Cloning and sequence analysis of llama cytokines related to cell-mediated immunity

In order to characterize the T helper 1 (Th1) cytokines of llama, we have cloned several llama cytokine genes and compared them to those of other mammalian species. The cDNAs encoding for interleukin (IL)-2, interferon (IFN)gamma, IL-12p35 and IL-12p40 were amplified using specific primers designed from reported sequences of bovine cytokine genes. The cDNAs for llama IL-2, IFN-gamma, IL-12 p35 and IL-12p40 were found to be 465, 501, 669 or 993 bp in length, with open reading frames encoding 154, 166, 222 or 330 amino acids, respectively. Homology analyses of nucleotide and deduced amino sequences of llama IL-2, IFN-gamma, IL-12p35 and IL-12p40 and phylogenetic analysis based on their nucleotide sequences indicated the close relationship in these cytokine genes between llama and eutherian mammalian order Artiodactyla, which includes pig and cattle.     

Complete cDNA sequences and phylogenetic analyses of the Th1 and Th2 cytokines of the bactrian camel (Camelus bactrianus)

The complementary DNAs of the Th1 (IL-2, IL-12p35, and IFN-gamma) and Th2 (IL-4, IL-10 and IL-13) cytokine genes of the bactrian camel (Camelus bactrianus) were cloned, sequenced, and analyzed. IL-2, IL-4, IL-10, IL-12p35, IL-13, and IFN-gamma were found to have 465, 402, 537, 669, 411, and 501 bp length open reading frames with 154, 133, 178, 222, 136, and 166 amino acid encodings, respectively. The homology ranged from 58.8% to 100% between the nucleotide sequences of the camel cytokine genes and the published sequences of other mammalian genes, including the llama, pig, cow, horse, human, and mouse. The cDNA had highest homology with orders Artiodactyla (pigs and cattle) and Perissodactyla (horses), especially to the recently cloned llama sequences.     

Molecular cloning and phylogenetic analysis of inflammatory cytokines of Camelidae (llama and camel)

We cloned, sequenced and analyzed the cDNAs encoding Camelidae inflammatory cytokines, including llama (lama glama) interleukin (IL)-1alpha, IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha and camel (Camelus bactrianus) IL-6 and TNF-alpha. The similarity levels of the deduced amino acid sequences of IL-1alpha, IL-1beta, IL-6 and TNF-alpha from llama (camel) to those from other mammalian species, ranged from 60.7% to 87.7%, 52.8% to 75.3%, 41.4% to 98.6%, and 72.9% to 99.6%, respectively. Phylogenetic analyses based on nucleic acid sequences showed that llama IL-1alpha, IL-1beta, IL-6 and TNF-alpha were more closely related to those of camel, pig, cattle, sheep and horse than to those of human, dog, cat, mouse and rat.     

Cloning and sequence analysis of IL-2, IL-4 and IFN-γ from Indian Dromedary camels (Camelus dromedarius)

The cDNAs of three cytokines, viz., IL-2, IL-4 and IFN-γ from Dromedary camels were amplified by PCR using Bactrian camel sequences and subsequently cloned for sequence analysis. Relationship based on amino acid sequences revealed that Dromedary camel IL-2 shared 99.5% and 99.3% identity at the nucleotide and amino acid levels with Bactrian camel IL-2. In the case of IL-4, the identity of Dromedary camel was 99.7% and 99.2% at the nucleotide and amino acid levels, respectively with that of Bactrian camel. The Dromedary camel IFN-γ shared 100% identity both at nucleotide and amino acid levels with Bactrian camel IFN-γ. Phylogenetic analysis based on amino acid sequences indicated the close relationship in these cytokine genes between the Dromedary camel and other camelids.     

Molecular cloning and sequence analysis of interferon-gamma and interleukin-6 from Tibetan macaque (Macaca thibetana)

The cloning and sequence analysis of Tibetan macaque IFN-(gamma) and the IL-6 cDNAs are described. The Tibetan macaque IFN-gamma and IL-6 cDNAs were found to be 498 and 639 bp in length, with open reading frames encoding 165 and 212 amino acids, respectively. Homology analyses indicated that the identity levels of nucleotide and deduced amino acid sequences of IFN-gamma among primates ranged from 93.4 to 99.2%, and 87.3 to 99.4%, respectively, and that of IL-6 ranged from 92.6 to 99.8%, and 85.4 to 99.5%, respectively. Phylogenetic analysis based on amino acid sequences showed that the Tibetan macaque is most closely related to Old World monkeys, as compared to Hominoidea and New World monkeys. These findings provide insights into the evolution of primate IFN-gamma and IL-6 and additional valuable information regarding amino acid residues essential for their biological activity.     

Cloning and phylogenetic analysis of Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) from Indian dromedaries (Camelus dromedarius)

The cDNAs of two proinflammatory cytokines viz., IL-6 and TNF-α from dromedarian camels were amplified by PCR using bactrian camel sequences and subsequently cloned for sequence analysis. Relationship based on amino acid revealed that dromedarian camel IL-6 shared 99.5% identity both at nucleotide and amino acid level with bactrian camel IL-6 and in case of TNF-α, the identity of dromedarian camel was 99.4% and 99.1% at nucleotide and amino acid level, respectively with that of bactrian camel. Phylogenetic analysis based on their amino acid sequences indicated the close relationship in these cytokine genes between dromedarian camel and other members of camelids.     

Molecular cloning of feline interferon-gamma-inducing factor (interleukin-18) and its expression in various tissues

Interleukin-18 (IL-18) is a cytokine with potent interferon-gamma-inducing activity, and plays an important biologic role in the enhancement of the activity of natural killer cells and cytotoxic T-lymphocytes. In this study, feline IL-18 cDNA was cloned and characterized to establish a basis for the prospective cytokine therapy in small animal practice. The nucleotide sequence of feline IL-18 cDNA obtained in this study was 712bp long and contained its entire open reading frame encoding 192 amino acid residues. The predicted amino acid sequence of feline IL-18 cDNA showed 77.2, 84.8, 60.2 and 62.6% similarity with those of human, dog, rat and mouse counterparts, respectively. The feline IL-18 cDNA included a putative cleavage site of IL-1beta-converting enzyme (ICE) and IL-1 signature-like sequences identified in human and mouse IL-18 cDNAs. Expression of IL-18 mRNA was detected in various tissues including spleen, liver and cerebrum in the cat.     

Development of reagents to study the turkey's immune response: cloning and characterisation of two turkey cytokines, interleukin (IL)-10 and IL-13

The cDNAs of two turkey cytokines, interleukin (IL)-10 and IL-13, were cloned using oligonucleotide primers designed from their chicken orthologues. The coding regions of the chicken and turkey genes are highly conserved, with IL-10 and IL-13 exhibiting 94.1% and 90% nucleotide and 92% and 79.9% amino acid identity respectively. Both showed consistent mRNA expression in turkey lymphoid and gut tissues. Expression in non-lymphoid tissues was more variable but generally highest in the skin and trachea. Recombinant turkey IL-10 was expressed and bioactivity demonstrated by inhibition of IFN-γ synthesis from activated splenocytes. Chicken and turkey IL-10 cross-reacted in functional assays.     

广西沼泽型水牛IL-4和IL-5基因的克隆及其序列分析

从刀豆素A(Concanavalin A,ConA)刺激的广西沼泽型成年水牛外周血单个核细胞(Peripheralblood mononuclear cells,PBMCs)中提取总RNA,用逆转录聚合酶链式反应(RT-PCR)方法扩增出白细胞介素-4(Interleukin-4,IL-4)和白细胞介素-5(IL-5)基因,分别克隆到pMD18-T载体上,进行序列分析。结果表明,从培养9 h、17 h的单个核细胞中扩增得到的IL-4基因在核苷酸水平上与GenBank上登录的印度水牛IL-4 cDNA序列同源性为98.8%,与牛、绵羊、猪和马的同源性分别为99.3%、94.1%、84.8%和80.6%;氨基酸水平的同源性分别为96.3%、98.5%、90.4%、78.4%和59.6%。从培养12 h的单个核细胞中扩增得到的IL-5基因在核苷酸水平上与GenBank上登录的牛、绵羊、猪和马IL-5 cDNA序列同源性分别是99.0%、96.5%、89.6%和89.1%;氨基酸水平的同源性分别为97.8%、96.2%、85.9%和86.7%。本研究为进一步了解水牛IL-4和IL-5的结构和水牛免疫学特性奠定了基础。     

Molecular cloning of canine IL-13 receptor alpha chain (alpha1 and alpha2) cDNAs and detection of corresponding mRNAs in canine tissues

This communication reports the cloning of cDNAs encoding two canine IL-13 receptor alpha chains (caIL-13Ralpha1 and caIL-13Ralpha2). As described for the members of type-I cytokine receptors, both caIL-13Ralpha1 and caIL-13Ralpha2 were found to contain the highly conserved motifs, such as cysteine and tryptophan residues in their N-terminal portion and the WSXWS at C-terminus. The isolated caIL-13Ralpha1 cDNA contains 1547 nucleotides with an open reading frame that encodes 405 amino acid residues. Canine IL-13Ralpha1 is 82.0 and 69.3% identical to human and mouse IL-13Ralpha1s, respectively, at the amino acid level. Canine IL-13Ralpha1 has an almost identical cytoplasmic domain to its human and mouse counterparts. The isolated caIL-13Ralpha2 cDNA contains 1454 nucleotides and encodes an open reading frame of 386 amino acid residues. Canine IL-13Ralpha2 is 62.6 and 47.5% identical to its human and mouse counterparts, respectively, at the amino acid level. Using RT-PCR with caIL-13Ralpha1 and caIL-13Ralpha2 specific primers, mRNAs of caIL-13Ralpha1 and caIL-13Ralpha2 were detected in most dog tissues. In addition, RT-PCR detected caIL-13Ralpha1 mRNA in one of two canine mastocytoma (C2 but not Br) cell lines and in a canine macrophage-derived cell line (DH82). CaIL-13Ralpha2 mRNA was detected in all three canine cell lines.     

Molecular cloning and sequences of interleukin-10 in the Djungarian (Phodopus sungorus), Chinese (Cricetulus griseus), and Syrian (Mesocricetus auratus) hamster

Interleukin 10 (IL-10) genes of Djungarian, Chinese, and Syrian hamsters were cloned. The clones of IL-10 consisted of 537 bp nucleotides and 178 amino acids in full length, and the nucleotide and amino acid sequences exhibited a high degree of homology with those of the mouse and human. Since the number and position of signal sequences, N-glycosylations and cysteine sites in the IL-10 amino acid sequences of the hamsters were the same as those of the mouse, we suggest that the IL-10 molecular structures of the hamster are closer to that of the mouse than human.     

广西巴马小型猪IL-4和IL-10基因的克隆和序列分析

为研究猪白细胞介素4（IL-4）和白细胞介素10（IL-10）在机体免疫应答中的作用,从ConA刺激的巴马小型猪的外周血淋巴细胞提取总RNA,用RT-PCR方法扩增出IL-4和IL-10基因,将其分别克隆到PMD18-T 载体上,进行序列分析.结果表明,克隆的IL-4和IL-10基因的核苷酸和氨基酸序列与GenBank上登录的其它猪种的IL-4和IL-10基因的核苷酸和氨基酸序列同源性分别为98.8%～100%、97.0%～99.3%和99.2%、97.7%.     

白来航鸡IL-18和GM-CSF基因克隆与序列分析

根据GenBank中鸡白细胞介素18(IL-18)和粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因的序列设计2对特异引物,以白来航鸡脾淋巴细胞总RNA为模板进行RT-PCR扩增,克隆白来航鸡IL-18和GM-CSF工基因并进行序列分析.测序结果表明,白来航鸡IL-18基因开放阅读框为597 bp,编码199个氨基酸;序列分析表明,所获得的白来航鸡IL-18基因与GenBank中鸡IL-18的核苷酸同源性为99.0%～99.8%,氨基酸同源性为97.5%～99.5%.白来航鸡GM-CSF基因开放阅读框为435 bp,编码145个氨基酸;序列分析表明,所获得的白来航鸡GM-CSF基因与GenBank鸡GM-CSF的核苷酸同源性为99.3%～100%,氨基酸同源性为99.3%～100%,生物信息学分析表明,IL-18与GM-CSF基因编码的蛋白具有亲水性,都有很强的抗原性,IL-18共有2个潜在的N-糖基化位点,可能存在4个蛋白激酶C磷酸化位点,存在3个酪蛋白激酶Ⅱ磷酸化位点;GM-CSF共有1个潜在的N-糖基化位点,可能存在1个蛋白激酶C磷酸化位点,存在4个酪蛋白激酶Ⅱ磷酸化位点.     

禽呼肠病毒P10、P17非结构蛋白基因的克隆及序列分析

根据GenBank上的禽呼肠病毒(ARV)S1基因序列,设计并合成了一对跨越P10和P17非结构蛋白基因的特异性引物,对13个ARV毒株进行RT-PCR扩增、克隆及序列测定。结果显示,13个ARV毒株的P10蛋白基因ORF全长均为297bp,编码98个氨基酸;P17蛋白基因ORF全长为441bp,编码146个氨基酸。这13个ARV毒株P10、P17蛋白基因核苷酸同源性分别在96.6%~100%和95.2%~99.3%之间,推导的氨基酸同源性分别在98.2%~100%和91.9%~99.0%之间。将这13个ARV毒株与GenBank上其他正呼肠病毒毒株,包括番鸭株(DRV)和飞狐上分离到的内尔森海湾病毒(NelsonBayvirus,NBV)及两个澳洲分离株(ARM-1和SOM-4)进行同源性比较和遗传进化树分析,结果表明,呼肠病毒有地域和种类的差别。     

Molecular cloning and phylogenetic analysis of inflammatory cytokines of the ferret (Mustela putorius furo)

The present study determined the cDNA and deduced amino acid sequences of ferret (Mustela putorius furo) inflammatory cytokines, interferon (IFN)-gamma, interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis factor (TNF)-alpha. The homologies of the nucleotide sequences of IFN-gamma, IL-1beta, IL-6, IL-8 and TNF-alpha of the ferret to those from other mammalian species ranged from 64.3-92.9%, 73.0-83.9, 58.1-84.8%, 58.1-89.7% and 79.0-95.0%, respectively. As distinctive amino acid residues constituting various motifs and ligand-binding sites and cysteine residues were highly conserved in ferret inflammatory cytokine proteins, ferret cytokines may have fundamentally similar functions to those of other mammals. Phylogenetic analyses based on the deduced amino acid sequences revealed that all ferret inflammatory cytokines were more closely related to those of the Carnivora order, specifically dog and cat, than to other species.     

罗曼鸡胚脾细胞白介素-18基因的克隆与序列分析

根据国外已发表的鸡白细胞介素 18(IL- 18) c DNA基因序列设计了 1对特异性引物 ,应用 RT- PCR技术 ,从鸡新城疫 系病毒接种 4 8h左右的罗曼鸡胚脾细胞中扩增出鸡 IL - 18全基因 ,并进行了序列测定。结果表明 ,扩增片段全长 5 94 bp,共编码 198个氨基酸的前体蛋白 ,其中含有表达完整功能蛋白所必需的起始密码子和终止密码子。该序列与国外报道的鸡 IL - 18全基因核苷酸序列及推导的氨基酸序列的同源性分别为 99.8%和 10 0 % ;序列中编码成熟蛋白的这段基因与国内报道的源自白来航鸡编码 IL- 18成熟蛋白的基因核苷酸序列及推导的氨基酸序列的同源性分别为 99.8%和 99.4 %。本研究为鸡 IL - 18的扩增及其他细胞因子的扩增提供了一种简便易行的新方法 ,为进一步研究IL- 18基因的结构、功能、表达及表达产物的应用奠定了良好基础