MHC-Ⅰ细胞内抗体重组腺相关病毒载体的构建

The construction of MHC-Ⅰ intrabody recombinant AAV vector

目的构建Ⅰ型主要组织相容性复合体(MHC-Ⅰ)重组腺相关病毒载体(rAAV)。方法合成内质网滞留型MHC-Ⅰ细胞内抗体的基因片段,测序正确后酶切获取该胞内抗体的基因片段,将该基因片段插入质粒pSSHG/CMV的EcoR I-BamH I位点构建pSSHG/MHC-Ⅰ。用磷酸钙共沉淀法将腺病毒辅助质1粒pFG140、包装质粒pAAV/Ad及已构建的pSSHG/MHC-Ⅰ三质粒在293细胞系中同源重组包装rAAV。采用地高辛标记的斑点杂交方法测定rAAV滴度。结果成功构建了重组病毒质粒pSSHG/MHC-Ⅰ及人类MHC-Ⅰ胞内抗体重组腺相关病毒载体(rAAV-MHCI),经蔗糖梯度离心法获得rAAV组分,梯度稀释测得滴度为6.88×1010PFU/mL。结论通过分子克隆体外重组技术成功构建了rAAV-MHCI,为下一步人体细胞实验及移植免疫的基因治疗奠定了基础。

Objective To construct major histocompatibility complex-Ⅰ （MHC-Ⅰ ） recombinant adeno-associated virus （rAAV） vector. Methods The gene fragment of endocytoplasmic reticulum stagnation MHC-Ⅰ intrabody was synthe- sized followed by sequencing. The intrabody gene fragment was then obtained by restriction enzyme. The intrabody gene fragment was inserted into the EcoR I-BamH I site of the plasmid pSSHG/CMV to construct pSSHG/MHC-Ⅰ. The rAAV viral stock was packaged in 293 cell lines through co-transfecting with the pSSHG/MHC-Ⅰ, pAAV/Ad and pFG140 instead of adenovirus by calcium phosphate precipitation. Digoxin labeled dot blot hybridization was used to determine the rAAV titer. Results The recombinant viral stock vector of plasmid pSSHG/MHC-Ⅰ was successfully constructed. The results of dot blot hybridization showed that the rAAV stocks of high titre 6.88 × 1010 PFU/mL had been obtained. Con- clusion rAAV-MHC-Ⅰ is successfully constructed by molecular cloning and recombination in vitro techniques, which can serve as the foundation of further human cell experiments and gene therapy of transplantation immunity.