内毒素受体TLR4基因多态性“Asp299Gly”的分型检测

Genotype analysis of lipopolysaccharide receptor TLR4 polymorphism “Asp299Gly”

目的 :建立一种用于内毒素受体 TL R4基因多态性“Asp2 99Gly”检测的方法 ,并在中国汉族人中进行该位点的检测。 方法 :以等位基因特异性 PCR原理为基础 ,在两个等位基因特异性引物的 3′端第 3位引入不配对碱基以增加特异性 ,与共同的下游引物分别构成两个 PCR反应体系。采用 PCR定点突变方法得到含和不含“Asp2 99Gly”突变位点的 DNA片段插入质粒作为标准模板 ,用以验证这一方法的鉴别效果 ,并采用 PCR- RFL P分析和测序加以验证。 结果 :该方法用于“Asp2 99Gly”检测的结果 ,与 PCR- RFL P分析的结果及测序的结果完全相符 ,采用我们建立的已知基因型的突变型和野生型重组质粒作模板验证 ,结果也完全相符。采用该方法共检测 92份汉族人群 DNA样本 ,未发现“Asp2 99Gly”的携带者。结论 :以等位基因特异性 PCR原理为基础的检测方法 ,是一种可用于 TL R4基因多态性“Asp2 99Gly”检测的简便、快速、准确、适合于一般实验室的好方法。中国汉族人群携带这一突变的基因频率可能很低。

Objective:To establish a simple,accurate and practical method for TLR4 polymorphism “Asp299Gly” genotyping based on allele specific PCR (ASPCR),and to assay this polymorphism in Chinese people. Methods:Two allele specific primers,with a mismatch introduced at the third 3′-termminal base,were both included in PCR system. The recombinant plasmid carrying TLR4 polymorphism “Asp299Gly” or wild type TLR4 gene segment constructed through PCR site-directed mutagenesis were used as template to verify the specificity of “Asp299Gly”. PCR-RFLP was also used to verify the method. Results: The results assayed by ASPCR were the same to those obtained with PCR-RFLP,and were confirmed by the recombinant plasmid carrying TLR4 polymorphism “Asp299Gly” or wild type TLR4 gene segment. The “Asp299Gly” allele of the TLR4 gene was not detected in any of the 92 Chinese DNA specimens with this method. Conclusion:ASPCR is a rapid and simple technique for TLR4 polymorphism “Asp299Gly” genotyping and suitable for routine laboratories and large-scale population studies. The “Asp299Gly” allele of the TLR4 gene may be very rare in Chinese.