Asymmetric expression of CA2 and CA13 linked to calcification in the bilateral mandibular condyles cause crossed beaks in chickens

Crossed beak is a complex mode of inheritance with prevalence ranging from 0.2 to 7.4% in at least 12 chicken strains worldwide.To reveal the intrinsic factors causing crossed beaks,genes expression patterns in bilateral mandibular condyle between affected and normal birds were characterized by RNA sequencing analysis in the present studies.Crossed beak was induced by short length of unilateral mandibular ramus,and a total of 110differentially expressed genes were up-or down-regulated in the affected(short)mandibular condyle side as compared to the normal side.Carbonic anhydrase 2(CA2)and Carbonic anhydrase 13(CA13)were enriched in the carbonate dehydratase activity,and high-expressed in mandibular condyle and osteoblasts(P<0.05).However,both were low-expressed in short mandibular condyle side of affected birds(P<0.05).The carbonate dehydratase inhibitor experiments confirmed that there is positive association between the calcification and carbonic anhydrase isoenzymes.Quantitative analysis with cetylpyridinium chloride showed a decrease in calcification when the cells were transfected with an anti-CA13 shRNA.Our research suggested that CA2 and CA13 are down-calcified in shortside mandibular condyle,and caused mandibular ramus to grow slowly.CA2 and CA13 have the critical role in crossed beaks by regulating calcification of mandibular condyle.

Significant genomic introgression from grey junglefowl(Gallus sonneratii)to domestic chickens(Gallus gallus domesticus)

Background Chicken is one of the most numerous and widely distributed species around the world,and many studies support the multiple ancestral origins of domestic chickens.The research regarding the yellow skin phenotype in domestic chickens(regulated by BCO2)likely originating from the grey junglefowl serves as crucial evidence for demonstrating the multiple origins of chickens.However,beyond the BCO2 gene region,much remains unknown about the introgression from the grey junglefowl into domestic chickens.Therefore,in this study,based on wholegenome data of 149 samples including 4 species of wild junglefowls and 13 local domestic chicken breeds,we explored the introgression events from the grey junglefowl to domestic chickens.Results We successfully detected introgression regions besides BCO2,including two associated with growth trait(IGFBP2 and TKT),one associated with angiogenesis(TIMP3)and two members of the heat shock protein family(HSPB2 and CRYAB).Our findings suggest that the introgression from the grey junglefowl may impact the growth performance of chickens.Furthermore,we revealed introgression events from grey junglefowl at the BCO2 region in multiple domestic chicken breeds,indicating a phenomenon where the yellow skin phenotype likely underwent strong selection and was retained.Additionally,our haplotype analysis shed light on BCO2 introgression event from different sources of grey junglefowl into domestic chickens,possibly suggesting multiple genetic flows between the grey junglefowl and domestic chickens.Conclusions In summary,our findings provide evidences of the grey junglefowl contributing to the genetic diversity of domestic chickens,laying the foundation for a deeper understanding of the genetic composition within domestic chickens,and offering new perspectives on the impact of introgression on domestic chickens.

Expression of B Locus Beta 2 (<i>BLB2</i>) Gene at Cytolytic and Latent Immune Response Stages of Immunocompetence in Nigerian Indigenous Chickens

An economically-important trait in poultry for which gene identification continues to be a challenge is immune response. The objective of the study was to quantitate the expression of major histocompatibility complex (MHC) class II BLB2 gene at cytolytic and latent immune response stages in Nigerian indigenous chickens. A total of 108 Nigerian indigenous chickens (NIC) were sourced across the South-western states in Nigeria. The birds were inoculated with sheep red blood cells (SRBC), after which blood samples were obtained (5 days post-inoculation) and antibody haemagglutination test was carried out to place the birds into groups of high and low antibody titre levels. The categorisation of the birds resulted in six groups of normal feather high, normal feather low, naked neck high, naked neck low, frizzle feather high and frizzle feather low antibody groups. A total of 48 chicks were selected from the progeny for gene expression studies. Surgical excision of thymus and spleen was carried out for the detection of cytolytic and latent responses of the birds. β-actin was used as the endogenous control and the critical threshold method (2–ΔΔCт) was carried out for the determination of fold change. The fold change of spleen tissue expression at cytolytic immune response of the birds was 30,362.44 compared to latent response 294.07;and the fold change of thymus expression at cytolytic immune response of the birds was 51.98 compared to latent response 5.24. At both cytolytic and latent stages of immune response to SRBC antigen, BLB2 expression in the spleen was comparatively higher than in the thymus and the height of transcriptional activity was associated with the cytolytic stage. The birds of high titre at both the cytolytic and the latent responses had higher mRNA expression. This study concluded that BLB2 gene expression in the Nigerian indigenous chicken was induced at the cytolytic stage and repressed at the latent stage. During avian infections, the category of high immune response birds would perform better than the low immune response counterpart;and the protective response that BLB2 gene offers will be repressed from one time point to the other.

鸡白细胞介素-2基因在Pichia methanolica酵母中的表达

鸡白细胞介素-2(chIL-2)是近年来新发现的一种禽类细胞因子.为建立用甲醇型酵母Pichiamethanolica(P.methanolica)表达鸡白细胞介素-2基因(chIL-2)的新途径,将chIL-2基因插入到P.methanolica分泌型表达载体pMETαA构建了重组PMAD11表达菌株(pMETαA/rchIL-2).经SDS-PAGE、Western印迹测定,证实chIL-2基因在P.methanolica中表达成功,rchIL-2的分子量约为16kDa,其表达量占分泌总蛋白的35%.

普洱茶茶渣对茶花鸡2号肉品质的影响

试验旨在探讨饲粮中添加不同水平普洱茶茶渣对茶花鸡2号肉品质的影响。选取144只茶花鸡2号,随机分为3组,每组6个重复,每个重复8只。对照组饲喂基础饲粮,1%和2%茶渣组分别在基础饲粮上添加1%和2%的普洱茶茶渣,试验期28 d,试验结束进行鸡肉品质测定。结果表明:①与对照组相比,1%和2%茶渣组胸肌和腿肌肉品质物理特性(L^(*)值、a^(*)值、b^(*)值、pH4_(5min)、pH_(24h)、失水率、蒸煮损失率、剪切力)和常规营养成分(粗蛋白、粗脂肪和粗灰分含量)无显著差异(P>0.05),1%茶渣组胸肌各氨基酸含量以及2%茶渣组腿肌各氨基酸含量无显著差异(P>0.05)。②与对照组相比,2%茶渣组胸肌Asp、Thr、Ser、Glu、Ala、Cys、Val、Ile、Leu、Tyr、Lys、UAA和TAA含量极显著降低(P<0.01),Met、Phe、His、Arg、SAA、BAA、EAA和NEAA含量显著降低(P<0.05),Gly和Pro含量无显著变化(P>0.05),1%茶渣组腿肌Tyr含量极显著降低(P<0.01),Asp、Thr、Ser、Glu、Ala、Val、Ile、Leu、Phe、Lys、Arg、UAA、SAA、BAA、EAA、NEAA和TAA含量显著降低(P<0.05),Gly、Cys、Met、His和Pro含量无显著变化(P>0.05)。由此可见,饲粮中添加1%和2%普洱茶茶渣分别降低了茶花鸡2号腿肌和胸肌氨基酸含量,降低了肌肉风味。

鸡γ-干扰素与补体受体2蛋白相互作用的研究

【目的】研究鸡γ-干扰素(chicken interferonγ,chIFN-γ)与鸡补体受体2(chicken complement receptor 2,chCR2)蛋白的相互作用,为研究chIFN-γ和chCR2新功能奠定基础。【方法】通过同源重组方法构建chIFN-γ相关表达质粒,利用免疫共沉淀、激光共聚焦和表面等离子共振技术鉴定chIFN-γ与chCR2蛋白的相互作用,并通过分子对接技术模拟chIFN-γ与chCR2蛋白的相互作用模式。【结果】成功构建了pCMV-Myc-chIFN-γ表达质粒。将pCMV-Myc-chIFN-γ和pCMV-HA-chCR2-ΔTM质粒共转染HEK-293FT细胞,结果表明chIFN-γ蛋白可以与chCR2蛋白进行相互作用;将pCMV-Myc-chIFN-γ和pCMV-HA-chCR2-ΔTM质粒共转染DF-1细胞,结果表明chIFN-γ蛋白与chCR2蛋白可以在DF-1细胞中进行共定位。表面等离子共振试验结果显示chIFN-γ与chCR2蛋白之间的平衡解离常数(KD)值为0.362μmol/L,二者之间有较高的亲和力。分子对接试验结果显示,chIFN-γ与chCR2蛋白之间的相互作用模式为Asp23-His150间形成1组盐桥作用,Asn37-Ser143、Glu57-Gly179和Ile93-Tyr184间形成3组氢键作用。【结论】本研究结果证明了chIFN-γ与chCR2蛋白可以进行相互作用,且二者之间的作用模式为1组盐桥和3组氢键作用。

Comparison of Five Expression Vectors for the Ha Gene in Constructing a DNA Vaccine for H6N2 Influenza Virus in Chickens

A number of eukaryotic expression vectors have been developed for use as DNA vaccines. They showed varying abilities to initiate immune responses;however, there is little data to indicate which of these vectors will be the most useful and practical for DNA vaccines in different species. This report examines the use of five expression vectors with different promoters and Kozak sequence to express the same hemagglutinin (HA) protein of an H6N2 avian influenza virus for DNA vaccination in chickens. Although intramuscular vaccination with seven DNA constructs elicited no or limited measurable H6 HA antibody responses in Hy-Line chickens, variable reduction in virus shedding for either oropharyngeal or cloacal swabs post-virus challenge were observed. This indicated that all DNA constructs generated some levels of protective immunity against homologous virus challenge. Interestingly, lower dose (50 or 100 μg) of plasmid DNAs consistently induced better immune response than higher dose (300 or 500 μg). In the transfection experiments there appeared to be a hierarchy in the in vitro expression efficiency in the order of pCAG-optiHAk/ pCAG-HAk > pCI-HAk > VR-HA > pCI-HA > pCI-neo-HA > pVAX-HA. Since the level of in vitro expression correlates with the level of immune response in vivo, in vitro expression levels of the DNA constructs can be used as an indicator for pre-selection of plasmid vaccines prior to in vivo assessment. Moreover, our results suggested that the Kozak sequence could be used as an effective tool for DNA vaccine design.

Association of Bone Morphogenetic Protein-2 Gene with Skeletal Traits in Chickens

Bone morphogenetic protein-2 （BMP-2） plays a key role in bone formation and maintenance. BMP-2 can stimulate longitudinal bone growth by increasing the growth plate chondrocyte proliferation, hypertrophy, and cartilage matrix synthesis. The current study was designed to investigate the associations of the BMP-2 gene polymorphism with chicken skeletal traits. Northeast Agricultural University F2 resource population was used in this study. Body weight and body composition traits were measured in F2 population. Polymorphism between parental lines was detected by DNA sequencing, and PCR-fragment length polymorphism method was then developed to screen the population. The results showed that the BMP-2 gene polymorphism was associated with skeletal traits in F2 population. This research suggests that BMP-2 gene may be a candidate locus or linked to a major gene that affects skeletal traits in chickens.

High expression circRALGPS2 in atretic follicle induces chicken granulosa cell apoptosis and autophagy via encoding a new protein

Background The reproductive performance of chickens mainly depends on the development of follicles.Abnor-mal follicle development can lead to decreased reproductive performance and even ovarian disease among chick-ens.Chicken is the only non-human animal with a high incidence of spontaneous ovarian cancer.In recent years,the involvement of circRNAs in follicle development and atresia regulation has been confirmed.Results In the present study,we used healthy and atretic chicken follicles for circRNA RNC-seq.The results showed differential expression of circRALGPS2.It was then confirmed that circRALGPS2 can translate into a protein,named cir-cRALGPS2-212aa,which has IRES activity.Next,we found that circRALGPS2-212aa promotes apoptosis and autophagy in chicken granulosa cells by forming a complex with PARP1 and HMGB1.Conclusions Our results revealed that circRALGPS2 can regulate chicken granulosa cell apoptosis and autophagy through the circRALGPS2-212aa/PARP1/HMGB1 axis.

Correlation between Egg Production Traits and Broodiness Traits in Shiqiza,Langya and Luqin B2 Chickens

[ Objective] To investigate the relationship between the egg production traits and broodiness of Shiqiza, Langya and Luqin B2 chickens. E Method] Three chicken breeds, Shiqiza chickens （a breed preserved by Shandong Academy of Agricultural Sciences）, Langya chickens （a local breed in Shandong Province） and Luqin B2 chickens （a cultivated breed） were selected. Then their broodiness traits were observed and the egg productions were analyzed. Finally, the relationships between the egg production traits and broodiness were analyzed by SAS software. [ Result] The Shiqiza, Langya and Luqin B2. chickens were broody and their broodiness rate was 15.80%, 2.24% and 6.95%, respectively. Compared with the non-broody chickens, the egg productions of Shiqiza, Langya and Luqin B2 chickens were respectively decreased by 17.16%, 1.53% and 10.8% at the age of 43 weeks. The three breeds had significant differences in repeats of broody cycles （ P 〈 0.05）, and extremely significant differences in the broodiness duration, the the first day of broodiness, the age at the first egg, total amount of abnormal eggs and amount of double- yolked eggs （P 〈 0.01 ）. [Conclusion] The broodiness is a main factor affecting egg production traits. The degree of effects of broodiness on egg production traits is decreased successively in Shiqiza chicken, Luqin B2 chicken and Langya chicken.

鸡白细胞介素-2抗家禽病毒病作用及机制研究进展

家禽病毒病严重危害我国养殖业的发展和公共卫生安全。鸡白细胞介素-2(Chicken interleukin-2,ChIL-2)是一类具有免疫活性的细胞因子,广泛参与非特异性免疫和特异性免疫应答,发挥重要的抗病毒作用,在动物疾病防治、新型免疫佐剂研发、提高动物抗病能力等方面具有广阔的应用前景。文章对ChIL-2的结构、ChIL-2介导的免疫信号通路及其在家禽病毒病的应用进行综述,旨在为研发新型家禽抗病毒药物和疫苗佐剂提供理论参考。

H9N2亚型禽流感病毒A／Chicken／Gansu／2／99株基因组的分子特征

目的测定H9N2亚型禽流感病毒A/Chicken/Gansu/2/99(CK/GS/2/99)分离株的基因组序列并与参考毒株进行同源性分析,阐明该毒株的遗传变异及分子特征。方法采集发病鸡泄殖腔样品,经鸡胚尿囊腔接种分离病毒,采用RT-PCR方法对CK/GS/2/99株的8个分节段基因进行扩增,分别将其克隆到pGEM-T easy载体后进行序列测定与同源性分析。结果CK/GS/2/99株PB2、PB1、PA、HA、NP、NA、M、NS基因的开放阅读框分别由2280、2274、2151、1683、1497、1401、759、864个碱基组成,分别编码759、757、716、560、498、466、252/97、231/122个氨基酸残基。该毒株HA上HA1和HA2裂解位点序列为PARSSR↓GLF,具有典型的低致病性禽流感病毒的特征。同源性分析显示,CK/GS/2/99株与1998-2002年间大部分中国大陆分离株遗传关系较近,尤其与CK/NX/4/99和CK/HB/31/00株遗传关系密切。结论CK/GS/2/99与大部分流行于中国内陆的H9N2亚型毒株均来源于共同的祖先毒株CK/BJ/1/94,这为了解中国H9N2亚型禽流感病毒的分子流行病学提供了资料。

鸡Toll样受体2的克隆表达及多克隆抗体的制备

试验旨在制备鼠抗鸡Toll样受体2(chTLR2)的多克隆抗体。采用RT-PCR方法从活化的鸡外周血淋巴细胞中扩增其胞外区片段chTLR2(chTLR2t1和chTLR2t2),并构建这两个片段的原核表达重组质粒,优化蛋白质的表达条件,SDS-PAGE和Western-blot法检测融合蛋白,将融合蛋白作为免疫原免疫Balb/c小鼠,利用相应的原核表达的全长蛋白检测特异性抗体滴度。结果显示,成功获得chTLR2的胞外区片段,构建的原核表达重组质粒经IPTG诱导能成功表达预期的重组蛋白,免疫小鼠可产生特异性抗体,抗体效价分别为1∶16 000和1∶10 000。表明制备的鼠抗chTLR2多抗具有良好的特异性,为进一步研究chTLR2的功能提供了重要材料。

枸杞多糖对雏鸡淋巴细胞体外增殖及分泌IL-2的影响

为探讨枸杞多糖(LBP)对雏鸡T、B淋巴细胞体外增殖的影响,将不同浓度的LBP分别加入到用植物血凝素(PHA)刺激的体外培养的雏鸡胸腺、外周血、脾脏淋巴细胞中,以及用细菌脂多糖(LPS)刺激的体外培养的雏鸡法氏囊、外周血、脾脏淋巴细胞中,分别用MTT比色法和ELISA法测定淋巴细胞增殖的变化以及IL-2的分泌量。结果显示:(1)LBP在一定浓度范围内时,均能刺激T、B淋巴细胞的增殖和促进IL-2的分泌,且随着多糖浓度的增加而增加;(2)LBP对同一器官来源的T、B淋巴细胞的OD值随着雏鸡日龄的增长均呈现增长趋势;(3)同浓度LBP对相同日龄雏鸡胸腺T淋巴细胞和法氏囊B淋巴细胞的OD值均高于外周血和脾脏的T、B淋巴细胞OD值。结果表明LBP能够促进雏鸡淋巴细胞的增殖和IL-2的分泌,并且对淋巴细胞增殖的促进作用有一定的量效、时效关系和组织相关性。

鸡α干扰素/白细胞介素2基因的融合表达及活性研究

【目的】研究高效广谱鸡基因工程重组复合抗病毒制剂以对鸡的病毒性疾病进行防治。【方法】采用重叠延伸PCR(splicing by overlap extension-PCR,SOE-PCR)方法通过一基因柔性接头将鸡α干扰素(chicken interferon alpha,ChIFN-α)与鸡白细胞介素2(chicken interleukin-2,ChIL-2)基因构建成ChIFN-α-linker-ChIL-2嵌合基因并克隆入pGEM-T Easy载体,将嵌合基因亚克隆入pQE-30表达载体中进行原核表达。通过尿素变性、低浓度蛋白复性液复性、PBS溶液透析等步骤对表达的重组融合蛋白(rChIFN-α-linker-ChIL-2)进行纯化。采用细胞病变抑制法检测rChIFN-α-linker-ChIL-2蛋白在细胞上抑制水泡性口炎病毒(VSV)和传染性法氏囊病毒(IBDV)增殖活性。采用ChIL-2ELISA试验方法检测rChIFN-α-linker-ChIL-2蛋白与抗ChIL-2单抗发生特异性免疫反应的活性。分别测定rChIFN-α-linker-ChIL-2蛋白在SPF鸡胚上对新城疫病毒(NDV)和禽流感H9N2亚型病毒(AIV H9N2)的抑制活性以测定其在鸡胚内抗病毒活性。分别测定rChIFN-α-linker-ChIL-2蛋白在鸡体内对NDV活疫苗和灭活疫苗的免疫增强作用以测定其在鸡体内的活性。【结果】成功构建并克隆了ChIFN-α-linker-ChIL-2嵌合基因。嵌合基因在大肠杆菌中表达的rChIFN-α-linker-ChIL-2蛋白分子量大小约35.9kD,蛋白经纯化后纯度在96%以上。rChIFN-α-linker-ChIL-2蛋白在CEF细胞上具有明显抗病毒活性,其抗VSV和IBDV活性明显高于单一rChIFN-α的抗病毒活性;rChIFN-α-linker-ChIL-2蛋白可以和抗ChIL-2单抗发生特异性免疫反应。IFN-α活性单位为200IU的rChIFN-α-linker-ChIL-2蛋白在SPF鸡胚内可明显降低NDV和AIV H9N2所引起的鸡胚死亡和胚体出血,并能显著延长鸡胚存活时间,但过高剂量的rChIFN-α-linker-ChIL-2蛋白抑制鸡胚死亡和出血能力有所下降。合适剂量的rChIFN-α-linker-ChIL-2蛋白在鸡体内具有显著的抗病毒活性和免疫增强活性。【结论】rChIFN-α-linker-ChIL-2蛋白在鸡体内外均具有ChIFN-α和ChIL-2蛋白的双重生物学活性,这为进一步研究以rChIFN-α-linker-ChIL-2蛋白为主要成分的基因工程抗病毒制剂在鸡体内抗病毒活性研究奠定了基础。

中药复方对鸡免疫器官指数及IL-2表达的动态影响

将300只1日龄公雏鸡随机分为3组,两组分别给予中药方Ⅰ和方Ⅱ(由石膏、苍术、黄柏、藿香分别按质量比1∶1∶1∶1和0.5∶1∶1∶1比例组成,按体重0.3 mg/kg的剂量饮水),对照组按照常规方法饲养。分别于雏鸡第7、21、35、49日龄时,每组随机抽取5只鸡剖杀,取腔上囊、脾、胸腺测定免疫器官指数和IL-2平均阳性表达率,以探讨中药复方对雏鸡免疫器官指数及其IL-2表达的动态影响。结果显示,随着用药时间的增加,中药方Ⅰ和方Ⅱ可显著(P<0.05)或极显著(P<0.01)提高腔上囊、脾和胸腺等免疫器官指数及其IL-2的平均阳性表达率,方Ⅰ对各免疫器官IL-2阳性表达率的影响最为显著(P<0.05)。证实该中药复方可促进雏鸡细胞免疫水平,增强其非特异性免疫功能,并促进免疫器官的发育。

鸡白细胞介素-2基因的克隆与酵母表达质粒的构建

从经ConA活化的4周龄岭南黄鸡脾脏淋巴细胞中分离提取总RNA,经过反转录PCR(RT PCR)扩增出鸡白细胞介素 2(IL 2)cDNA片断.PCR产物克隆至pGEM TEasy载体,重组质粒命名为IL 2 T,经菌落PCR与限制酶切鉴定,然后测序,结果表明克隆片断长度为432bp,与预期大小一致,为鸡IL 2基因的编码区全长.将IL 2 T克隆重组质粒进行双酶切(ClaI与XbaI),回收IL 2片断插入同样酶切的毕赤酵母表达质粒pPICZα C,构建重组IL 2 C表达质粒,为鸡IL 2在毕赤酵母中的表达奠定基础.

bcl-2基因在成年和胚胎鸡免疫器官中的表达及其与细胞凋亡的关系

用一步法从17 日龄来克亨SPF鸡胚和90 日龄成年鸡的法氏囊、脾脏和胸腺组织中提取总RNA(TRNA),用鸡B-细胞淋巴瘤/白血病-2基因 (bcl-2) cDNA探针进行Northern blot杂交。发现bcl-2 在成年和胚胎鸡的不同免疫器官中表达量不同, 成年鸡bcl-2 的表达量多少依次为胸腺、脾脏、法氏囊, 而胚胎鸡则是胸腺、法氏囊、脾脏。同时用流式细胞法测定这些器官的细胞凋亡情况, 胚胎鸡法氏囊、脾脏和胸腺的细胞凋亡率分别是0.40% , 0.68% 和0.38% ; 成年鸡则是6.98% , 1.17% 和0.61% 。结果表明, 鸡bcl-2 基因通过阻抑细胞凋亡直接调控正常免疫器官的发育, 在淋巴细胞发育过程中bcl-2是免疫细胞亚型选择的重要调节物。

白细胞介素2对固始鸡十二指肠黏膜免疫增强作用

以父母代固始鸡为试验材料,用添加不同剂量的白细胞介素2(IL-2)佐剂的鸡志贺氏菌苗对试验鸡进行分组免疫,应用免疫组织化学技术,通过Qwin图像处理系统对鸡十二指肠内分泌型免疫球蛋白A(SIgA)阳性细胞及其分泌物的分布和阳性面积进行系统分析。结果表明,SIgA阳性细胞主要分布于十二指肠肠黏膜的固有膜及肠腺腔中,在志贺菌组中添加IL-2能够提高SIgA细胞的分泌,从而提高动物体的免疫能力,且以IL-2的添加剂量在50μg时对增加志贺菌苗的免疫性效果最佳。

鸡IL-2在新城疫病毒F基因疫苗免疫中的作用

利用国内首次克隆成功的鸡 IL- 2基因真核表达质粒与新城疫病毒 (NDV) D2 6株 F基因真核质粒 (F基因疫苗 )联合免疫 SPF雏鸡 ,观察了其诱导的免疫应答及免疫保护作用 ,以及不同免疫方式对其免疫作用的影响。结果证实了国内克隆鸡 IL- 2基因的免疫增强作用 ,以及作为免疫佐剂应用于禽类基因免疫的可行性 ,为鸡 IL- 2这一新型佐剂的实际应用提供了重要的试验依据。