| Previous studies have shown that gene immunization against somatostatin can promote the production of anti-somatostatin antibodies in animals,neutralize the endogenous somatostatin,block the interaction between somatostatin and its receptors,and promote the secretion and release of growth hormone（GH）,insulin-like growth factors-1（IGF-1）and other hormones,thereby promoting the growth of animals and improving their production performance.The laboratory has constructed a non-antibiotic resistance DNA vaccine C500（pVGS/2SS-asd）with pVGS/2SS-asd carried by attenuated salmonella choleraesuis live bacteria,which has the advantages of low preparation cost,easy operation and good safety,and has achieved good growth-promoting effect in clinical trials on a variety of animals.Clinical trials on pigs have been completed in both small experiment and pilot experiment,and it is necessary to carry out a field experiment to provide scientific basis for the transgenic biosecurity assessment and the application of new veterinary medicine.In this study,we electrotransformed pVGS/2SS-asd which stored in laboratory and identified correct intoΔasdΔcrp double deletion attenuated salmonella choleraesuis live bacteria C500 strain to prepare non-antibiotic resistance DNA vaccine with pVGS/2SS-asd carried by attenuated salmonella choleraesuis live bacteria（hereinafter referred to as DNA vaccine）,identifyed the stability of DNA vaccine and selected the best generation vaccine engineering bacteria as fermentable seeds for vaccine preparation.After the experimental growing pigs were immunized with DNA vaccine,the weight gain,backfat thickness and immune response were observed,and the antibody correlation analysis was conducted to comprehensively evaluate the growth-promoting effect of the vaccine.At the same time,the vaccine security related items including the body temperature and reaction observation of the experimental animal,the recombinant gene fragments spread of the vaccine environment release,the DNA integration analysis,and the histopathological observation was monitored after vaccination to evaluate the vaccine security.The purpose of this study was to explore the immunogenicity,immune response and growth-promoting effect of DNA vaccine during the growth period and observe and monitor the safety of DNA vaccine in a larger scope under more a sample size and combining with actual production conditions.The main research contents and results are as follows:1.Stability identification of DNA vaccineThe pVGS/2SS-asd plasmid preserved in the laboratory was amplified,identified by double restriction enzyme digestion analysis and first generation sequencing,and then electrotransformed into attenuated salmonella choleraesuisΔasdΔcrp C500 strain to prepare DNA vaccine C500（pVGS/2SS-asd）.After the vaccine engineering bacteria which identified correct are continuously cultured for 10,20,30,40,50 and 60generations,detected the key genes of the vaccine by colony PCR,it was found that the key genes GS/2SS,Δasd,invA andΔcrp were stable existence during consecutive cultures for 60 generations,suggesting that the vaccine engineering bacteria have good genetic stability,the plasmid is not lost,have invasiveness and the toxicity does not recover during the culture process.Each generation of vaccine engineering bacteria was cultured at a constant temperature in LB medium for 24 hours,sampled every 1 h to determine the growth curve of vaccine engineering bacteria by measuring OD 600,it was found that each generation of vaccine bacteria grew to the logarithmic phase at 5h,entered the stationary phase at the 10h,reached the maximum at 12h.The 20 and 30 generation vaccine engineering bacteria grew better than other generations.The 12h bacteria volume was significantly higher than other generations（P<0.05）,suggesting that the 20 and 30generations are the best generation of vaccine fermentation preparation.2.The immune response of DNA vaccine on growing pigsA total of 147 DLY crossbreeding pigs 50 days-age were selected and divided into 4groups,which were nasally inoculated with a somatostatin DNA vaccine baterina C500（pVGS/2SS-asd）in high(4.5×1010,n=39),medium（4.5×109,n=35）,low（4.5×108,n=35）and 0-dose（with PBS,as a negative control,n=38）at 65 days-age,respectively.The immunization was performed twice a day for three consecutive days,and boosted once after 45d.Blood was collected to separate serum samples fordetections of antibodies,hormones and immune cytokines by use of ELISA methods.The results showed that all three immune doses can induce the body to produce somatostatin antibodies.The positive rates of somatostatin antibodies in animals immunized with high,medium and low doses of DNA vaccine were 30.8%,37.1%,and 40%,respectively for the initial immunization.The antibody positive rates at the time of slaughter were 20.5%,14.3,and 20.0%,respectively.The concentrations of GH,IGF-1,IL-4 and IFN-γin blood of animals with somatostin antibodies were higher than those in animals without somatostatin antibodies,although the difference was not significant（P>0.05）.3.The growth-promoting effect of DNA vaccine on growing pigsThe pigs in each group were weighed head by head on d 45（110 days-age）after the first immunization and slaughtering time（185 days-age）.The result shewed that the whole period body weight gain in the immune group（0.72±0.01 kg/d）was significantly higher than that in the control group（0.69±0.01 kg/d）（P<0.05）,the weight gain of the immune group at d 50 to d 110（0.73±0.01 kd/d）was significantly higher than that of the control group（0.69±0.01 kd/d）（P<0.05）,but there was no significant difference between the immune group（0.72±0.01 kg/d）and the control group（0.69±0.01 kg/d）in body weight gain at d 110 to d 185（P>0.05）.By comparing the dose relationship,it was found that the low-dose group（0.73±0.01 kg/d）showed better weight gain than the other dose groups.Body weight gain in the antibody positive group was significantly higher than that in the antibody negative group at all period（P<0.05）,In the antibody positive group,the body weight gain was increased by 15.14%at d 50 to d 110,increased by 4.33%at d 110to d 185 and increased by 9.75%at the whole period.There was a significant positive correlation between whole period weight gain and antibody levels at all periods（r=0.396,r=0.446）.4.The improvement of meat quality of growing pigs by DNA vaccineBackfat thickness was measured head by head at d 185,the result shewed that the backfat thickness of the antibody positive group was significantly negatively correlated with the antibody level at 110 days-age（r=-0.406）.The meat quality analysis for longissimus dorsi after slaughter showed that antibody positive pig intermuscular fat（EE）was significantly lower than antibody negative group（P<0.05）,the others measured meat quality indicators were not significantly different from antibody negative group（P>0.05）.5.The safety assessment of DNA vaccineThe body temperature of each experimental pig was measured on day 1,3,5,7,10,12 and 15 after vaccination and the behavioral reaction was observed.No significant change in body temperature was found before and after vaccination（P>0.05）;the mental state,food intake and defecation of the experimental pigs did not change obviously.At day 3,5,7,10,15 and 30d after inoculation,the soil samples at 1,3,5m and 7m vents in the east,west,north and south of the experimental barn,and the water samples,the fecal samples in the experimental corral were collected respectively for DNA vaccine fusion fragment GS/2SS PCR amplification,it was found that the recombinant fragments were detected in the water samples on the 5d and the feces samples on the 3d and 5d,but the recombinant fragments were not detected in the samples after 5d.Blood samples of all experimental pigs,muscle,lung and liver tissue samples of antibody positive individuals were collected after slaughter,blood and tissue genomic DNA was extracted for DNA vaccine fusion fragment GS/2SS amplification,results showed no target bands were detected in all samples within the PCR sensitivity range.HE pathological sections of muscle,lung and liver tissue samples of antibody positive individuals were prepared and observed,no pathological changes were found. |
