猪LFA-1和CTLA-4实时荧光定量PCR检测方法的建立

摘要：【研究目的】建立猪LFA-1和CTLA-4实时荧光定量PCR检测方法。【方法】根据GenBank中核苷酸序列设计猪淋巴细胞功能相关抗原-1(LFA-1)和细胞毒性T淋巴细胞相关抗原4(CTLA-4)的特异性引物，经RT-PCR扩增、目的片段与载体连接转化以及重组质粒的鉴定，并对重组质粒标准品和样品cDNA进行检测，构建LFA-1和CTLA-4荧光定量RT-PCR标准曲线。【结果】以1×109~1×102拷贝/μL不同稀释水平的标准品进行荧光定量PCR扩增后，统计学分析显示标准品浓度的对数与Ct值之间存在良好的线性关系（LFA-1：RSq =0.992；CTLA-4：RSq =0.994）；样品检测显示LFA-1与CTLA-4引物的扩增曲线图和熔解曲线图的特异性。【结论】所构建的质粒标准品具有线性关系好、重复性好、敏感性高等特点，以及其引物在样品检测中的可应用性，为分析猪免疫细胞在感染病毒前后LFA-1和CTLA-4表达的变化以及评估猪体免疫功能，提供必要的技术平台。

Development of Real-time Fluorescent Quantitative PCR for Detection of Porcine LFA-1 and CTLA-4

Abstract: 【OBJECTIVE】To construct the detection method of Real- time Fluorescent Quantitative PCR (Real-time FQ-PCR) for LFA-1/CTLA-4 partial genes. 【METHOD】The nucleotide sequence from GenBank was used for design of specific primers for LFA-1 and CTLA-4, and the standard curves of fluorescent quantitative RT-PCR for LFA-1 and CTLA-4 were established through amplification of RT-PCR, ligation of target genes and vector and transformation, identification of the recombinant plasmids and detection of cDNA sample. 【RESULTS】Statistic analysis on the basis of Real-time FQ-PCR amplification of different standard plasmids for serial dilutions of 1×109~1×102 copies /μL showed that there is a good linear relationship between Ct value and the logarithm of their concentrations（LFA-1: RSq =0.992; CTLA-4: RSq =0.994）, and that specificity existed in the amplification curves and the melting curves for LFA-1 and CTLA-4 primers. 【CONCLUSION】 The constructed plasmid samples are of high linearity, reproducibility and sensitivity, which, together with the application of the primers in the sample detections, lay a necessary technologic platform for analyzing the expression variations of LFA-1 and CTLA-4 in porcine immune cells and estimating porcine immune function.