新冠病毒BA.5高免疫原性RBD抗原制备及抗体开发

doi:10.3969/j.issn.1006-5725.2025.17.007

Abstract

Abstract:

Objective To develop high-immunogenicity antigens targeting the BA.5 variant of SARS-CoV-2 and to screen for monoclonal antibodies with high neutralizing and blocking activity， providing new strategies for the development of vaccines and diagnostic reagents. Methods The ZP protein gene fragment from zebrafish vitellogenin and the HIS protein tag were inserted into the pCDNA3.4 plasmid to construct the recombinant plasmid pCDNA3.4-RBD（BA.5）-ZP-HIS， which was then transfected into 293F cells to express the RBD-ZP protein. The expression of the protein was verified by SDS-PAGE and its binding capabilities to ACE2 receptor molecules and aluminum adjuvant were detected. The immunogenicity of the fusion protein was evaluated using a BALB/c mouse model， and monoclonal antibodies were prepared through hybridoma technology. Monoclonal antibodies with strong neutralizing and blocking activity were screened and their neutralizing activity was detected by blocking ELISA. Results The ZP gene and HIS protein tag sequence were successfully inserted into the pCDNA3.4 vector and the RBD-ZP protein with a molecular weight of 50 kDa was successfully expressed. The immunogenicity test results showed that the ZP protein effectively enhanced the immunogenicity of the RBD protein and improved its binding capability to the ACE2 receptor. After immunizing mice with the RBD-ZP protein， 8 monoclonal antibodies that specifically bind to both the mutant and wild-type strains were cloned and screened through hybridoma technology， among which 3 could effectively block the binding of the SARS-CoV-2 RBD protein to the human ACE2 receptor. Conclusion This study successfully expressed the RBD-ZP fusion protein， which significantly enhanced the immunogenicity and receptor binding capability of the RBD protein. Three monoclonal antibodies with high neutralizing and blocking activity were screened out， providing strong support for the development of COVID-19 vaccines and diagnostic reagents..

Key words: SARS-CoV-2, RBD-ZP protein, antigen preparation, monoclonal antibody screening

CLC Number:

Q812

Fan WU,Hongni QIN,Yuzhen XIE,Baoyong REN,Dongsheng DAI. Study on the preparation of high immunogenicity RBD antigen and antibody development of COVID⁃19 BA.5[J]. The Journal of Practical Medicine, 2025, 41(17): 2653-2660.

Figures/Tables 10

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References 26

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	1	2	3	4	5	6	7	8	9	10	11	12
A	0.513	0.482	0.632	0.106	0.256	0.431	0.823	0.732	0.429	3.113	0.793	空白
B	0.421	1.328	0.024	1.297	1.022	0.123	0.085	0.218	4.069	0.694	0.731	空白
C	0.386	1.432	0.025	0.943	0.174	0.351	0.839	0.431	0.621	0.632	0.072	空白
D	0.451	0.205	0.091	0.027	0.523	2.659	0.071	0.301	0.742	3.612	0.924	空白
E	0.027	1.376	0.172	0.029	3.213	0.382	3.915	0.727	0.116	0.942	0.215	0.069
F	0.623	0.096	1.382	0.328	0.536	0.351	0.106	0.086	0.173	0.814	0.076	0.083
G	0.821	0.843	0.043	0.779	0.518	0.245	1.285	0.943	2.649	0.327	0.843	0.163
H	0.643	0.076	0.785	0.144	0.116	0.583	0.082	0.432	0.525	0.834	0.536	3.831

	1	2	3	4	5	6	7	8	9	10	11	12
A	1.954	2.152	1.984	1.635	1.448	1.746	1.853	1.859	1.884	3.236	1.826	空白
B	1.956	2.184	1.265	2.147	1.994	1.954	1.287	1.893	4.082	1.885	1.853	空白
C	2.054	2.132	1.432	1.867	1.302	1.342	2.054	1.934	1.646	1.942	1.327	空白
D	2.208	1.749	1.946	1.084	1.459	2.810	1.229	1.363	1.947	3.632	2.001	空白
E	2.231	2.339	1.845	1.726	3.362	1.532	3.963	1.756	1.593	2.013	1.632	1.538
F	1.967	1.394	2.094	1.584	1.735	1.541	1.535	1.236	1.874	1.987	1.523	1.632
G	1.950	1.847	1.352	1.943	1.832	1.493	2.344	1.935	2.703	1.638	1.969	1.739
H	1.849	1.864	1.945	2.047	1.295	1.462	1.322	1.642	1.536	1.864	1.743	3.853

顺序	抗体名称	总体积/μL	浓度/（mg/mL）	抗体总量/mg
1	B1-2-3H12	300	0.849	0.2547
2	B1-2-1G9	300	0.954	0.2862
3	B1-2-3B9	300	0.867	0.2601
4	B3-2-2D10	300	1.002	0.3006
5	B3-2-E7	300	1.863	0.5589
6	B1-2-2E5	300	1.013	0.3039
7	B1-2-3D6	300	2.787	0.8361
8	B1-2-3A10	300	1.471	0.4413

Study on the preparation of high immunogenicity RBD antigen and antibody development of COVID⁃19 BA.5

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