Preparation of Monoclonal Antibody to Vasorin in Tree Shrew and Exploration of Its Application

doi:10.12300/j.issn.1674-5817.2025.027

Abstract

Abstract:

Objective To obtain tree shrew Vasorin (VASN) recombinant protein through prokaryotic expression and purification, prepare monoclonal antibody against tree shrew VASN by immunizing mice with this protein, and preliminarily evaluate its application value. Methods Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify the full-length sequence of tree shrew VASN gene in vitro. The tree shrew VASN gene fragment was inserted into pET-30a vector to construct pET-30a-VASN recombinant plasmid. The recombinant plasmid was subjected to double digestion with BamHⅠ and SalⅠ for identification, and its correctness was further verified by sequencing. The recombinant plasmid with correct sequencing was transformed into BL21 (DE3) competent cells, and isopropyl β-D-thiogalactoside (IPTG) was used to induce expression of VASN recombinant protein. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the VASN recombinant protein was purified by KCl. Purified recombinant protein was used to immunize BALB/c mice for four times, and serum antibody titer was detected by enzyme-linked immunosorbent assay (ELISA). Splenocytes from mice with serum antibody titer above 1:10 000 were used for cell fusion with myeloma cells. Hypoxanthine-aminopterin-thymidine (HAT) culture medium was first used to screen hybridoma cells. ELISA was used to screen positive hybridoma cell lines that could secrete specific antibodies, and monoclonal hybridoma cell lines were obtained by limiting dilution method. VASN monoclonal antibodies were prepared in large quantities by ascites induction method, purified using rProtein G, and the affinity and in vitro reaction specificity of the monoclonal antibodies were detected by ELISA and Western blotting. Results The full-length sequence of the tree shrew VASN gene was successfully amplified and the recombinant plasmid vector of tree shrew pET-30a-VASN was constructed. The sequence obtained by sequencing of the recombinant plasmid vector was identical to the tree shrew VASN target gene sequence. Recombinant protein VASN mainly existed in the form of inclusion bodies, and the purity after purification reached 90%, meeting the requirements of subsequent immunization experiments. After four immunizations with recombinant protein VASN, mouse serum antibody titer reached 1:729 000. Monoclonal positive hybridoma cell lines were obtained through ascites induction and purification, and the constant affinity value of monoclonal antibodies measured by ELISA reached 2.59x10⁷ L/mol. Western blotting results showed that the tree shrew VASN monoclonal antibody could bind to tree shrew VASN recombinant protein, but it showed no binding reaction with porcine retinol-binding protein 4 recombinant protein, human VASN-leucine rich repeat recombinant protein, or bovine serum albumin. Anti-tree shrew VASN monoclonal antibody could specifically recognize VASN protein in tree shrew heart, liver, spleen, lung, kidney and muscle, with clear bands and clean background. Immunohistochemical detection results showed that this monoclonal antibody could recognize VASN protein in tree shrew spleen, lung, and tree shrew immortalized fibroblasts with high VASN mRNA expression levels, and the detection results were positive. Conclusion Monoclonal antibody against tree shrew VASN is successfully prepared. This antibody can be used for immunohistochemical detection of tree shrew immortalized fibroblasts, spleen tissue, and lung tissue, providing an important tool for further research on the function of VASN in tree shrew models.

Key words: Vasorin, Tree shrew, Monoclonal antibody, Prokaryotic expression, Hybridoma fusion

CLC Number:

Q95-33

OU Meizhen,LI Yongfeng,WEN Sha,et al. Preparation of Monoclonal Antibody to Vasorin in Tree Shrew and Exploration of Its Application[J]. Laboratory Animal and Comparative Medicine, 2025, 45(5): 611-622. DOI: 10.12300/j.issn.1674-5817.2025.027.

Figures/Tables 4

Figure 1 Construction of the pET-30a-VASN recombinant plasmidNote： A， VASN gene amplification product （M， DNA Marker； 1， VASN amplification product）； B， PCR identification of monoclonal colonies （1-5， different colonies）； C， Recombinant plasmid double digestion identification （1-2， pET-30a-VASN double digestion product）.

Figure 2 Expression and purification of VASN recombinant proteinNote： A， Expression of VASN recombinant protein （M， Protein marker； 1， Uninduced pET-30a-VASN supernatant； 2， Uninduced pET-30a-VASN precipitate； 3， Induced pET-30a-VASN superna-tant； 4， Induced pET-30a-VASN precipitate）； B， Purification of VASN recombinant protein （M， Protein marker； 1， Before purifi-cation of VASN recombinant protein； 2， After purification of VASN recombinant protein； 3， Protein loading buffer）.

Figure 3 Preparation of monoclonal antibody to tree shrews VASNNote： A， Mouse serum titer； B， Fusion process of mouse spleen cells and SP2/0 myeloma cells； C， Monoclonal antibody ascites purified （M， Protein marker； 1， Purified TV5-B11）； D， Monoclonal antibody affinity measurement； E， Antibody specificity identification （M， Protein marker； 1， tree shrews VASN recombinant protein； 2， porcine retinol-binding protein 4 recombinant protein； 3， human VASN-leucine rich repeat recombinant protein； 4 bovine serum albumin standard protein）； F， Antibody gradient concentration identification （M， Protein marker； 1， 0.5 μg； 2， 1.0 μg； 3， 1.5 μg； 4， 2.0 μg）.

Figure 4 Application of tree shrews VASN antibodyNote：A， The protein expression level of VASN in various tissues of tree shrew was identified by Western blotting（M， Protein marker； 1， Heart tissue； 2， Liver tissue； 3， Spleen tissue； 4， Lung tissue； 5， Kidney tissue； 6， Muscle tissue）； B， Relative expression levels of VASN mRNA in various tissues of tree shrews detected by qRT-PCR （Compared with lung tissue，???P<0.001，????P<0.000 1； compared with spleen tissue， ###P<0.001，####P<0.000 1）； C， Amplification results of the VASN gene（M， DNA Marker； 1， Positive control； 2， VASN amplification product； 3， Blank control）； D， Antibodies were used in immunohistochemistry to identify the expression of VASN protein in immortalized fibroblasts of tree shrews； E， Antibodies were used in immunohistochemistry to identify the expression of VASN protein in spleen tissue； F， Antibodies were used in immunohistochemistry to identify the expression of VASN protein in lung tissue.

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