[Construction and expression of regulatable recombinant adenovirus vector for mouse PLP-Ig chimeric protein].
OBJECTIVE
To construct a regulatable recombinant adenovirus vector expressing mouse proteolipid protein (PLP)-Ig.


METHODS
Total RNA was extracted from WT-1 hybridoma cells to clone Ig heavy chain Fc fragment gene. PCR was carried out to link PLP(139-151) gene and signal peptide with Fc fragment gene. pTRE-shuttle vector was used to ligate PLP-Ig gene and the backbone of the replication-incompetent adenoviral vector. After confirming the desired recombinant adenovirus by PCR and restriction endonuclease digestion analysis, it was packaged and propagated in HEK 293 cells. Tetracycline was used to regulate the expression of PLP-Ig, and Western blot was used to detect the gene expression.


RESULTS
The expression of PLP-Ig was confirmed by Western blot and its expression could be regulated by tetracycline.


CONCLUSIONS
The regulatable recombinant adenovirus vector for mouse PLP-Ig was constructed and expressed successfully, which lays the foundation for further study on gene therapy of EAE and induction of immune tolerance.
Zhang
Yan
Y
Department of Laboratory diagnosis, Changhai Hospital, Second Military Medical University, Shanghai 200433, China. zhangyansh@sina100.com
Liu
Meng-lei
ML
Shen
Qian
Q
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004622:Embryo, Mammalian; D005822:Genetic Vectors; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics* / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D051379:Mice; D018991:Myelin Proteolipid Protein / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D014162:Transfection
D007141:Immunoglobulin Fc Fragments; D007143:Immunoglobulin Heavy Chains; D018991:Myelin Proteolipid Protein; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C435023:myelin proteolipid protein (139-151)