Abstract:

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.