【摘要】：荷包豬屬于我國(guó)地方優(yōu)質(zhì)品種豬,是經(jīng)過(guò)原始進(jìn)化而成的物種,具有重要的科學(xué)研究?jī)r(jià)值。豬的DR基因座屬于豬主要組織相容性復(fù)合體(maj or histocompatibility complex,MHC),亦即豬白細(xì)胞抗原(swine leukocyte antigen,SLA)II類分子重要功能基因,包括α鏈(DRA)和p鏈(DRB),是物種分子進(jìn)化的標(biāo)記。為研究荷包豬SLA-DR基因,以荷包豬脾臟組織為材料,采用RT-PCR技術(shù),分別以引物DRAF1/DRAR1擴(kuò)增sLA-DRA、以引物DRBF1/DRBR1擴(kuò)增sLA-DRB。擴(kuò)增產(chǎn)物分別與pMD18-T載體連接,并轉(zhuǎn)化宿主菌JM109。EcoR I和Hind Ⅲ雙酶切篩選陽(yáng)性克隆。最后利用生物軟件DNAMAN和Mega 5等進(jìn)行比對(duì)、分析及構(gòu)建分子進(jìn)化樹(shù)。結(jié)果顯示,成功克隆3個(gè)荷包豬SLA.DRA.HB01-03和3個(gè)SLA.DRB.HB01-03等位基因。序列測(cè)定結(jié)果顯示,α鏈3個(gè)等位基因SLA.DRA.HB0l-03長(zhǎng)度為779 bp,編碼252個(gè)氨基酸,分別獲得序列號(hào)LC002668.LC002669和LC002670；p鏈3個(gè)等位基因SLA-DRB-HB01-03為836bp,編碼266個(gè)氨基酸,分別獲得序列號(hào)LC20542、LC20543和LC20544。氨基酸序列對(duì)比分析發(fā)現(xiàn),α鏈等位基因SLA-DRA.HB01.03與其他SLA-DRA等位基因相比較,變異位點(diǎn)較少。對(duì)于p鏈基因SLA-DRA-HB01-03.與其他SLA-DRB等位基因相比較變異位點(diǎn)較多。一致性分析顯示,SLA-DRA-HB01-03與其他SLA-DRA等位基因一致性為98.7%-99.6%,而SLA-DRB-HB01-03與其他SLA-DRB等位基因一致性為90.3%-99.8%。分子進(jìn)化樹(shù)分析結(jié)果顯示,SLA-DRA-HB01-03自成一體系,但與其他等位基因的距離均較近；而荷包豬SLA-DRB-HB01-03進(jìn)化樹(shù)也自成一系,與其他等位基因相比較進(jìn)化距離較遠(yuǎn),進(jìn)化更加原始。本研究成功克隆了荷包豬DR基因座α鏈和p鏈,證明荷包豬DR基因座α鏈基因比較保守,而β鏈基因多態(tài)性較豐富,進(jìn)化上更具有特征性。本研究為今后進(jìn)一步研究荷包豬SLA-DR基因座α鏈和β鏈基因結(jié)構(gòu)和功能奠定基礎(chǔ)。
[Abstract]:The purse pig belongs to the local high quality pig of our country. It is a species of primitive evolution and has important scientific research value. Porcine DR loci belong to the porcine major histocompatibility complex (maj or histocompatibility complex,MHC), that is, porcine leukocyte antigen (swine leukocyte antigen,SLA) II class important functional genes, including 偽 -chain (DRA) and p-chain (DRB), is the molecular evolution of species markers. In order to study the SLA-DR gene of the purse pig, the spleen tissue of the purse pig was used as the material, the sLA-DRA, was amplified by the primers DRAF1/DRAR1 and the sLA-DRB. was amplified by the primer DRBF1/DRBR1 using RT-PCR technique. The amplified products were ligated with pMD18-T vector respectively, and the positive clones were screened by double digestion of JM109.EcoR I and Hind 鈪，

本文編號(hào)：2238088