论文部分内容阅读
[目的]为研究以鼠兔LDH-C4为靶蛋白的节育型鼠药奠定基础。[方法]黑唇鼠兔属于兔形目,鼠兔属,在GenBank数据库里没有兔形目物种LDH-C基因的相关序列,故采用设计简并引物的方法进行克隆。设计的3条简并引物序列为:D-Ps:5′-atgtcmacygtcaaggagcagct-3′:D-Pa1:5′-gcagayacabtgtaatcttttcc-3′;D-Pa2:5′-ttacarccacttccratyac-3′。用反转录生成的cDNA作为模板,采用巢式PCR法克隆LDH-C基因的EST,再根据EST序列设计了2条基因特异引物,采用3′RACE技术克隆LDH-C基因的3′端,2条基因特异引物为Pika-Ps1:5-cttgcccttgttgatgttgcaga-3和Pika-Ps2:5-ggatcttcaacatggcagtct-3。核酸序列的分析、拼接和相似性搜索采用Vector NTI Suite 9软件,系统发生树的构建用Mega 4.0软件,采用邻接法。[结果]黑唇鼠兔的LDH-C基因的cDNA全长1 498 bp,其中ORF长996 bp,编码332个氨基酸,3′UTR长486 bp。ORF序列比对显示LDH-C基因在大的分类单位上均很保守。系统树分析显示黑唇鼠兔的LDH-C基因和灵长类及偶蹄目的遗传距离较近,和啮齿目较远。[结论]成功的克隆得到了黑唇鼠兔的LDH-C基因的cDNA序列。
[Objective] The research aimed to lay the foundation for the study of contraceptive rodent with LDH-C4 as the target protein. [Method] The black-lip mouse is a member of the genus Rabbit and the genus of the genus Rabbit. There is no related sequence of the LDH-C gene in the GenBank database, so it was cloned by designing the degenerate primer. The designed three degenerate primer sequences were: D-Ps: 5’-atgtcmacygtcaaggagcagct-3 ’: D-Pa1: 5’-gcagayacabtgtaatcttttcc-3’; D-Pa2: 5’-ttacarccacttccratyac-3 ’. The cDNA of LDH-C was cloned by nested PCR using reverse transcriptase-generated cDNA as a template. Two gene-specific primers were designed according to the EST sequence. The 3’RACE was used to clone the 3’-end of LDH-C gene. The two gene-specific primers were Pika-Ps1: 5-cttgcccttgttgatgttgcaga-3 and Pika-Ps2: 5-ggatcttcaacatggcagtct-3. Nucleotide sequence analysis, splicing and similarity search using Vector NTI Suite 9 software, phylogenetic tree construction using Mega 4.0 software, using the adjacency method. [Result] The cDNA of LDH-C gene of black-lip mouse was 1 498 bp in length, of which the ORF was 996 bp, encoding 332 amino acids and the length of 3’UTR was 486 bp. ORF sequence alignment revealed that the LDH-C gene is conserved across large taxa. Phylogenetic tree analysis showed that the LDH-C gene of black-lip mouse was close to the primate and cloven-hoofed, and far from rodents. [Conclusion] The cDNA sequence of LDH-C gene in black-lip mouse was obtained by successful cloning.