Construction,Evaluation And Mechanism Of A Novel DNA Vaccine Dually Expressing Somatostatin And Cortistatin

Dual eukaryotic expression system has great advantage over single expression systems,as it can lead to 2-fold increase in the expression of exogenous genes.Double eukaryotic expression vector,which contains Somatostatin(SS)and Cortistatin(CST)fragments can stimulate the body’s immune response.So,in theory the vaccines for dually expressed SS and CST genes may further boost the immune response which will ultimately promote animal growth.This study aims to construct pIRES plasmid having hepatitis B surface antigen 3’end,and contains IRES component in which CST-14 peptide and S/2SS fragment are cloned for the double expressions of Somatostatin(SS)and Cortistatin(CST)plasmid,and is named as pIRES-S/CST14-S/2SS(hereafter referred to as a dually expressing plasmid).After that,this plasmid was transfected into GH3 and Hela cells to observe the regulatory mechanism of these genes.Then,these vectors were injected intramuscularly for immunization for double expressions of somatostatin and cortistain in 4 weeks old female Kuming mice.In another group immunization against SS DNA vaccine(pVGS/2SS-asd)was achieved by oral or intramuscular route in 4 weeks old female BALB/c mice.All of these experiments will help to find out the safe and efficient route of immunizations against somatostatin DNA vaccine.1.The construction and expression of the dually expressing plasmidIn the synthetic CST-14 segments of hepatitis B surface antigen S and the existed S/2SS segment,the two fragments were cloned to plasmid pIRES at MCSA and MCSB sites for double expressions of SS and CST plasmid and was named as pIRES-S/CST14-S/2SS.After that,double enzyme digestion and sequencing of S/CST14(729bp)and S/2SS fragments(789bp)showed that the gene insertion point,direction and sequence were inserted correctly.Then,the plasmid was transfected into Hela cells by using LTX and the expressions of these fragments were confirmed by using RT-PCR,Western blotting and immunefluorescence after 48h transfection.Immunofluorescence result showed the positive expressions and western blotting confirmed the protein fragments of fusion protein S/CST14(26KDa)and S/2SS(28KDa).These results indicate that this plasmid can be used for successful expressions in eukaryotic cells.2.The regulative mechanism of dually expressing plasmidIn this study,pIRES-S/CST14-S/2SS plasmid was transfected into GH3 cells to observe regulatory functions of SS and CST14.The results showed higher level of cAMP and somatostatin receptors(SSTR1-5)and therefore indicated that overexpression of SS and CST could decrease the concentration GH and PRL by regulating these receptors(P<0.05).Western blotting results showed that SSTR2 protein was lower pIRES-S/CST14-S/2SS plasmid than single expressed cortistatin plasmid(pIRES-S/CST14).This could be due to the double expression of CST14 and SS,which attenuate their effects.These results may indicate that GH3 cells can be used in an animal model to detect the function of the somatostatin DNA vaccine.3.Effects of the dually expressing plasmid immunization of somatostatin and cortistatin on growth and immune responses in miceIn this study,180 Kunming female mice(4 weeks old)were divided into nine groups including three groups of double doses of expression vectors,three other groups of SS or CST plasmid and their dual expression vector,a positive control group of somatostatin(SS)DNA vaccine delivered by attenuated Salmonella choleraesuis(pVGS/2SS-asd),blank plasmid pIRES and a group injected with NaCl.After this,levels of anti-SS antibody(IgG),growth hormone and CST were measured.After 8 weeks of totalimmunization,total weight gain in low dose of dually expressing plasmid vaccine group(10μg/100μl)was 8.8%higher than positive control group.Interestingly,positive detection of anti-SS antibody in low dose of dually expressing plasmid group was 15%lower than positive group,however,average and highest levels of anti-SS antibody was higher than positive control.The results showed that the low dose of dually expressing plasmid vaccine could produce the same level immune response and weight gain as positive control,but with the precaution that further studies are required,how to improve the positive rate of the vaccine immunization?4.The safety of the dually expressing plasmid in miceAt the end of immunization experiment(part 3),180 mice were killed by cervical dislocation,and heart,lungs,liver,spleen,kidneys,brain,small intestine,ovaries,muscle at the site of immunization were collected for total DNA.Then,the DNA samples wereanalyzed by PCR.The results showed that no evidence of integration(7.5 × 10-8)was found in the DNA of mice tissues.The sensitivity of PCR was about 10 copy/μg.5.To explore the determinants which improve the somatostatin DNA vaccine immunizationComparison of biodistribuion,immune response and growth promoting effect of a somatostatin(SS)DNA vaccine delivered by attenuated Salmonella choleraesuis(pVGS/2SS-asd)were made between oral and intramuscular administrations to explore an effective way of improving DNA immunoreactions.In the present study,forty mice were divided into two groups(n=20 in each)and immunized via oral or intramuscular(i.m.)routes with pVGS/2SS-asd plasmid encoding two copies of SS.DNA samples were collected from different tissues and organs on Day 7,Day 21,Day 35 and Day 49 to determine the plasmid biodistribution and quantification(by real time PCR).In another experiment,after immunization using same vector(n=10 each),weight gain and immune response was measured.The results showed that,i.m.vaccination induced significant increase in specific total IgGs(IgQ IgG1,IgG2a and IgA)antibodies,promoted weight gain and showed a bias(IgG1 to IgG2a ratio>1)typical of the Th2-phenotype in both groups.Biodistribution studies showed that C501(pVGS/2SS-asd)plasmid was persistent in the muscles surrounding the site of injection in both groups for 7 weeks.Furthermore,intramuscular vaccination exhibited a quick surge(4 weeks)and higher DNA copy numbers during whole experimental period compared to oral immunization.These results indicate that the immune and growth promoting effect can be improved by increase of plasmid in the the muscle of mice immunized against Somatostatin DNA Vaccine delivered by attenuated Salmonella choleraesuis.