Construction And Evaluation Of A Co-Expression DNA Vaccine Against Somatostatin And Cortistatin Without Antibiotic Resistance Gene

Our previous research had constructed two plasmids co-expressed both cortistatin（p IRES-S/CST14-S/2SS）and somatostatin（p IRES-S/2SS-S/CST14）.These vaccines have been identified effective to improve growth of mice,respectively.However,there were limitations in the efficiency caused by the IRES element of p IRES vector,and the antibiotic resistance genes contained in the plasmid,affecting the biological safety and ease of operation of these two vaccine.In this study,2A peptide strategy was used to construct co-expression vaccine named p VGS/2SS-2A-S/CST14-asd without antibiotics gene and transform it to attenuated Salmonella typhimurium C500 by electro-transformation.The mice were immunized with three doses of recombinant attenuated Salmonella typhimurium C500.The objective of this study was to compare its efficacy from immune efficacy,growth promoting effect and safety,and identify the best immune dose.The main contents and results are as follows:1.Construction of p VGS/2SS-2A-S/CST14-asd plasmid.The synthesized 2A-S/CST14 fragment was inserted into the downstream of S/2SS in the constructed vector p VGS/2SS-asd,named p VGS/2SS-2A-S/CST14-asd.Double enzyme digestion and sequencing results showed that the plasmids were successfully constructed.2.Expression and identification of double expressed vaccines in eukaryotic cellsPlasmids p VGS/2SS-2A-S/CST14-asd and p VAX-asd were transfected to He La cell48 h to extract protein respectively.The results of Western Blot show that p VGS/2SS-2A-S/CST14-asd can efficiently express GS/2SS and S/CST14 fusion protein in eukaryotic cells.The expression of up-stream genes was same with the down-stream.3.Study on the stability of recombinant attenuated Salmonella C500The p VGS/2SS-2A-S/CST14-asd co-expression plasmid was electrotransformed into attenuated Salmonella typhimurium C500.The 40 generation was continuously passaged for the detection of stability in vitro.The results showed that the growth rhythm of the second,fifth,tenth,20th,30th and 40th generations of C500（p VGS/2SS-2A-S/CST-asd）and C500 strains were the same,and did not change significantly because of loading plasmids.The crp,inv A,and GS/2SS-2A-S/CST genes were amplified by PCR,which proved that the recombinant bacteria could maintain stability during the passage process and did not recover the virulence.4.Evaluation of the best dose and effect of new vaccine immunized miceKM female mice at age of 4 weeks were intramuscularly immunized twice at an interval of 2 weeks with three doses(0.4×10¹⁰CFU,0.4×10⁹CFU,0.4×10⁸CFU)of the co-expression vaccine so as to explore the best immune dose of the vaccine.The results show that all the experimental group of mice induced by a strong immune response,the positive rate of each dose group 4 weeks after vaccination was 100%.8 weeks after immunization,the positive rate in the high and middle dose groups was 100%,and the positive rate in the low dose group was 90%.The middle dose group were significantly higher than the other two groups in the level of antibodies,antibody positive rats body weight,total weight gain was significantly higher than control group（P<0.01）,weight gain increased by 24.29%in comparison with the control group.5.Safety evaluation of co-expression vaccineAt the end of the experiment,ten organs or tissues of mice were collected,including the brain,heart,liver,spleen,lung,kidney,small intestine,ovary,stomach and muscle of injection site.The results showed that there was no integration between plasmid and genomic DNA by PCR tests and no tissue lesion was found by making paraffin sections.The results suggested that the novel co-expression vaccine was relatively safe.