Recombinant expression of rabies single chain antibody fusing shuttle peptide in E. coli and neutralization activity analysis
10.3760/cma.j.cn112866-20200205-00018
- VernacularTitle:融合穿梭肽的狂犬病单链抗体在大肠杆菌中的重组表达及中和活性分析
- Author:
Song XIE
1
;
Zongqin YU
;
Wenrui ZHAO
;
Xuexin LU
Author Information
1. 山东省第二人民医院，济南 250022
- Keywords:
Single-chain variable fragment;
Neutralizing antibody;
Rabies;
Shuttle peptide
- From:
Chinese Journal of Experimental and Clinical Virology
2021;35(2):172-175
- CountryChina
- Language:Chinese
-
Abstract:
Objective:The recombinant shuttle peptide-single chain antibody with neutralizing activity is expected to be obtained by combining the shuttle peptide sequence with rabies single chain antibody and optimizing the expression conditions in E. coli.Methods:In this study, the SO57 single-stranded antibody sequence was recoded and the shuttle peptide of Arg 12 was fused at the N-terminal to construct pET22 (b)-rSO57 expression vector, which was then expressed in E. coli. The growth density OD 600, induction time, induction temperature and inducer concentration were optimized to obtain a higher expression effect. The recombinant protein was purified by immobilized metal chelating chromatography, and the relative affinity of the recombinant single-chain variable fragment (scFv) was determined. The neutralization effect of the recombinant scFv was verified by rSO57/CVS-11 virus strain mixed with infected cells and rapid fluorescent focus inhibition test (RFFIT). Results:The recombinant rSO57 was successfully constructed and induced in BL21 (de3). The optimal expression condition was that when the cell density of OD 600 was 0.8, IPTG of 0.4 mmol/ml was used for induction. After induction at 16 ℃ for 24 hours, rSO57 was expressed in soluble form, accounting for 19.8% of the soluble protein in the cell. After chromatography purification, the recombinant rSO57 with a purity of 84% was obtained. ELISA showed that the relative affinity coefficient Ka of rSO57 was 4.3×10 5. When rSO57 was mixed with CVS-11 strain, the infection rate of cells increased with the increase of dilution, indicating the neutralization activity of recombinant antibodies. Using the RFFIT, 50 μg rSO57 was equivalent to 2.17 IU of rabies neutralizing serum. Conclusions:In this study, the recombinant scFv fusion with shuttle peptide was expressed, which could neutralize rabies virus, in order to prepare specific and targeted antiviral drug carriers for rabies treatment.
