rPA/SP-B 的融合表达和纤维蛋白溶解活性。

Fusion Expression and Fibrinolytic Activity of rPA/SP-B.

机构信息

Department of Microbiology and Biochemical Pharmacy, College of Pharmacy and Bioengineering, Chongqing University of Technology, Bananqu, Chongqing 400054, China.

Molecular Pathology Lab, Shantou University Medical College, Shantou, Guangdong 515041, China.

出版信息

Protein Pept Lett. 2021;28(9):1033-1042. doi: 10.2174/0929866528666210301151302.

DOI:10.2174/0929866528666210301151302

PMID:33645472

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.

摘要

背景

肺表面活性物质功能障碍是急性呼吸窘迫综合征（ARDS）和肺纤维化（PF）的重要病理因素。

目的

本研究旨在探讨重组成熟肺表面活性物质蛋白 B（SP-B）和瑞替普酶（rPA）融合蛋白在急性肺损伤细胞模型中保持良好肺表面活性和 rPA 纤维蛋白溶解活性的特点。

方法

我们研究了 SP-B 融合表达的特点，克隆了 rPA 基因和 rPA/N 端 SP-B/C 端 SP-B 共表达基因，并将其构建到真核表达载体 pEZ-M03 中，获得重组质粒 pEZ-rPA 和 pEZ-rPA/SP-B。将重组质粒转染中国仓鼠卵巢（CHO）K1 细胞，通过 Western Blot 分析表达产物。用脂多糖（LPS）诱导 CCL149（肺泡上皮细胞系）细胞损伤模型。用与 pEZ-M03 配套的增强型绿色荧光蛋白（eGFP）对 rPA 和 rPA/SP-B 进行荧光染色；用细胞喇曼光谱技术分析 rPA/SP-B 融合蛋白与 CCL149 细胞膜磷脂结构的相互作用。用纤维蛋白-琼脂糖平板法测定融合蛋白中 rPA 的酶活性。