双表达去势DNA疫苗的构建及其稳定性研究

doi:10.16431/j.cnki.1671-7236.2021.05.026

Abstract

Abstract: The aim of current study was to construct a safer and more effective new type of immune castration DNA vaccine.With the help of the self-cleavage function of the 2A peptide and introducting of a balanced lethal system instead the resistance gene screening process,the hypot halamic-pituitary-gonadal axis (HPG) upstream regulatory gene kissin 1 (KISS1) and hormone-releasing hormone (GnRH) being selected as the target were successfully transferred into the non-resistant screening plasmid pVAX-asd.Enzyme digestion verification and sequencing comparison verification on the insertion direction and sequence of the target gene were completely correct.When recombinant plasmid being transfected into HeLa cells,the results of amplification of the target gene after reverse transcription showed that the recombinant plasmid could be transcribed normally in eukaryotic cells,ensuring that the recombinant plasmid could be expressed normally after being introduced into the body,thereby causing a specific immune response.The successfully constructed plasmid was transformed into the attenuated Salmonella choleraesuis C500 to obtain a live vector vaccine that could be directly orally immunized.The results of enzyme digestion and sequencing showed that the co-expression recombinant plasmid was successfully introduced into the engineered bacteria.The live bacteria vaccine was continuously passaged in vitro for 50 times,and strains of 0,2,5,10,20,30,40,and 50 generations were selected for stability study.The growth curve test results showed that there was no significant change in its growth characteristics when the engineered bacteria were continuously passaged in vitro for 50 times,and it was consistent with the growth characteristics of attenuated Salmonella choleraesuis C500.There also was no significant changes were found when it carrying plasmids.At the same time,Salmonella marker genes (invA) and virulence genes (crp) was used to amplified with each generation of bacterial fluid,the results showed that the engineered bacteria still had the characteristics of Salmonella after multiple passages,and their attenuation characteristics had not changed.Direct enzyme digestion verification of bacterial liquid with various generations showed that multiple passages did not affect the stability of the plasmid,and the recombinant co-expression plasmid could maintain normal copy function in Salmonella C500.In summary,both the recombinant plasmid and the engineered bacterial vaccine had good stability,which could be directly applied to the study of animal immunocastration.

Key words: DNA vaccine; KISS1; GnRH; 2A peptide; immune castration; stability

CLC Number:

S852.4

XU Xuelin, YANG Bo, JING Huansong, HU Yinghong, WANG Fenghua, SUN Peihao, XIONG Jiajun. Construction and Stability Study of Co-expression Castration DNA Vaccine[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(5): 1745-1754.

References

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Construction and Stability Study of Co-expression Castration DNA Vaccine

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