Molecular cloning and sequencing of the silver chimaera (Chimaera phantasma) interleukin-8 cDNA
Molecular cloning and sequencing of the silver chimaera (Chimaera phantasma) interleukin-8 cDNA
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DOI：
10.1016/s1050-4648(02)00158-4
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发表时间：
2003-09-01
影响因子：
4.7
通讯作者：
Nakanishi, T
中科院分区：
文献类型：
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作者：
Inoue, Y;Endo, M;Nakanishi, T
最近，炎症细胞因子如IL-1β已在小斑点猫鲨（Scyliorhinus canicula）[12]和带状角鲨（Triakis scyllia）（AB 074142）中克隆，IL-8已在带状角鲨中克隆[13]。七鳃鳗（Lampetra fluviatilis）[14]、比目鱼[15]和虹鳟鱼[16]的IL-8也有报道。带状角鲨IL-8含有四个半胱氨酸残基，这是CXC亚家族细胞因子的特征，并且缺乏存在于七鳃鳗、比目鱼和虹鳟鱼中的ELR基序。为了研究IL-8的多样性是否存在于整个软骨鱼纲中，我们分析了银嵌合体（Chimaera phantasma）的IL-8基因，并与其他脊椎动物的IL-8基因进行了比较。phantasma）获自相模湾（神奈川，日本）。收集脾细胞并用含有10%胎牛血清（EQUITECH-BIO）、3 mM β-谷氨酰胺（WAKO）、350 mM尿素（和子）、100 IU ml-1青霉素（MEIJI）和100 µg ml-1链霉素（MEIJI）的RPMI 1640（NISSUI）洗涤两次。将脾细胞悬浮于上述含有100μ g ml-1脂多糖（LPS：大肠杆菌026：B6）（Sigma）和300 ng ml-1佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）（Sigma）的培养基中，调节至1107个细胞ml-1，然后在24孔微量培养板中于20 ℃孵育4小时，然后提取RNA。根据制造商的说明书，使用Trizol试剂（Gibco BRL）从脾细胞提取总RNA。使用SMARTe PCR cDNA合成试剂盒（Clontech）按照制造商的方案合成cDNA。用于扩增银嵌合体IL-8基因的引物是：5 #-AAC AAC GCA GAG TAC GCG GG-3 #（5 #-F）和5 #-TCA CCC AGG SGG CWT CAG G-3 #（S= G或C; W= A或T）（IL-8-R），并预期得到约300-bp的片段，其包括约50%的IL-8编码序列。5 #-F引物由cDNA合成引物改造而成，IL-8-R引物选自七鳃鳗[14]和斑角鲨[13] IL-8基因编码序列的高度保守区。通过在1.5%琼脂糖凝胶中电泳分离PCR产物，然后通过凝胶提取（Qiagen）部分纯化。为了获得完整的IL-8序列，使用基于以上确定的部分IL-8序列的引物进行3#-RACE。用IL-8正向引物（IL-8-F）和cDNA合成引物（3#-end-R）分别作为3#-端特异性反向引物5#-TCA TTG CCA CGC TTA AAA GC-3#和5#-AAC AAC GCA GAG TAC TTT TT-3#进行PCR扩增。用这些引物，扩增出约600 bp的片段。根据制造商的说明书将扩增的片段亚克隆到pGEM-T Easy载体（Promega）中。从转化的MOSBlue感受态细胞中纯化选择的克隆。根据制造商的说明书，在ABI PRISM 3100遗传分析仪（Applied Biosystems）上使用T7和SP 6引物和BigDyee Terminator 3.0版循环测序试剂盒（Applied Biosystems）测序的几个单独克隆的两条链上确认了银嵌合体IL-8的核苷酸序列。使用Genbank和SWISS-PROT数据库结合Genbank和SWISS-PROT第5版（Genetic Information Processing Software）程序进行预测的蛋白质序列和多重比对，以与其它已知基因序列进行比较。系统发育树是通过相邻连接方法[17]生成的，并使用自举1000方法生成。
Recently, inflammatory cytokines such as IL-1β have been cloned in the small spotted catshark (Scyliorhinus canicula)[12] and the banded dogfish (Triakis scyllia)(AB074142), and IL-8 was cloned in the banded dogfish [13]. IL-8 of the lamprey (Lampetra fluviatilis)[14], the flounder [15] and the rainbow trout [16] have also been reported. The banded dogfish IL-8 contained four cysteine residues, which are characteristic of CXC subfamily cytokines and lacked on ELR motif, which is present in the lamprey, flounder and rainbow trout. To investigate whether the diversity of IL-8 is present in the entire class chondrichthyes, we analyzed an IL-8 gene of the silver chimaera (Chimaera phantasma) as compared with other vertebrate IL-8 genes.Live specimens of silver chimaera (C. phantasma) were obtained from the bay of Sagami (Kanagawa, Japan). Splenocytes were collected and washed twice with RPMI 1640 (NISSUI) containing 10% fetal bovine serum (EQUITECH-BIO), 3 mM-glutamine (WAKO), 350 mM urea (WAKO), 100 IU ml 1 penicillin (MEIJI), and 100 µg ml 1 streptomycin (MEIJI). Splenocytes were suspended in the above medium containing 100µgml 1 lipopolysaccharide (LPS: Escherichia coli 026: B6)(Sigma) and 300 ng ml 1 phorbol 12-myristate 13-acetate (PMA)(Sigma), adjusted to 1107 cells ml 1 and then incubated in 24-well microplates for 4 h at 20 (C before RNA extraction. Total RNA was extracted from the splenocytes using Trizol reagent (Gibco BRL) according to the manufacturers’ instructions. cDNA was synthesized using a SMARTe PCR cDNA Synthesis Kit (Clontech) following the manufacturers’ protocol. The primers used to amplify the silver chimaera IL-8 gene were: 5#-AAC AAC GCA GAG TAC GCG GG-3#(5#-F) and 5#-TCA CCC AGG SGG CWT CAG G-3#(S= G or C; W= A or T)(IL-8-R) and expected to give an approximate 300-bp fragment, which included about 50% of the coding sequence of IL-8. The 5#-F primer was modified from the cDNA synthesizing primer and the IL-8-R primer was chosen from the highly conserved region of the coding sequences of the lamprey [14] and banded dogfish [13] IL-8 genes. The PCR products were separated by electrophoresis in a 1.5% agarose gel and then partially purified by gel extraction (Qiagen). To obtain the complete IL-8 sequence, 3#-RACE was performed using primers based on the partial IL-8 sequence determined above. The cDNA was amplified with PCR using the first IL-8 forward primer (IL-8-F) and modified cDNA synthesizing primer (3#-end-R) as 3#-end specific reverse primer 5#-TCA TTG CCA CGC TTA AAA GC-3# and 5#-AAC AAC GCA GAG TAC TTT TT-3#, respectively. With these primers, fragments of approximately 600 bp were amplified. The amplified fragments were subcloned into pGEM-T Easy vector (Promega) according to the manufacturers’ instructions. The selected clones were purified from transformed MOSBlue competent cells. The nucleotide sequence of the silver chimaera IL-8 was confirmed on both strands of several separate clones that were sequenced on an ABI PRISM 3100 genetic analyzer (Applied Biosystems) using T7 and SP6 primers and a BigDyee Terminator version 3.0 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturers’ instructions. The predicted protein sequence and multiple alignments were carried out using the- version 5 (Genetic Information Processing Software) program in combination with the Genbank and SWISS-PROT databases for comparison with other known gene sequences. The phylogenetic tree was generated by the neighbor-joining method [17] and bootstrapped 1000 using …