CAJ | 학술논문

[目的] 采用RT-PCR法克隆与分析瘦素受体Ob-Rb cDNA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-Rb mRNA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-T easy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-Rb cDNA序列用Blast程序与在GenBank中发表的猪Ob-Rb cDNA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.
[목적] 채용RT-PCR법극륭여분석수소수체Ob-Rb cDNA서렬. [방법]종160일령초정기적소강저적하구뇌중제취조직총RNA, 근거GenBank중저Ob-Rb mRNA전서렬설계인물, 용반전록-취합매련식반응(RT-PCR)진행cDNA확증, 획득1조343 bp적편단, 장PCR산물극륭우pGEM-T easy재체후진행측서분석.[결과] 용자외분광광도계검측소제취적RNA질량,OD260/OD280=1.9,증명소제RNA적질량교호,무강해화단백질오염;용1%적경지당검측소제RNA,유3조청석적조대,분별시28S、18S、5S,28S여18S농도비약위2: 1,설명소제적RNA적완정성교호.소획Ob-Rb cDNA서렬용Blast정서여재GenBank중발표적저Ob-Rb cDNA서렬(AF092422)비교표명,기동원성위100%,설명소확증적서렬시정학적.[결론]해연구위진일보탐색Ob-Rb기인조직표체화작용궤리전정료이론기출.
[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 concn. 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 cDNA 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.