【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。更多还原

【Abstract】 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 Ⅰ α chain extracellular domains gene and β2m mature protein gene were amplified by RT-PCR respectively.There are 15 neucleartides overlap between reverse primer of the first 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 α Ⅰ 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 MHCⅠ α chain extracellular domain gene and β2m mature protein gene through a 45 nucleotides linker.Electrophoresis analysis showed that the specific single sequence of the two target products could be amplified 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 Ⅰ α 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 MHC Ⅰ in vitro.更多还原