禽致病性大肠杆菌Hcp2b对雏鸡气管黏膜细胞因子-细胞因子受体相互作用通路的影响

T6SS （type VI secretion system）是革兰阴性菌中常见的一种分泌系统,其效应蛋白Hcp2b作用机制迄今仍未明晰。本研究以禽致病性大肠杆菌（avian pathogenic Escherichia coli,APEC） Hcp2b蛋白为研究主体,旨在探究Hcp2b蛋白在APEC感染鸡气管黏膜过程中发挥的作用及机制。采用Red重组方法以质粒pKD3为模板构建hcp2b缺失株,pSTV-28质粒连接hcp2b目的片段构建重组载体,导入感受态Δhcp2b菌株构建回复株,并对Δhcp2b菌株的生长曲线进行测定。7日龄雏鸡气管感染hcp2b缺失株及野生株,感染后12、24 h收集鸡气管黏膜细胞进行转录组学测序及生物信息学分析。结果表明,hcp2b缺失株及回复株构建成功,hcp2b基因缺失对菌株的生长性能无影响。hcp2b基因缺失株感染气管黏膜后,mRNA的表达谱发生变化,感染后12 h,有144个基因表达量发生变化（上调差异基因87个,下调57个）;感染后24 h,有135个基因表达量发生变化（上调差异基因79个,下调56个）。生物信息学分析发现差异基因富集在Cytokine-cytokine receptor interaction、Protein processing in endoplasmic reticulum等信号通路。hcp2b基因缺失未影响APEC的生长特性,hcp2b基因缺失后影响雏鸡气管黏膜Cytokine-cytokine receptor interaction通路基因mRNA转录水平的变化,IL-1β、IL-12b的表达均上调。该结果为APEC的致病机制研究提供了理论依据。     

5-Aza-dC对牛成肌细胞MyoD1启动子甲基化及mRNA表达的影响

旨在探究5-氮杂-2-脱氧胞苷（5-Aza-2’-deoxycytidine,5-Aza-dC）对牛成肌细胞的增殖和肌分化因子（myogenic differentiation 1,MyoD1）启动子甲基化以及mRNA表达的影响。本研究复苏了前期冻存的关岭牛成肌细胞,并进行培养,待其生长到对数生长期,再通过不同浓度的5-Aza-dC对牛成肌细胞进行处理,利用流式细胞仪检测细胞凋亡和周期;结合高通量检测方法检测MyoD1启动子甲基化水平,并利用qRT-PCR检测MyoD1及相关基因的表达水平。研究发现,0.1 μmol·L^-15-Aza-dC为最适浓度,该浓度下细胞抗凋亡因子Bcl的表达极显著降低（P<0.01）,促凋亡因子Bax的表达极显著提高（P<0.01）,促凋亡因子Caspase-9的表达显著提高（P<0.05）;周期因子Cyclin A2的表达显著提高（P<0.05）,而细胞因子Cyclin B1、Cyclin D的表达无显著变化;检测空白组和试验组MyoD1启动子甲基化水平发现,试验组甲基化水平极显著低于空白组（P<0.01）;而mRNA的表达水平极显著高于空白组（P<0.01）。5-Aza-dC能够通过改变Caspase-9、Bcl、Bax、Cyclin A2等基因的表达来调节关岭牛成肌细胞的增殖和凋亡,并能够有效降低MyoD1基因启动子的甲基化水平（P<0.01）;极显著提高其mRNA的表达量（P<0.01）。低浓度的5-Aza-dC能通过促进细胞凋亡及调控关岭牛MyoD1的甲基化水平来调控MyoD1的表达;同时推测,MyoD1基因启动子的甲基化水平能够影响关岭牛成肌细胞的生长发育,可为遗传标记辅助关岭牛的改良提供理论参考。     

犏牛和牦牛ESCO2基因的序列特征及其在睾丸中的表达对比分析

旨在克隆犏牛和牦牛姐妹染色单体内聚建立蛋白2（establishment of sister chromatid cohesion N-acetyltransferase 2,ESCO2）基因,并分析其在不同发育阶段睾丸中的表达与定位,为进一步解析ESCO2在减数分裂过程中的作用机制提供理论依据。本研究以健康雄性犏牛及牦牛为试验动物,根据年龄分为胎牛组（5~6月龄）、幼年组（1~2岁）和成年组（3~4岁）,每组各3头。通过RT-PCR技术克隆犏牛和牦牛ESCO2基因并进行生物信息学分析,采用实时荧光定量PCR （quantitative real-time PCR,qRT-PCR）检测ESCO2基因在犏牛不同组织中的表达谱,比较分析ESCO2在犏牛和牦牛不同时期睾丸中的表达规律,利用免疫组织化学（immunohistochemistry,IHC）技术检测ESCO2蛋白的细胞定位和表达差异。结果显示,犏牛ESCO2基因（GenBank登录号：MW198470） CDS区为1 833 bp,编码610个氨基酸,与牦牛相比,犏牛ESCO2序列第301~319位多19个氨基酸,另有3个氨基酸突变;犏牛ESCO2蛋白序列与黄牛的同源性高于其他哺乳动物;ESCO2可能与SMC3、SMC1A、PDS5A、PDS5B、STAG2等蛋白相互作用,互作蛋白功能与姐妹染色单体凝聚、减数分裂细胞周期、DNA修复、细胞分裂和染色体重构等生物学过程相关。ESCO2在犏牛各组织中均有表达,但在睾丸中的相对表达水平显著高于其它组织（P<0.05）;在犏牛睾丸中的表达随年龄增长呈上升趋势,幼年和成年时期犏牛睾丸中ESCO2的表达显著低于同时期牦牛（P<0.05）;IHC染色结果发现,雄性犏牛减数分裂阻滞于初级精母细胞,ESCO2蛋白在犏牛初级精母细胞中无表达并与牦牛存在差异。本研究结果表明,犏牛与牦牛的ESCO2基因、蛋白序列差异较大,且在睾丸发育过程中的表达模式差异显著,这可能是引起雄性犏牛减数分裂阻滞及不育的原因之一,其具体作用机制有待进一步研究。     

猪ADAR2基因全长cDNA克隆、序列特征及表达模式分析

旨在克隆猪作用于RNA的腺苷脱氨酶2基因（ADAR2）全长cDNA序列,同时对该基因在猪不同组织中的表达规律进行探索。利用RACE （rapid-amplification of cDNA ends）对大白猪ADAR2基因mRNA全长序列进行克隆,并进行生物信息学分析;用荧光定量PCR方法检测35日龄大白猪心、肝、肺、肾、脾、脑、小肠、背最长肌和背部脂肪9种组织中ADAR2的表达水平。结果表明,猪ADAR2基因cDNA全长6 305 bp,共包含12个外显子,编码704个氨基酸,与人、黑猩猩、猕猴、长臂猿、黄牛、山羊和绵羊的CDS区核酸序列和氨基酸序列的一致性均在84%以上。该基因编码的蛋白含有2个双链RNA结合基序和一个脱氨酶结构域。猪ADAR2在检测的各组织中均表达,其中在肺中的表达量最高。综上所述,本研究成功克隆了猪ADAR2基因全长cDNA序列,并且发现其在猪体内广泛表达,为深入研究ADAR2的功能奠定了良好的基础。     

Identification of the core motif of the BRCA2 C-terminal RAD51-binding domain by comparing canine and human BRCA2

Mammary tumors are the most common tumors in women and non-spayed female dogs. One of the reasons for mammary tumors is mutations of the tumor suppressor gene, BRCA2. BRCA2 participates in homologous recombination repair by interacting with the RAD51 recombinase. BRCA2 has two RAD51-binding domains, consisting of BRC repeats and the C-terminal RAD51-binding domain, respectively. Although several studies have addressed the function of the C-terminal RAD51-binding domain of human BRCA2, the amino acid sequences required for the RAD51-interaction activity remain unclear. In this study, the C-terminal RAD51-binding domains of canine and human BRCA2 were compared; the canine domain displayed a weaker interaction with RAD51. This difference was attributed to the C-terminal portion of the domain via a comparison between canine and human domains. Furthermore, peptides shorter than those previously reported displayed RAD51-interacting activity, and a core motif of this domain consisting of 25 amino acids was identified. Since a mutation (S3323N) was reported in the core motif of this domain, the effect of this mutation was evaluated. The mutant exhibited similar RAD51-binding activity as that of the wild-type protein, suggesting that the mutation was functionally neutral. These data suggested that the C-terminal portion of the BRCA2 C-terminal RAD51-binding domain influenced its RAD51-interaction activity, and a minimum core motif of 25 amino acids was identified in this domain. These data may help clarify BRCA2 function, as well as the tumorigenic effects of BRCA2 mutation.     

1株猫杯状病毒的分离鉴定及其ORF2序列的遗传演化分析

为了解猫杯状病毒形态特征及遗传演化情况,采用F81细胞从患病宠物猫的鼻拭子样品中分离获得1株猫杯状病毒(feline calicivirus,FCV),命名为SH1。经电镜观察,病毒粒子呈球形,无囊膜,符合FCV的形态特征。采用RT-PCR方法扩增了该毒株的全基因组,并进行了序列测定和衣壳蛋白基因(ORF2)序列的分析。结果显示:分离株与国内外参考株的ORF2序列的核苷酸和氨基酸同源性分别为74.1%~79.7%和84.5%~90.9%;ORF2基因的遗传演化分析显示,30株FCV毒株形成两大分支,即基因群Ⅰ和Ⅱ,分离株属于基因群Ⅰ;进一步分析发现,基因群Ⅰ和Ⅱ主要在377、539和557氨基酸位点存在差异,基因群Ⅰ和Ⅱ分别为N、A、G和K、V、S。研究结果为FCV感染的防控提供科学依据。     

Single oral β-cryptoxanthin administration increases its serum concentration and enhances peripheral blood neutrophil function in Holstein cattle

We investigated the effect of oral administration of β-cryptoxanthin (β-CRX) on its serum concentration and peripheral neutrophil functions by the chemiluminescence (CL) response in Holstein cattle. A single oral administration of β-CRX was performed for serum β-CRX concentration (0, 0.05, 0.1, or 0.2 mg/kg body weight [BW]) and for peak CL response of peripheral neutrophils (0.2 mg/kg BW). The serum β-CRX concentration was peaked on 2 days after, similar to peak CL response on 3 days after β-CRX administration. Therefore, a single oral administration of β-CRX (0.2 mg/kg BW) induces higher serum concentration and concurrently enhances bactericidal ability of peripheral neutrophils in Holstein cattle.     

Molecular characterization of Ascaridia galli from Bangladesh and development of a PCR method for distinguishing A. galli from Heterakis spp.

We analyzed the nuclear ribosomal internal transcribed spacer (ITS) 1 and ITS2 sequences for Bangladesh isolates of Ascaridia galli, and we determined that the sequences were unreliable as molecular markers for distinguishing A. galli from other Ascaridia species, because the sequences showed high identity with that of A. columbae. However, the ITS1 sequences were available for designing PCR primers distinguishable between Ascaridia galli and Heterakis spp. Bangladesh isolates of A. galli constituted a monophyletic clade along with other geographical isolates in the cytochrome c oxidase subunit I (COI) phylogenetic tree, however, we could not clarify the phylogenetic relationships between A. galli and other Ascaridia spp., because their available sequences in GenBank were very few. The developed PCR method using DNA from A. galli and Heterakis spp. eggs would enable differential diagnosis of the individual infections in the future.     

Immunohistochemical expression of HER - 2 and Ki - 67 in granulosa cell tumor in bitches

Granulosa cell tumour, an ovarian neoplasm of stromal origin, is an important tumour related to oestrogenic dominance syndrome and cystic endometrial hyperplasia–pyometra complex. In order to analyse ovarian tumour´s malignant potential, immunohistochemical markers can be used, such as anti-HER2 and anti-Ki-67. The aim of this study was to evaluate the expression of immunohistochemical markers HER-2 and Ki-67 in granulosa cell tumour from bitches´ ovaries. In HER-2 immunomarker analysis using the HercepTest^® method, most tumours were classified as 2+ (moderate labelling). Concerning Ki-67 immunomarker, only one case was described as having a high proliferative index. An association was found between immunostained cell percentage by anti-HER-2 antibodies and high pleomorphism, represented by the pattern of follicular/trabecular tumour arrangement. There was no correlation between anti-Ki-67 and anti-HER-2 antibody immunostaining intensities, probably due to only one case with a high Ki-67 index. With an effective protocol for HER-2 and Ki-67 immunohistochemical identification in granulosa cell tumours in bitches, it was possible to characterize this neoplasm proliferation profile.