Effect of thioredoxin on the immunogenicity of the recombinant P32 protein of lumpy skin disease virus

Vet World. 2022 Oct;15(10):2384-2390. doi: 10.14202/vetworld.2022.2384-2390. Epub 2022 Oct 11.


Background and aim: The rapid spread of lumpy skin disease (LSD) globally poses a serious threat to the agricultural sector. The timely and accurate diagnosis of the disease is crucial to control LSD. This study aimed to determine the effect of thioredoxin on the immunogenicity of the recombinant P32 (rP32) protein of LSD virus (LSDV). Since the P32 protein is poorly soluble, it is often expressed by adding an auxiliary sequence of a highly soluble partner protein such as thioredoxin.

Materials and methods: The P32 gene fragment was amplified using a polymerase chain reaction from genomic DNA used as a template. The resulting DNA fragments were cloned into the pET32a vector, and transformed into Escherichia coli BL21 (DE3) cells through electroporation. Purification of the rP32 protein was performed using a HisTrap column. Purified rP32 protein fused with thioredoxin (rP32Trx) was characterized by western blotting, liquid chromatography with tandem mass spectrometry and indirect enzyme-linked immunosorbent assay (ELISA).

Results: Indirect ELISA revealed that, despite the lower molecular weight, the main part of the antibodies in the serum of immunized mice was directed against thioredoxin and not the target P32 protein. Thus, the antibody titers against rP32Trx were 1:102400, whereas antibody titers against heterologous recombinant 3BTrx and PD1Trx proteins were 1:25600 and 1:51200, respectively. Concurrently, the antibodies did not bind to the heterologous recombinant PD1 protein, which did not contain thioredoxin.

Conclusion: The results showed that the rP32 protein fused with the partner protein thioredoxin could not be used to obtain polyclonal and monoclonal antibodies. However, the recombinant fusion protein rP32Trx can be used to develop a serological test to detect antibodies, since antibodies against thioredoxin were not detected in the animal sera.

Keywords: P32 protein; immunogenicity; lumpy skin disease virus; recombinant antigen; thioredoxin.