实验用西藏小型猪CYP3A46基因扩增梯度PCR方法的建立

Establishment of gradient PCR amplification method for CYP3A46 gene of laboratory Tibet minipigs

目的:建立西藏小型猪CYP3A基因梯度PCR扩增方法。方法:以西藏小型猪肝脏总cDNA为模板,基于CYP3A46基因引物,对CYP3A46进行梯度PCR扩增(设计温度梯度55-65℃)。PCR产物进行琼脂糖凝胶电泳,获得特异性好的最佳退火温度。结果:各退火温度均能特异的扩增出1500bp左右的目的条带。结论:本反应体系及各温度梯度均可特异性扩增西藏小型猪CYP3A46基因,为进一步克隆表达奠定了基础。

Objective ： Establishing gradient PCR amplification method for CYP3A46 gene of Tibet minipigs. Methods ：Based on CYP3A46 primers, using total c DNA of Tibet minipig liver as template, CYP3A46 was amplified by gradient PCR, temperature gradients were set as 55-65 ℃. PCR fragments was checked by DNA agarose gel electrophoresis,trying to get the best annealing temperature. Results ： A specific band of 1500 bp corresponding to the target band could be amplified by any temperature from 55 to 65 ℃. Conclusions ： CYP3A46 gene of Tibet minipigs could be specifically amplified by this reaction system and temperature gradients, laying a foundation for the further cloning and expression.