猪Ob-Rb基因的克隆与分析

Cloning of Ob-Rb Gene of Porcine and Its Sequence Analysis

[目的]采用RT-PCR法克隆与分析瘦素受体Ob-RbcDNA序列。[方法]从160日龄初情期的苏姜猪的下丘脑中提取组织总RNA,根据GenBank中猪Ob-RbmRNA全序列设计引物,用反转录-聚合酶链式反应(RT-PCR)进行cDNA扩增,获得1条343 bp的片段,将PCR产物克隆于pGEM-Teasy载体后进行测序分析。[结果]用紫外分光光度计检测所提取的RNA质量,OD260/OD280=1.9,证明所提RNA的质量较好,无降解和蛋白质污染;用1%的琼脂糖检测所提RNA,有3条清晰的条带,分别是28S、18S、5S,28S与18S浓度比约为2∶1,说明所提的RNA的完整性较好。所获Ob-RbcDNA序列用Blast程序与在GenBank中发表的猪Ob-RbcDNA序列(AF092422)比较表明,其同源性为100%,说明所扩增的序列是正确的。[结论]该研究为进一步探索Ob-Rb基因组织表达和作用机理奠定了理论基础。

[ Objective ] The study aimed to clone and analyze the Ob-Rb cDNA sequence of leptin receptor by using RT-PCR method. [ Method] The total RNA of porcine was extracted from hypothalamus of 160-day-old Sujiang pig at puberty. A pair of primers was designed according to the Ob-Rb mRNA whole sequence reported in GenBank previously. A cDNA fragment in length 343 bp was synthesized and amplified by RT-PCR. The PCR product was cloned into pGEM-T easy vector and then sequenced. [ Result] When the extracted RNA was detected by ultraviolet spectrophotometer, OD260/OD280 was 1.9, proving that the extracted RNA had good quality without degradation and protein pollution. When the extracted RNA was detected by 1% agarose, it had 3 clear bands, being 28S, 18S and 5S with the eoncn, ratio of 28S and 18S of 2：1, showing that the extracted RNA had good integrity. The comparison on the obtained Ob-Rb cDNA sequence with reported Ob-Rb eDNA sequence of porcine from GenBank（AF092422） by Blast procedure indicated that its homology was up to 100%, demonstrating that this amplified sequence was correct. [ Conclusion] This study laid the theoretical base for further exploring the tissue expression and action mechanism of Ob-Rb gene.