Cloning and identification of the CTLA-4IgV gene and functional application of vaccine in Xinjiang sheep

Huifang Kong; Shangqi Zhao; Jia Zheng; Bin Liu; Yanxia Zhou; Yanmin Li; Wentao Zhou; Xiaotao Zhou

doi:10.1515/biol-2022-0524

Cloning and identification of the CTLA-4IgV gene and functional application of vaccine in Xinjiang sheep

Open Life Sci. 2022 Nov 23;17(1):1555-1567. doi: 10.1515/biol-2022-0524. eCollection 2022.

Authors

Huifang Kong¹, Shangqi Zhao¹, Jia Zheng¹, Bin Liu², Yanxia Zhou¹, Yanmin Li¹, Wentao Zhou³, Xiaotao Zhou^{1

4}

Affiliations

¹ Department of Immunology, Basic Medical College, Xinjiang Medical University, Urumqi, China.
² Department of Clinical Laboratory, The First Affiliated Hospital of Xinjiang Medical University, Urumqi, China.
³ Department of Respiratory Medicine, The Fifth Affiliated Hospital of Xinjiang Medical University, Urumqi, China.
⁴ Department of Immunology, College of Basic Medicine of Xinjiang Medical University, Urumqi, China.

Abstract

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is an important surface molecule of activated T cells that has a strong affinity with the B7 molecule on the surface of antigen-presenting cells. Among these molecules, the CTLA-4 extracellular region (CTLA-4 IgV) may be used as a novel immune adjuvant molecule for delivering antigens and inducing strong humoral and cellular immune responses. In this study, bioinformatics analysis was performed to determine and clone the extracellular region of Xinjiang sheep CTLA-4 (NM_001009214). The CTLA-4 IgV gene was amplified and ligated into the pMD19-T vector, and the positive bacteria were screened by blue-white spots for sequencing and comparison. The correctly sequenced CTLA-4 IgV was digested and then ligated into the prokaryotic expression vector pET-30a(+). The plasmid pET30a-CTLA-4 IgV was constructed to induce the expression of the recombinant protein CTLA-4 IgV. Thereafter, CTLA-4 IgV was identified. Clustal X multiple sequence alignment revealed that the protein sequence of Xinjiang sheep CTLA-4 IgV was different from that of the known CTLA-4 extracellular region. The 3D protein structure of Xinjiang sheep CTLA-4 IgV was constructed via the bioinformatics method. Subsequently, molecular docking between the Xinjiang sheep CTLA-4 IgV protein and the B7 molecule was conducted. Results revealed multiple binding sites in the extracellular region of Xinjiang sheep CTLA-4, and two multiple interactions ensured stable binding after docking. The functionality of the Xinjiang sheep CTLA-4 IgV protein was further verified by fusing the CTLA-4 extracellular V region with EgG1Y162, a protective protein from Echinococcus granulosa, and the purified recombinant protein CTLA-4 IgV-EgG1Y162 was expressed with the mouse bone marrow-derived. The addition of the Xinjiang sheep CTLA-4 IgV protein at the amino terminus promoted the binding of EgG1Y162 to dendritic cells (DCs) and increased the maturation rate of these cells, further indicating that the protein could effectively improve the antigen presentation ability of DCs. The CTLA-4 extracellular domain protein of Xinjiang sheep is unique and has the potential to promote the presentation of the fusion protein by DCs as an adjuvant. The cloning and expression of this gene provide new measures and ideas for the preparation of the Xinjiang sheep vaccine to prevent zoonotic diseases.

Keywords: Bioinformatics; CTLA-4 extracellular domain; Xinjiang sheep; dendritic cells.