重叠延伸PCR法体外重建鸡主要组织相容性复合体Ⅰ  

Reconstruct Chicken Major Histocompatibility Complex Ⅰ in vitro Using Splicing Overlap Extension PCR Method

作　　者：李新生[1] 闫若潜 高风山[1] 方勤美[1] 郝惠芳[1] 李云岗[1] 陈红英[3] 夏春[1] 

机构地区：[1]中国农业大学动物医学院农业部预防兽医学重点实验室,北京100094 [2]河南省兽医防治站,郑州450002 [3]河南农业大学牧医工程学院,郑州450002

出　　处：《畜牧兽医学报》2006年第10期1047-1052,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金资助项目(30371098)

摘　　要：本研究旨在体外重建鸡主要组织相容性复合体Ⅰ类分子(MHCⅠ)重链(α链)胞外区与轻链(β2m)成熟肽的重组嵌合分子。采用RT-PCR法分别扩增鸡MHCⅠ的重链胞外区和轻链的成熟肽序列;采用重叠延伸PCR(Splicing overlap extension PCR method,SOE-PCR)把通过45个碱基的链连接的鸡MHCⅠ重链胞外区基因和轻链成熟肽基因重组到可溶性表达质粒pMAL-p2X进行可溶性表达。经琼脂糖凝胶电泳证明RT-PCR可分别扩增出鸡MHCⅠα链胞外区基因和β2m成熟肽基因,大小符合预期。采用MHCⅠα链胞外区序列的反义引物与β2m成熟肽基因正义引物有15个碱基重叠的两对引物,以MHCⅠα链胞外区序列与β2m成熟肽序列PCR产物的混合物作为模板,进行重叠延伸获得了预期大小的连接片段;测序显示重组质粒上MHCⅠα链胞外区序列与轻链β2m成熟肽基因的靶序列由一柔性的linker相连,阅读框正确且无移码。本研究表明SOE-PCR是体外重构鸡MHCⅠ的一种简捷可行方法。To reconstruct a hybrid molecule composed of chicken major histocompatibility complex heavy chain（α chain） extracelular domains gene and β2-microglobulin （β2m） mature protein gene in vitro, chicken MHC Ⅰα chain extracellular domains gene and β2m mature protein gene were ampliβed by RT-PCR respectively. There are 15 neucleartides overlap between reverse primer of the βrst pair of primers for α chain extracellular domains gene and upper primer of the second pair of primers for β2m mature protein gene. Then, using MHC αⅠ chain extracellular gene PCR product and β2m mature peptide gene PCR product mixture as a template, we reconstructed soluble expression plasmid pMAL-p2X which contained major histocompatibility complex Ⅰ by linking MHC Ⅰα chain extracellular domain gene and β2m mature protein gene through α 45 nucleotides linker. Electrophoresis analysis showed that the speciβc single sequence of the two target products could be ampliβed by RT-PCR, and the product of interest was accord with expectation. Sequencing result proved that expression reading frame of recombinant plasmid was composed of the expected MHC Ⅰα chain extracellular domains sequence of interest and β2m mature protein gene target sequence, which linked by a limp linker, and there was no base malposition. In conclusion, splicing overlap extension by PCR is a convenient method to obtain recombinant chicken MHCⅠ in vitro.

关 键 词：鸡 主要组织相容性复合体Ⅰ(MHCⅠ) 重链 轻链 重叠延伸PCR 

分 类 号：S852.4[农业科学—基础兽医学] R392.13[农业科学—兽医学]