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目的:对TRAF2的新剪切体进行鉴定与表达分析。方法:以人脑库为模板,利用PCR扩增,电泳确定新剪切体的存在。再提取不同细胞与组织的总RNA,逆转录得到cD-NA后作为模板对两种不同剪切体的表达进行比较分析。结果:利用P1与P2引物对从人脑库扩增出1条约1 500 bp的条带,而利用P3与P4引物对则得到2条明显电泳带,通过测序表明其中1条包含TRAF2的第6外显子,另1条缺失了TRAF2的第6外显子。通过对新剪切体TRAF2Δ6与全长剪切体TRAF2的表达量比较表明在T47D中全长剪切体表达占优势;而在Hep3B、GC-1与MCF7中TRATΔ6剪切体表达占优势;在HepG2、HBL100、A549与HeLa细胞中未发现两种剪切体的明显表达;在PANCI与SW480中两种剪切体表达量相近。在胎大脑与一级神经胶质瘤中TRAF2Δ6表达占优,而二级与三级神经胶质瘤中则全长剪切体TRAF2表达占优势。结论:人体TRAF2基因除可表达全长TRAF2 mRNA外,还可通过可变剪切表达缺失第6外显子的TRAF2Δ6 mRNA,而且这两种剪切体的表达存在细胞与组织特异性。
OBJECTIVE: To identify and express TRAF2 in vitro. Methods: The human brain bank was used as a template to determine the existence of the new splicing by PCR amplification and electrophoresis. Total RNA was extracted from different cells and tissues. The expression of cD-NA was reverse transcribed and used as a template to analyze the expression of two different kinds of splices. Results: One 1 500 bp band was amplified from the human brain bank by using P1 and P2 primers. Two clear electrophoresis bands were obtained by using P3 and P4 primer pairs. Sequencing showed that one of the 6 bands containing TRAF2 Exon, the other one is missing exon 6 of TRAF2. The comparison between the expression of TRAF2Δ6 and the full-length TRAF2 showed that the expression of full-length was dominant in T47D, while the expression of TRATΔ6 was predominant in Hep3B, GC-1 and MCF7. No obvious expression of the two kinds of splicing was found in HepG2, HBL100, A549 and HeLa cells. The expression of two kinds of splicing was similar in PANCI and SW480. TRAF2Δ6 expression predominates in fetal brain and primary glioma while TRAF2 expression predominates in secondary and tertiary gliomas. CONCLUSION: In addition to the expression of full-length TRAF2 mRNA, human TRAF2 gene can also express TRAF2Δ6 mRNA of exon 6 by deletional mutagenesis. Furthermore, the expression of TRAF2 gene in these two kinds of spliceosomes is cell-and tissue-specific.