[Construction and stable expression of intracellular antibodies to glycoprotein of hantavirus]
Tao Wang1, Jian-dong Li, Chuan Li
1Institute for Viral Disease Control and Prevention, China Centers for Disease Control and Prevention, Beijing 100052, China.
Objective:
To understand the molecular mechanisms of hantavirus assembly and maturation by stably expressing the intracellular antibodies to hantavirus glycoprotein G1 and G2 in endoplasmic reticulum (ER) and cytoplasm (Cyto) of Vero E6 cell.
Methods:
The genes of VH and VL of antibodies against glycoprotein of hantavirus were amplified by PCR and cloned into pOPE 101-215 (Yol) vector. The G1 and G2 proteins specific ScFv genes were first expressed in E.coli and the function and binding properties were identified. The gene of ScFv were further inserted into intracellular expression vectyrs pEF/ myc/ ER and pEF/ myc/ CYTO vector and transfected Vero E6 cell. The clonal cell line which stabl expresses ScFv were isolated under the pressure of G418.
Results:
The ScFv genes of hantavirus G1 and G2 specific antibodies were successfully expressed in subcellular compartment ER and Cyto of Vero E6 cells and specifically targeted G1 and G2 protein after virus infection of the cells.
Conclusions:
The recombination of intrabody to Hantann virus glycoprotein was constructed successfully, and it may provide basic material for the studying antiviral gene therapy and the molecular mechanism of viral replication and infection.
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