Abstract: 【Objective】 This experiment was aimed to construct different types of novel immune castration DNA vaccines, including KISS1, neurokinin B (NKB) single expression gene vaccines, and gonadotropin releasing hormone (GnRH), KISS1 and NKB three genes co-expression gene vaccines.After injection into animals, they could stimulate immune responses and produce corresponding antibodies, thereby achieving the goal of reducing androgens by regulating the reproductive axis.【Method】 By selecting GnRH and its upstream regulatory genes KISS1 and NKB on the hypothalamic pituitary gonadal axis as targets, and introducing a balanced lethal system instead of antibiotic screening, the recombinant plasmids was constructed.The three expression plasmids were co-expressed with the target gene through the self-cleavage function of the 2A peptide.After transfecting the recombinant plasmids into 293T cells, the cell RNA was extracted to verify its normal expression in the body.Subsequently, the successfully constructed plasmids were electrically transferred to attenuated Salmonella Choleraesuis C500 competent cells to obtain recombinant live bacterial vaccines.The constructed engineered bacteria were continuously passaged 50 times in vitro, and the stability studies were conducted on strains from the 0, 2nd, 5th, 10th, 20th, 30th, 40th and 50th generations.【Result】 Recombinant plasmids pVAX-2A/KISS1-asd, pVAX-2A/S-NKB-asd and pVAX-S/GnRH-2A/S-NKB-2A/KISS1-asd were digested to obtain bands of 498, 1 146 and 2 622 bp, respectively, consistent with the target gene.After comparing the sequencing results with the target sequence, it was found that the size and direction were consistent.The recombinant plasmids were transfected into 293T cells and subjected to RT-PCR to obtain target bands of 498, 1 146 and 1 349 bp, respectively, indicating that the target gene could be transcribed normally in eukaryotic cells.After the above recombinant plasmids were electrically transferred into attenuated Salmonella Choleraesuis C500 competent cells, the extracted plasmids were digested and the band sizes were 498, 1 146 and 2 622 bp, respectively.The sequencing results were consistent with the target sequence, indicating that the recombinant plasmids were successfully introduced into attenuated Salmonella Choleraesuis C500.The D_{600 nm} values of recombinant engineering bacteria and attenuated Salmonella Choleraesuis C500 bacteria detected every 2 h were similar, and the difference was not significant.The growth curve test results showed that during the process of 50 consecutive passages in vitro, the logarithmic growth period of both engineering bacteria and attenuated Salmonella Choleraesuis C500 bacteria were around 0-10 h, with a growth plateau period of 10-14 h.After about 16 h, entering the platform phase, the growth characteristics of the engineering bacteria were consistent with those of the attenuated Salmonella Choleraesuis C500, and did not change due to carrying plasmids.At the same time, the amplified bands of the Salmonella genus marker gene invA and virulence gene crp in each generation of bacterial fluid were 580 and 599 bp, respectively.The results showed that the recombinant engineered bacteria still possessed the characteristics of Salmonella after multiple passages, and their detoxification characteristics remained unchanged.The results of plasmid digestion in each generation were 498, 1 146 and 2 622 bp, indicating that multiple passages did not affect the stability of the plasmid, and the recombinant plasmid was able to maintain normal copy function in Salmonella Choleraesuis C500.【Conclusion】 The constructed recombinant plasmids and engineered bacterial vaccines had good stability and could be expressed normally after being introduced into the body, making them suitable for research on animal immune castration.

Key words: gene vaccine; immune castration; GnRH; KISS1; NKB