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目的:扩增TLR7主要编码区外显子3(exon 3)的全长基因片段并进行基因多态性分析,筛选健康人群中TLR7基因的主要SNP位点。方法:采用酚-氯仿抽提方法从28例健康女性血样中提取基因组DNA,采用长片段扩增方法分别扩增TLR7 exon 3区的3个片段,测序、拼接后进行多态性分析。结果:采用LA-Taq酶体系成功扩增TLR7 exon 3区基因片段;经与Genbank数据库中TLR7参考序列比较,我国女性TLR7基因序列高度保守,仅出现了4个点突变,并发现1个SNP位点RS3853839,表现为GG、CG和CC三种基因型。结论:建立了TLR7编码区基因扩增方法,筛选到1个TLR7 SNP位点RS3853839,可为分析TLR7多态性与多种病毒感染性疾病的关系提供参考。
OBJECTIVE: To amplify the full-length gene fragment of exon 3 in the major coding region of TLR7 and analyze the gene polymorphism to screen the major SNP loci of TLR7 gene in healthy population. Methods: Genomic DNA was extracted from blood samples of 28 healthy women by phenol - chloroform extraction method. Three fragments of TLR7 exon 3 region were amplified by long fragment amplification method. Sequencing and splicing were performed to analyze the polymorphism. Results: The gene fragment of TLR7 exon 3 was successfully amplified by LA-Taq enzyme system. Compared with the TLR7 reference sequence in Genbank database, the TLR7 gene sequence of Chinese women was highly conserved. Only 4 point mutations were found and one SNP locus Point RS3853839, manifested as GG, CG and CC three genotypes. Conclusion: The method of gene amplification of TLR7 coding region was established. One TLR7 SNP locus RS3853839 was screened, which could provide a reference for the analysis of the relationship between TLR7 polymorphism and various viral infectious diseases.