| 人天然免疫基因BCL10的猪同源物的识别、克隆与初步表达分析 |
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| 引用本文： | 何建锋,张彦明,段会娟,曹金锁,张德礼.人天然免疫基因BCL10的猪同源物的识别、克隆与初步表达分析[J].遗传,2008,30(6):747-754. |
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| 作者姓名： | 何建锋 张彦明 段会娟 曹金锁 张德礼 |
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| 作者单位： | 西北农林科技大学动物医学院预防兽医学系病毒免疫与生物信息研究室, 杨凌 712100 |
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| 基金项目： | 西北农林科技大学引进人才基金，国家自然科学基金 |
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| 摘 要： | 采用电子克隆方法克隆到大小为925 bp的人天然免疫蛋白BCL10的猪同源基因完整cDNA序列(GenBank登录号：EU088132), 并利用RT-PCR方法从猪的全血中扩增出包含702 bp的完整开放读码框架(ORF)的cDNA片段。经核酸测序, 证明与电子克隆结果相符。利用NCBI BLAST分析该cDNA包含3个大小为57 bp、289 bp和356 bp的外显子, 并且定位于猪的4号染色体上。采用半定量PCR技术检测基础水平猪各组织BCL10基因mRNA表达丰度, 并将该基因构建到带有绿色标签的真核表达载体pEGFP-C1中, 采用脂质体转染法将该基因转入PK-15细胞, 通过绿色荧光标记和RT-PCR方法检测实验组的BCL10蛋白表达。研究结果表明, BCL10基因mRNA在脾脏中表达最高; 胸腺、大脑和淋巴结表达次之, 而肝脏只有微量表达, 肾脏没有检测到表达; 同时BCL10基因在PK-15细胞中得到了有效表达。
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| 关 键 词： | 真核表达 表达分析 克隆 BCL10基因 |
| 收稿时间： | 2007-12-25 |
| 修稿时间： | 2007年12月25 |
In silico identification, molecular cloning and verification of a novel pig gene homologous to human BCL10 of in-nate immunity and its preliminary expression profiles in pigs |
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| HE Jian-Feng,ZHANG Yan-Ming,DUAN Hui-Juan,CAO Jin-Suo,ZHANG De-Li.In silico identification, molecular cloning and verification of a novel pig gene homologous to human BCL10 of in-nate immunity and its preliminary expression profiles in pigs[J].Hereditas,2008,30(6):747-754. |
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| Authors: | HE Jian-Feng ZHANG Yan-Ming DUAN Hui-Juan CAO Jin-Suo ZHANG De-Li |
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| Affiliation: | Division of Virology, Immunology & Bioinformatics, Department of Preventive Veterinary Medicine, College of Veterinary Medicine , Northwest A & F University, Yangling 712100, China |
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| Abstract: | We identified and characterized a novel swine gene Bcl10 with GenBank (Accession No. EU088132) which was homologous to human BCL10 (B-cell CLL/lymphoma 10) gene. The full-length cDNA of 925 bp for swine BCL10 was in silico cloned, and then its ORF of 702 bp coding 233 amino acid residues was verified by RT-PCR and DNA sequencing. NCBI BLAST assay indicated that this cDNA contained three extrons with a length of 57, 289 and 356 bp respectively, and it was located on the chromosome 4 of pig genome. Using semi-quantitative PCR, preliminary expression profiles of swine BCL10 were verified in different swine tissues. The expression of swine BCL10 was verified by GFP marker and RT-PCR assay. We found that, BCL10 expressed in high level in swine spleen, and with a modest level in thymus, brain and lymph node; low level mRNA was expressed in liver and not detectable level in kidney. The swine BCL10 gene was cloned to the GFP-containing eukaryotic expression vector pEGFP-C1 and transfected to PK-15 cell line by lipofectin. BCL10 was ex-pressed effectively in PK-15 cells. In summary, we cloned a novel swine BCL10 gene, and investigated its expression char-acters. This will be the fundament of the future research on its function. |
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