibited cell cycle progression and downregulated the mRNA and protein levels of the above genes. Furthermore, overexpression of MAT2A and MAT2B activated the phosphorylation of ERK1/2. Moreover, the inhibitory effect of U0126 (a specific ERK1/2 inhibitor) on the ERK1/2 activities was partially recovered by overexpression of MAT2A and MAT2B in porcine intramuscular preadipocytes. Taken together, our findings suggested that MAT2A and MAT2B promote porcine preadipocyte proliferation by ERK1/2 signaling pathway.     

The therapeutic effects of SET/I2PP2A inhibitors on canine melanoma

Canine melanoma is one of the most important diseases in small animal medicine. Protein phosphatase 2A (PP2A), a well conserved serine/threonine phosphatase, plays a critical role as a tumor suppressor. SET/I2PP2A is an endogenous inhibitor for PP2A, which directly binds to PP2A and suppresses its phosphatase activity. Elevated SET protein levels have been reported to exacerbate human tumor progression. The role of SET in canine melanoma, however, has not been understood. Here, we investigated the potential therapeutic role for SET inhibitors in canine melanoma. The expression of SET protein was observed in 6 canine melanoma cell lines. We used CMeC-1 cells (primary origin) and CMeC-2 cells (metastatic origin) to generate cell lines stably expressing SET-targeting shRNAs. Knockdown of SET expression in CMeC-2, but not in CMeC-1, leads to decreased cell proliferation, invasion and colony formation. Phosphorylation level of p70 S6 kinase was decreased by SET knockdown in CMeC-2, suggesting the involvement of mTOR (mammalian target of rapamycin)/p70 S6 kinase signaling. The SET inhibitors, OP449 and FTY720, more effectively killed CMeC-2 than CMeC-1. We observed PP2A activation in CMeC-2 treated with OP449 and FTY720. These results demonstrated the potential therapeutic application of SET inhibitors for canine melanoma.     

泰妙菌素对人CYP3A4、CYP1A2、CYP2E1、CYP2D6酶活性的影响

本研究通过建立人肝微粒体体外孵育试验，考察泰妙菌素对4种CYP450亚酶的探针底物睾酮、非那西丁、氯唑沙宗、氢溴酸右美沙芬代谢的影响，以反映泰妙菌素对人CYP3A4、CYP1A2、CYP2E1、CYP2D6酶活性的作用。试验分为3组：药物试验组、阳性对照组（不含NADPH）、阴性对照组（不含CYP450抑制剂），孵育体系为100 μL，孵育试验在96孔板中进行。终止反应后使用高效液相色谱串联质谱仪（LC-MS/MS），以内标法检测96孔板中孵育液的剩余探针底物浓度。根据药物试验组与阴性对照组的探针药物代谢浓度之比，计算药物试验组的探针药物代谢率。使用Graphpad Prism 6.0软件，以药物试验组相对代谢率为纵坐标，药物浓度对数值为横坐标作图，计算试验组药物IC₅₀值。针对泰妙菌素与CYP3A4的孵育试验设置多个孵育时间点观察孵育时间对IC₅₀值的影响。试验结果显示，酮康唑对CYP3A4的IC₅₀值为0.044 μmol/L，α-萘黄酮对CYP1A2的IC₅₀值为0.030 μmol/L、4-甲基吡唑对CYP2E1的IC₅₀值为0.022 μmol/L、奎尼丁对CYP2D6的IC₅₀值为0.096 μmol/L。泰妙菌素对CYP1A2及CYP2D6的IC₅₀值均大于50 μmol/L，对CYP2E1的IC₅₀值为0.045 μmol/L，对CYP3A4的IC₅₀值为1.609 μmol/L。延长泰妙菌素与CYP3A4的孵育时间（10~50 min）后，泰妙菌素对CYP3A4的IC₅₀值由1.609 μmol/L增加至 8.657 μmol/L。本研究中4种亚酶常用抑制剂的IC₅₀值与参照值相近，表明所建立人肝微粒体体外孵育试验方法可靠。以IC₅₀值为指标显示泰妙菌素对CYP1A2和CYP2D6无抑制作用，对CYP2E1和CYP3A4存在强抑制作用，泰妙菌素可能是CYP3A4的可逆性抑制剂。     

Functions of histone H2A variants

One way that cells alter their chromatin structure is by incorporating histone variants into their nucleosomes. Unlike canonical histones, which are deposited behind the replication fork during the S‐phase of cell division, the accumulation of histone variants is independent of DNA replication. However, among the histone variants, H2A variants play an important role in DNA replication, damage repair, recombination and gene expression. This paper provides a review of H2A histone variants and their functions, exchange in the chromatin and depositional modes by specific chaperones.     

A型流感病毒M2蛋白研究进展

M2蛋白是A型流感病毒所特有的一种结构保守的非糖基化的跨膜蛋白,主要在病毒脱壳时酸化病毒粒子的内部环境,在表面血凝素糖蛋白合成过程中作为质子通道调节高尔基体跨膜转移通道的pH.其核苷酸序列高度保守,几乎不发生变异.其25位～43位氨基酸多肽是金刚烷胺类抗流感病毒药物的作用靶位,氨基酸极小变异都可能导致流感病毒抗药性的产生.M2蛋白胞外区域的氨基酸残基多肽(M2e)的特异性抗体可以在感染流感病毒的动物血清中检测出来,将M2e与相关佐剂联合使用可极太的提高机体对流感病毒的免疫力,在病毒感染中起重要作用,是一种潜在的交叉保护性抗原,常被用来探索具有广谱性和持久性的流感"通用疫苗".     

NR5A2基因研究进展

NR5A2属于是核受体超家族,是有配体激活的转录因子,它还属于NR5A亚家族,是一孤儿受体,也是肝受体同系物。一方面,在动物的肝脏、胰腺、肠及卵巢等脏器中NR5A2得到表达,尤其是在卵巢中为高表达;另一方面,NR5A2在动物胚胎发育及分化、体细胞编程、胆固醇或胆汁酸代谢、类固醇激素生成和生殖活动等方面发挥重要作用。     

Establishment of a Conditional Transgenic System Using the 2A Peptide in the Female Mouse Germline

Transgenic mice are essential research tools in developmental biology studies. The 2A peptide allows multiple genes to be expressed simultaneously at comparable levels in somatic cells, but there are no reports of it being used successfully in germ cells. We constructed a Cre/loxP-based conditional vector containing the 2A peptide to significantly enhance the expression of a reporter and target gene from a constitutive promoter in oocytes. Mice with a transgene insertion containing the chicken β-actin promoter, floxed EGFP-polyA cassette, mCherry reporter, 2A peptide and target gene DNA methyltransferase 3A2 (Dnmt3a2) were crossed with TNAP- or Vasa-Cre mice to produce offspring, in which mCherry and DNMT3A2 proteins were highly expressed in oocytes upon Cre-mediated removal of EGFP-polyA. This novel transgenic mouse line based on the 2A expression system can serve as a useful tool for examining gene function during oogenesis.     

FMDV2A介导的乳腺上皮细胞双基因表达载体构建及表达

构建FMDV 2A介导的人白细胞介素2基因(hIL-2)和增强型绿色荧光蛋白(EGFP)基因双顺反子乳腺特异性表达载体,并验证其在乳腺上皮细胞中的表达.克隆奶山羊β-酪蛋白启动子序列,将hIL-2基因序列置于启动子之后,然后利用口蹄疫病毒2A(FMDV 2A)自剪切序列连接EGFP基因,构建出乳腺特异性的双顺反子表达载体pFIENβ,并利用脂质体转染山羊乳腺上皮细胞,然后用RT-PCR技术和Western blot检测hIL-2基因与EGFP基因的表达.重组质粒pFIENβ经酶切鉴定后表明构建成功,转染质粒PFIENβ和pEGFP-C1的细胞均观察到绿色荧光;从转染pFIENβ的乳腺上皮细胞中扩增出hIL-2基因和EGFP基因,而转染pEGFP-C1的细胞中只扩增出EGFP基因;经Western blot检测,转染pFIENβ的细胞中均表达了hIL-2和EGFP蛋白.结果表明山羊β-酪蛋白启动子能同时启动hIL-2基因和EGFP基因在山羊乳腺上皮细胞中的表达,并且利用FMDV 2A元件实现了hIL-2基因和EGFP基因的非融合型表达.     

乳制品中A1 β-酪蛋白、A2 β-酪蛋白检测方法研究进展

随着消费者对A2 β-酪蛋白的关注度提升,A2 β-酪蛋白相关产品不断更新换代。A2 β-酪蛋白常用检测方法有反相高效液相色谱法（RP-HPLC）、液相色谱–高分辨串联质谱法、毛细管区带电泳法、试剂盒检测法等。本文对β-酪蛋白遗传变体A1 β-酪蛋白和原始形态A2 β-酪蛋白的功能特点、检测方法、定量分析进行综述,说明检测方法的原理和试剂药品的作用机理,加强对A1 β-酪蛋白、A2 β-酪蛋白检测方法的改进与优化,为A2 β-酪蛋白应用于功能性食品、婴幼儿配方食品提供理论依据。     

Prokaryotic Expression and Antigenic Analysis of H3N2 Swine Influenza Virus HA1 and HA2 Genes

Hemagglutinin protein plays an important role in disease prevention and treatment of the swine influenza virus (SIV).In order to clone and express subtype H3N2 SIV A/swine/Henan/1/2010 (H3N2) HA1 and HA2 genes with chicken embryo allantoic fluid containing the H3N2 SIV after extraction of RNA.The HA1 gene and HA2 gene were amplified from the total RNA using RT-PCR and they were inserted into prokaryotic expression vector pET-28a (+) to construct recombinant expression vector.Then the vector was transformed and expressed in E.coli BL21 (DE3) pLyS.Then the bacteria were induced by IPTG and their lysates were analyzed by SDS-PAGE and Western blotting,respectively.The monoclonal antibody 1C10,which was specific to HA protein,was used as primary antibody in Western blotting analysis.It was found that the expressed recombinant HA1 protein was 34.8 ku and recombinant HA2 protein was 23.2 ku analyzed by SDS-PAGE.As demonstrated by Western blotting,this HA1 expressed products showed the capacity of reacting with monoclonal antibody 1C10.All the results suggested that the expressed HA1 and HA2 proteins of H3N2 influenza virus would be very helpful in the development of rapid diagnosis method and vaccine development,which would facilitate further study on the function of HA at the same time.     

过表达JHDM2A对猪iPSCs诱导效率的影响

研究旨在探讨组蛋白赖氨酸去甲基化酶(JHDM2A)对猪成纤维细胞诱导为多能干细胞效率的影响,并对其内在的分子机制进行探究。以猪成纤维细胞为材料,采用多西环素(DOX)诱导的慢病毒生产诱导多能干细胞(iPSCs)体系,在此基础上过表达JHDM2A,通过碱性磷酸酶染色和绘制诱导时间轴检测其对诱导效率的影响;普通PCR技术检测克隆的多能性;免疫荧光检测多能因子的蛋白表达;实时荧光定量PCR检测JHDM2A在形成克隆后以及克隆分化过程中对多能因子、组蛋白甲基化相关基因表达的影响。结果表明,JHDM2A过表达组细胞在第3天发生形态变化,第8天形成iPSCs克隆,相比于对照组分别提前了1和2 d。碱性磷酸酶染色结果显示,与对照组相比,JHDM2A过表达组克隆形态明显改善,染色着色更深,诱导效率提高8倍。普通PCR结果显示,JHDM2A过表达组iPSCs、对照组iPSCs以及猪成纤维细胞(PFF)均表达内源性Oct4、Sox2、Klf4、c-Myc、Nanog,且iPSCs的Oct4表达量高于PFF。免疫荧光结果显示,JHDM2A过表达组iPSCs表达Oct4、Sox2、Stat3、JHDM2A,弱表达SSEA1、SSEA4。实时荧光定量PCR结果显示,与对照组和猪成纤维细胞相比,JHDM2A过表达组iPSCs的Sox2、Klf4、c-Myc、Nanog、Oct4、Tcl1的表达显著升高(P<0.05),Tfcp2l1和Zfp57的表达显著降低(P<0.05);JHDM2A过表达组P5代iPSCs培养基去除DOX后,随着代数的增加,Sox2、Nanog的表达逐渐降低,Klf4、c-Myc的表达先升高后降低,Oct4、Tcl1、Tfcp2l1、Zfp57的表达先降低后升高。以上结果表明,过表达JHDM2A通过促进组蛋白去甲基化提高猪成纤维细胞的诱导效率。     

H3N2亚型猪流感病毒HA1、HA2基因的原核表达及抗原性分析

血凝素（hemagglutinin,HA）蛋白是猪流感病毒（swine influenza virus,SIV）的一个重要蛋白,在疾病预防和治疗中具有重要作用。本试验旨在克隆和表达H3N2亚型猪流感病毒A/swine/Henan/1/2010（H3N2）的HA1和HA2基因。用含有H3N2亚型SIV的鸡胚尿囊液提取RNA,RT-PCR扩增后将目的基因定向克隆到pET-28a（+）原核表达载体上,并将其转入宿主菌BL21（DE3）pLyS进行表达,IPTG诱导后经SDS-PAGE检测并用该病毒重组HA蛋白制备的特异性单克隆抗体1C10作为一抗对两种蛋白进行Western blotting分析。SDS-PAGE结果显示,得到HA1和HA2大小分别为34.8、23.2 ku的重组蛋白,Western blotting结果表明,HA1蛋白与1C10单克隆抗体具有良好的反应原性,且1C10单克隆抗体表位在HA1蛋白上。本试验结果为进一步研究血凝素蛋白的结构和功能,以及建立快速诊断方法和基因工程疫苗提供材料。     

HSP27和Annexin A2蛋白对PRRSV感染的影响

猪繁殖与呼吸综合征病毒（PRRSV）侵染Marc145过程中HSP27和Annexin A2蛋白可参与病毒粒子的组装过程,且HSP27可起到抑制侵染早期细胞凋亡,增加病毒复制时间的作用。本研究分别构建HSP27和Annexin A2下调的Marc145细胞系,经PRRSV感染后发现,与空白对照相比,两者子代病毒TCID50值有明显下降。这一结果为研究PRRSV侵染过程及HSP27和Annexin A2调控机制奠定了基础,也为疫苗的研制提供了新的思路。     

猪氨基酸转运载体b0,+AT-2的分子克隆及其序列分析

本研究克隆了b0,＋AT-2的全长cDNA序列。基于NCBI发布的人和鼠b0,＋AT序列,应用生物学软件进行序列比对后在同源区设计引物,获取cDNA片段;再利用RACE技术克隆了其全长cDNA序列。生物信息学分析显示：猪b0,＋AT-2的全长1 488 bp,包括90 bp的3′非翻译区（UTR）和126 bp的5′UTR;编码区（CDS）编码423个氨基酸残基,分别和人、鼠b0,＋AT以及猪b0,＋AT-1有75.1%、71.9%和86.6%的同源性。     

共轭亚油酸和α-亚麻酸对HepG2细胞核转录因子NF-E2相关因子及谷胱甘肽硫转移酶A1表达的影响

本试验旨在研究共轭亚油酸(CLA)和α-亚麻酸(ALA)对于HepG2细胞核转录因子NF-E2相关因子(Nrf2)及其调控的谷胱甘肽硫转移酶A1(GSTA1)mRNA和蛋白质表达的影响。试验组采用浓度为0.2、0.5和1.0mmol/L的CLA和ALA作用HepG2细胞24h,采用荧光定量PCR法测定mRNA的表达量,蛋白质印迹法测定蛋白质的表达量。结果表明,CLA浓度分别为0.2和0.5mmol/L时,Nrf2和GSTA1mRNA和蛋白质表达量与对照组相比极显著增加(P0.01),并随CLA浓度增加呈下降趋势;采用ALA作用的细胞,Nrf2和GSTA1mRNA和蛋白质表达量与对照组相比,随ALA浓度的增加呈现极显著上升(P0.01)。由此可见,CLA在较低浓度时,诱导Nrf2和GSTA1mRNA和蛋白质表达量作用较强,而随ALA浓度增加Nrf2和GSTA1mRNA和蛋白质的表达量逐渐增多。     

赭曲霉毒素A对IPEC-J2细胞生长和氧化还原平衡的影响

实验以IPEC-J2细胞为材料,研究不同浓度和时间处理下赭曲霉毒素A(OTA)对仔猪空肠上皮细胞增殖存活的影响,并进一步考察不同浓度OTA对细胞的结构功能完整性和抗氧化防御系统的影响。结果表明:OTA暴露时间和浓度对细胞存活率均有影响(P<0.01),且二者存在显著交互效应(P<0.01)。在OTA暴露24 h条件下,细胞钠钾ATP酶(Na+,K+-ATPase)、谷胱甘肽过氧化物酶(GPx)和谷胱甘肽S转移酶(GST)活性以及总抗氧化能力(T-AOC)随OTA浓度增加呈线性或者二次曲线降低(P<0.01),过氧化氢酶(CAT)活性有降低趋势,丙二醛(MDA)含量呈线性增加(P<0.05);胞外乳酸脱氢酶(LDH)活性随OTA浓度增加呈线性或者二次曲线增加(P<0.01);OTA处理对超氧化物歧化酶(SOD)活性无明显影响(P>0.05)。结果显示,OTA破坏了IPEC-J2细胞的结构功能完整性,最终导致细胞的增殖或存活率降低,氧化应激可能是毒性效应发生的重要机制。