奶山羊催乳素受体基因CDS区的克隆和序列分析与腺病毒干扰载体的构建

Cloning, Sequence Analysis of Prl R CDS and Construction of Adenovirus Vector for RNA Interference in Dairy Goat

本文旨在克隆奶山羊催乳素受体（PrlR）基因CDS序列，在此基础上构建针对奶山羊PrlR的腺病毒干扰载体，为研究PrlR在奶山羊乳腺乳脂合成代谢中的功能与调控作用提供有效的RNA干扰工具。首先从西农萨能羊乳腺组织RNA中扩增得到完整CDS区序列，其CDS长为1746bp，包含581个氨基酸序列，GenBank登录号为JF966783；测序后进行序列分析和功能预测，奶山羊PrlR基因CDS区与其他反刍动物相应序列具有较高的同源性，其跨膜区为第236-258位氨基酸；然后根据测序得到的CDS序列，在线设计合成奶山羊PrlR的shRNA序列并成功构建重组干扰腺病毒载体；将这些载体在293细胞系中进行包装和扩繁，最终获取高滴度的腺毒，使用TCID50法检测病毒滴度为5．19×10^8PFU／mL。

To provide effective tool to study the role of PrIR in regulation of milk fat synthesis in mammary gland, the present study cloned the coding regions （CDS） of PrIR from mammary gland of Xinong Saanen goat and constructed RNA interfering adenovirus vectors targeting goat PrlR upon CDS sequence. First, complete CDS sequence was amplified from extracted RNA in mammary gland of Xinong Saanen goat and PrlR CDS （GenBank No. JF966783） is 1746 bp in length, coding 581 amino acid residues; Sequence analysis and functional prediction was performed after sequencing and found there was high homologies among goats and some other ruminants and the transmembrane region of PrlR locates at 236-258 amino acid residues; Then, shRNA sequences of PrlR gene was designed online and recombinant adenovirus vectors carrying shRNA targeting goat PrlR was successfully constructed according to the CDS sequences from sequencing; Finally, these adenovirus vectors were packaged and amplified in 293 cell line and high- titered adenovirus （5. 19× 10^8 PFU/mL） was obtained after multiple infection with TCID50 method.