北京鸭干扰素-α基因的分子克隆与表达

采用 PCR法从北京鸭 (Anas platyrhynchosdoestica L innaeus)基因组中克隆了其干扰素 - α基因 (BJ- Du IFN- α)。克隆片段长 4 86 bp,编码 1 6 1个 Du IFN-α成熟肽。与已报道的北京鸭干扰素 -α基因相比 ,BJ- Du IFN-α在 2 85位发生了同义变异。将 BJ-Du IFN-α插入原核表达载体 p QE3 0 ,在 E.Coli JM1 0 9工程菌中表达了重组蛋白。经 SDS- PAGE、Western Blot和抗病毒活性测定 ,表达产物占菌体总蛋白的 2 3 %,为重组鸭干扰素 -α(r Du IFN-α)。 r Du IFN-α抗滤泡性口炎病毒 (VSV)活性高达 7.9× 1 0 5U /mg。     

Molecular cloning and functional analysis of duck Toll-like receptor 5

Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in vertebrates. In this study, we cloned the single-exon TLR5 gene of the Maya breed of Common Shelduck (Tadorna tadorna). The TLR5 open reading frame is 2580 bp in length and encodes an 859-amino acid protein. The putative amino acid sequence of duck TLR5 consisted of a signal peptide sequence, 11 leucine-rich repeat domains, a leucine-rich repeat C-terminal domain, a transmembrane domain, and an intracellular Toll-interleukin-1 receptor domain. The duck TLR5 gene was highly expressed in the lung, bone marrow, spleen, and liver; moderately expressed in kidney, small intestine, large intestine, and brain. A plasmid expressing duck TLR5 was constructed and transfected into HEK293T cells, and expression was confirmed by indirect immunofluorescence assay. HEK293T cells transfected with duck TLR5- and NF-κB-luciferase-containing plasmids significantly responded to flagellin from Salmonella typhimurium, indicating that it is a functional TLR5 homolog.     

Molecular cloning and tissue distribution of a short form chicken leptin receptor mRNA

In mammals, alternative splicing of the leptin receptor (LEPR) produces several C-terminal truncated isoforms that are believed to play a role in the transport, cellular internalisation and degradation of the hormone leptin. The chicken leptin receptor (chLEPR) is similar to its mammalian counterparts in terms of its intron/exon structure and conserved motifs. However, it is unknown whether the chLEPR also undergoes alternative splicing. To test this, structural analysis of intron 19 of the chLEPR, equivalent to the intron in which alternative splicing occurs in mammals, was combined with 3'-rapid amplification of cDNA ends (3'-RACE) to search for chLEPR splice variants. A 44-amino acid alternative exon 20 was identified that is spliced to generate a short isoform of the chLEPR (chLEPR-SF). Comparative sequence analysis of intron 19 identified two regions that are highly conserved between the chicken and mammals, indicating their possible importance as intronic elements in the regulation of alternative splicing of the LEPR in vertebrates. Tissue expression of the chLEPR-SF was lower and more restricted than that of the chLEPR long isoform. Collectively these data demonstrate that the chLEPR is alternatively spliced to produce at least one short isoform, as is the case in mammals.     

鸭髓样分化因子MyD88部分cDNA克隆及组织表达图谱分析

本试验采用RT-PCR技术克隆出鸭MyD88部分cDNA序列,克隆到的序列长为195 bp;同源性分析结果显示,鸭MyD88与鸡、火鸡、珍珠鸟的同源性最高,为97％～100％;与人、鼠、牛、猪、狗、马、兔、鲤鱼的同源性为83％～84％;与绵羊的同源性为79％;系统进化树分析表明,鸭MyD88与鸡的亲缘关系最近,与火鸡、珍珠鸟的较为接近,与狗、鼠、兔、人、猪、马、绵羊、牛的亲缘关系依次渐远,与鲤鱼的亲缘关系最远.检测MyD88在鸭7种组织中的分布情况,结果表明,在鸭的心脏、肝脏、脾脏、肺脏、腔上囊中均检测出MyD88 mRNA的转录产物,而在肾脏、脑中未见.提示MyD88在不同种属及不同组织中具有生物多样性.     

猪ADSL基因克隆及其在部分组织中的mRNA定量表达

试验以通城猪为研究对象,在对猪ADSL基因全长编码区cDNA片段进行克隆、序列测定、与GenBank中已收录的其他14种动物的ADSL全长编码区序列进行同源性分析,并构建系统发生树的基础上,采用实时(相对)定量PCR方法对刚出生和65日龄两个不同生长发育时期的通城猪心、肝、肾脏、背最长肌4种组织的ADSL基因表达谱进行分析。结果表明,所克隆的ADSL基因片段大小为1 548 bp(GenBank登录号:GU249574),其中包含一长为1 455 bp的完整阅读框,通过核酸序列分析发现,该基因编码484个氨基酸;与GenBank中已收录的猪ADSL基因序列同源性最高,达99.9%;与恒河猴和黑猩猩的序列同源性最低,分别为66.3%和41.3%;15种动物的20条ADSL全长CDS序列聚类分布为两个主支。刚出生时的通城猪ADSL基因在心、肝、肾脏组织的mRNA表达水平均高于65日龄通城猪的心、肝、肾脏组织中的mRNA表达;然而,其背最长肌的ADSL基因mRNA表达水平则低于65日龄的通城猪。本结果为猪ADSL基因的生物学功能研究和遗传进化研究提供了分子依据。     

胡麻异质型ACCase亚基基因的克隆与表达分析

为研究胡麻异质型乙酰辅酶A羧化酶BCCP、BC、α-CT和β-CT四个亚基基因的生物学功能,采用基因克隆、生物信息学和RT-PCR技术分别对胡麻accA、accB、accC和accD 4个基因进行分析。结果发现陇亚10号accA基因编码α-CT亚基,CDS序列全长为2364 bp,编码787个氨基酸;accB基因编码BCCP亚基,CDS序列全长为1173 bp,编码275个氨基酸;accC基因编码BC亚基,CDS序列全长为1574 bp,编码527个氨基酸;accD基因编码β-CT亚基,CDS序列全长为1137 bp,编码378个氨基酸,且4个基因编码的蛋白都是脂肪酸系数较高的亲水性蛋白。RT-PCR结果表明,胡麻accA、accB、accC和accD 4个基因在3个胡麻品种(高含油量的张亚2号、中含油量的陇亚10号和低含油量的R2-17)的不同时期不同组织的表达模式不同,accA、accB、accC和accD四个基因在R2-17、陇亚10号和张亚2号3个胡麻品种所有时期的不同组织中均有表达,但在种子中的表达量均明显高于其他组织,尤其在种子发育10~25 d油脂快速积累时期,4个基因的表达量都迅速增加并达到最高峰。研究表明异质型乙酰辅酶A羧化酶基因可能调控胡麻种子发育前期油脂的合成积累。     

金黄地鼠（Mesocricetus auratus）朊蛋白基因的克隆及其原核表达

采用RT-PCR技术从金黄地鼠(Mesocricetus auratus)脑组织获得朊蛋白基因(Prion protein nucleic acid,PRNP)开放阅读框(Open reading frame,ORF),截去其N端信号肽(66 bp)和C端GPI锚定位点(69 bp)形成朊蛋白编码区PRNPx;将编码区重组于融合表达质粒Pet-DsbA中,并在大肠杆菌BL21(DE3)plysS中经IPTG诱导表达,并经Western-blot验证。结果表明,金黄地鼠PRNP基因ORF区全长为765 bp,编码254个氨基酸的前体蛋白,核苷酸序列与已发表金黄地鼠序列(M14054)同源性为99.87%,其第116个氨基酸发生了同义突变由GCT变为GCC;朊蛋白在大肠杆菌得到高效表达,产物是相对分子质量为47 000的融合蛋白。     

Cloning and expression analysis of prohibitin mRNA in canine mammary tumors

Prohibitin is an antiproliferative protein that is a product of a putative tumor suppressor gene. However, there is little information on prohibitins in companion animals. In this study, we cloned canine prohibitin mRNA using RT-PCR and 3′-RACE (Rapid Amplification of cDNA Ends). The sequence was well conserved compared with those of other mammals, including human. The deduced amino acid sequence translated from the open reading frame completely corresponded to the human sequence. Canine prohibitin mRNA was expressed in all normal mammary and tumor samples examined. These results suggest that this protein plays a vital role in cell growth mechanisms and may be related to the occurrence of canine mammary tumors.     

粘膜免疫对鸡十二指肠SS mRNA表达的影响

应用鸡新城疫弱毒疫苗进行消化道粘膜免疫,或在肌肉注射生长抑素(SS)亚单位疫苗的基础上应用鸡新城疫弱毒疫苗进行消化道粘膜免疫,通过RT PCR方法检测免疫鸡十二指肠中SS基因的表达.结果表明,首免后第3周,新城疫免疫组的SS mRNA表达高于SS亚单位苗组,但各试验组无显著差异;首免后第4周,新城疫免疫组SS mRNA的表达显著高于SS亚单位苗组(P＜0.05)并高于对照组.提示粘膜免疫可促进小肠粘膜SS mRNA的表达,SS亚单位苗可在基因水平上降低SS mRNA的表达.     

不同营养水平限制饲养对妊娠期蒙古绵羊生长激素受体(GHR)mRNA表达水平的影响

为了阐明不同营养水平限制饲养妊娠期母羊后,在不同生长阶段对蒙古绵羊生长激素受体(GHR)mRNA表达水平的影响,研究根据GenBank中绵羊的GHR序列,设计1对预计扩增产物为395 bp的引物,采用RT-PCR技术检测了蒙古绵羊在不同营养水平条件下肝脏和背最长肌中GHR mRNA表达量的变化。结果表明:在限制采食阶段,低营养水平组即营养限制组(RG1组)和阈值组(RG2组)肝脏的GHR mRNA表达水平低于正常营养的对照组(CG组),而背最长肌的GHR mRNA表达水平高于对照组,说明限制营养对绵羊GHR水平的影响具有组织特异性。在补偿生长结束阶段,RG1组和RG2组绵羊的肝脏和背最长肌中GHR mRNA表达水平均恢复到对照组水平。     

Molecular cloning of interleukin-12 receptor beta2 chain and its expression in dogs

This study cloned canine interleukin-12 receptor beta2 (IL-12Rbeta2). Its nucleotide sequences were determined. Canine IL-12Rbeta2 showed 85.4% homology at the nucleotide level and 76.8% homology at the amino acid level with human IL-12Rbeta2. Its structural motifs were well conserved. We also cloned cDNA with a 91-bp deletion including the transmembrane region, which produced a frame shift and an early stop codon. Examination of the expression of deleted canine IL-12Rbeta2 mRNA revealed that both deleted and intact mRNAs were expressed at a constant ratio in all the dogs. Results suggested that expression of the deleted mRNA was constitutive and produced by alternative splicing.     

生糖底物和神经内分泌因子对新生犊牛肝细胞PEPCK-C mRNA表达水平的影响

在原代单层培养的新生犊牛肝细胞培养液中分别加入不同浓度丙酸钠、丙酮酸钠、胰岛素、胰高血糖素和瘦蛋白，培养12h后，应用半定量RT-PCR方法检测体外培养的肝细胞PEPCK—C mRNA的丰度。结果显示，随着丙酸钠、丙酮酸钠浓度的升高，肝细胞PEPCK-C mRNA的丰度均先升高后下降（P〈0．01）；随胰岛素、胰高血糖素和瘦蛋白浓度的升高，肝细胞PEPCK-C mRNA的丰度分别剂量依赖性地降低、升高（P〈0．01）和无显著变化。表明，丙酸钠、丙酮酸钠能通过上调体外培养的新生犊牛肝细胞PEPCK—C mRNA的表达而促进肝糖异生代谢，但上调作用是有限的；胰岛素能通过下调体外培养的新生犊牛肝细胞PEPCK—C mRNA的表达而抑制肝糖异生代谢，且下调作用呈剂量依赖性；胰高血糖素与胰岛素作用刚好相反；瘦蛋白未起直接的调节作用。     

成年公猪睾丸及附睾中促黄体素受体mRNA表达的研究

试验采用FQ-PCR技术进行了成年公猪睾丸及附睾中促黄体素受体mRNA表达的研究,结果表明:促黄体素受体mRNA不仅在睾丸内表达,在附睾中也有表达;睾丸、附睾中促黄体素受体mRNA的表达量分别为(1.42±0.08)×109 copies/mL和(6.54±0.21)×109copies/mL,睾丸两组织间促黄体素受体mRNA表达量无明显差异(P>0.05).     

传染性法氏囊病病毒结构蛋白VP3基因的克隆与原核表达

利用PCR技术扩增出IBDV的结构蛋白VP3基因，将其克隆到表达载体pPRO^EX-HTa中，获得重组质粒命名为VP3／PA，经PCR、酶切和序列分析鉴定表明，插入的位置、大小和读码框均正确。SDS—PAGE检测表明，经重组质粒VP3／PA转化、诱导的受体菌E．coli DH5 α能表达结构蛋白VP3基因，约33 Ku，表达量达到了茵体总蛋白的33．9％，经Western blot等检测表明，诱导表达的抗原蛋白能与vvIBDV阳性血清发生特异性反应。这为IBDV血清学诊断方法的建立打下了基础。     

昆明小鼠与BALB/c小鼠MHC-Ⅱ分子恒定链的序列差异分析及多克隆抗体的制备

分离昆明小鼠与BALB/c小鼠脾脏淋巴细胞,经Con-A活化,提取细胞总RNA,RT-PCR获得小鼠Ii分子（P31、P41）基因,DNAStar软件分析序列。将BALB/c小鼠P31/P41分子插入到pGEX-KG多克隆位点构建原核表达载体,IPTG诱导并经GST标签蛋白纯化柱纯化表达蛋白,将GST-P31/P41蛋白分别与弗氏佐剂混合乳化后免疫家兔,采集血清。测序分析显示,两品系小鼠P31、P41均分别为648、840bp,编码215个、279个氨基酸;序列比对结果表明,昆明小鼠、BALB/c小鼠P31、P41分子与GenBank已登录核苷酸序列与/或氨基酸序列均存在多个位点差异,且所克隆的两品系小鼠P31分子之间核苷酸序列同源性为98.8%,氨基酸序列同源性为98.6%;两种小鼠P41分子之间核苷酸序列、氨基酸序列同源性均为99.3%;通过与人、鸡对应Ii分子进行比较,发现与人同源性较高,核苷酸序列同源性在79.0%以上,氨基酸序列同源性在73.0%以上,而与鸡同源性相对较低,核苷酸序列同源性在55.0%以上,氨基酸序列同源性在41%以上。IPTG诱导并纯化后获得GST-P31/P41蛋白,免疫家兔后所采集的血清经ELISA、Western-blotting证实获得了兔抗P31/P41多克隆抗体。结果表示,昆明小鼠与BALB/c小鼠Ii分子间存在差异位点,并成功制备了兔抗Ii分子多克隆抗体,为开展靶向性表位疫苗研究奠定基础。     

Developmental changes in mRNA expression in immune-associated cells of intestinal tract of broiler chickens after hatch and by dietary modification

Developmental changes in the immune‐related cells of the gut during the first 2 weeks post hatching were determined in broiler chickens fed a corn–soybean meal (CS) based diet, a semi‐purified diet with high fat (HF) and a high carbohydrate (HC) diet. The expression of CD3 and interferon‐gamma (IFN‐γ) mRNA increased during 3–7 days of age. When the chicks were fed the CS diet, the expression of interleukin‐2 mRNA in the foregut did not increase with age. The expression of iNOS mRNA was lowered at 3 days of age in the fore‐ and mid gut but the expression was constant at the other days of age. TLR‐2 mRNA expression increased in three section of the gut during 3–7 days of age, while TLR‐4 expression increased during 0–3 days of age. Chicks fed the HC diet showed higher interleukin‐2 but lower IFN‐γ mRNA expression than birds fed the CS diet. Feeding the HF diet tended to decrease the expressions determined. These findings suggest that the functional maturation of the T cell does not always correlate with an increase in T cell numbers, maturation of innate immunity may occur earlier than that of T cells and possibly the dietary composition modifies the developmental pattern.     

Myostatin in black Muscovy duck (Cairina moschata): full-length cDNA cloning and age-dependent mRNA expression compared with IGF-I

1. Insulin-like growth factor-I (IGF-I) and myostatin (MSTN) are a pair of critical positive and negative growth regulators. The aim of the current study was to examine the age-dependent and muscle-specific expression of IGF-I and MSTN mRNAs in black Muscovy ducks in order to understand their roles in regulating the postnatal muscle growth of domestic ducks.

2. The full-length cDNA of the black Muscovy duck MSTN gene was cloned and the age-dependent mRNA expression profile was compared with that of the IGF-I mRNA in skeletal muscles.

3. The cDNA sequence of the MSTN gene was 1128 bp in length and encodes 375 amino acids, with more than 94.9% homology with poultry MSTN genes, and 83.0–92.0% homology with that of human and mammals (accession: KR006339.1).

4. The IGF-I and MSTN mRNA expression exhibited opposite trends in age-dependency and in different muscles: IGF-I mRNA level was high in the early postnatal stage and low in the late mature stage, corresponding positively to growth; while the MSTN mRNA was low in the early stage, increased gradually and reached the highest level in mature muscles, and was negatively related to muscle growth. In the breast muscles, IGF-I mRNA was much higher than in the leg muscles; the opposite effect was seen in MSTN mRNA.

5. These data suggest that the relative expression levels of IGF-I and MSTN are essential determinants in the temporal and muscle-specific regulation of postnatal skeletal muscle growth in Muscovy duck and possibly in other poultry species as well.