Title:Fusion Expression and Fibrinolytic Activity of rPA/SP-B
Volume: 28
Issue: 9
Author(s): Yi-Shan Tang, Xiao-Jun Zhang, Wan-Neng Wang*, Ting Wang, Wu-Long Cao, Qiu-Han Zhang and Fu Chen
Affiliation:
- Department of Microbiology and Biochemical Pharmacy, College of Pharmacy and Bioengineering, Chongqing University of Technology, Bananqu, Chongqing 400054,China
Keywords:
Reteplase, pulmonary surfactant, fusion protein, acute lung injury, acute respiratory distress syndrome (ARDS),
pulmonary fibrosis (PF).
Abstract:
Background: Pulmonary surfactant dysfunction is an important pathological factor in
acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF).
Objective: In this study, the characteristics of recombinant mature surfactant protein B (SP-B) and
reteplase (rPA) fusion protein maintaining good pulmonary surface activity and rPA fibrinolytic activity
in acute lung injury cell model were studied.
Methods: We studied the characteristics of SP-B fusion expression, cloned rPA gene and N-terminal
rPA/C-terminal SP-B co-expression gene, and constructed them into eukaryotic expression vector
pEZ-M03 to obtain recombinant plasmids pEZ-rPA and pEZ-rPA/SP-B. The recombinant plasmids
was transfected into Chinese hamster ovary (CHO) K1 cells and the expression products were
analyzed by Western Blot. Lipopolysaccharide (LPS) was used to induce CCL149 (an alveolar
epithelial cell line) cell injury model. Fluorescence staining of rPA and rPA/SP-B was carried out
with the enhanced green fluorescent protein (eGFP) that comes with pEZ-M03; the cell Raman
spectroscopy technique was used to analyze the interaction between rPA/SP-B fusion protein and
the phospholipid structure of cell membrane in CCL149 cells. The enzyme activity of rPA in the fusion
protein was determined by fibrin-agarose plate method.
Results: The rPA/SP-B fusion protein was successfully expressed. In the CCL149 cell model of
acute lung injury (ALI), the green fluorescence of rPA/SP-B is mainly distributed on the CCL149
cell membrane. The rPA/SP-B fusion protein can reduce the disorder of phospholipid molecules
and reduce cell membrane damage. The enzyme activity of rPA/SP-B fusion protein was 3.42, and
the fusion protein still had good enzyme activity.
Conclusion: The recombinant eukaryotic plasmid pEZ-rPA/SP-B is constructed and can be expressed
in the eukaryotic system. Studies have shown that rPA/SP-B fusion protein maintains good
SP-B lung surface activity and rPA enzyme activity in acute lung injury cell model.