Abstract:

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 synthesized 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. Conclusion   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.

Key words: Major Histocmpatibility Complex-Ⅰ; Intrabody; Endoplasmic reticulum; Adeno-associated virus; Gene therapy