Construction of the expression plasmid pXK88ac3STaLT2
Using PCR site-directed mutagenesis technology, Three 60-bp STa genes with the TGT→AGT(Cys→Ser) mutation was amplified by using three pairs of STa PCR primers from E. coli C83902 plasmid. The three cloned STa mutant genes were connected in series into 180bp STa-STa-STa fusion gene. The 330-bp K88ac and 500-bp LT2 gene fragments were amplified separately using C83902 plasmid DNA as the template. The constructed STa-STa-STa fusion gene was connected with the K88ac and LT2 genes to construct the fusion gene K88ac-3STa-LT2 andcloned into pET-28b to generate the recombinant plasmid pXK88ac3STaLT2. It was transformed into the recipient bacteria BL21(DE3), and the recombinant strain BL21 (DE3) (pXK88ac3STaLT2) was constructed. Restriction enzyme digestion of pXK88ac3STaLT2 with Nco I/Not I confirmed the presence of the K88ac-3STa-LT2 gene. Moreover, the TGT→AGT(Cys→Ser) mutation was confirmed by sequence analysis (Fig. 1).
SDS-PAGE analysis and ELISA detection of K88ac-3STa-LT2 fusion protein
BL21(DE3)(pXK88ac3STaLT2) culture was induced with IPTG for 4 h at 37℃, and SDS-PAGE analysis showed that K88ac-3STa-LT2 was expressed highly in the E. coli strain BL21(DE3). The K88ac-3STa-LT2 proteins accounted for 73.53% of total cellular protein(Fig.2). The K88ac-3STa-LT2 fusion protein could be recognized by the STa monoclonal antibody, K88ac and LT2 antibody through STa, K88ac and LT2 ELISA detection kits.
Safety test of recombinant strain BL21(DE3)(pXK88ac3STaLT2)
In order to determine whether the K88ac-3STa-LT2 fusion protein expressed by the recombinant strain had lost the STa enterotoxin activity, Recombinant strain BL21(DE3)(pXK88ac3STaLT2) was inoculated to mice by intraperitoneal injection and oral administration. All the mice survived after 3 weeks without clinical symptoms and no pathological changes during necropsy, indicating that the strain was not pathogenic and very safe.
Mice and piglet challenge protection test
The test determined that the minimum lethal dose (1 MLD) for mice was 50 million live bacteria, and the minimum lethal dose (1 MLD) for piglets was 200 million live bacteria. The mice immunized with the crude inclusion body and the engineered strain inactivated vaccine were challenged with the E. coli C83902 and both obtained better immune protection.
With 1 MLD challenge, the protection rate of the inclusion body immunization group was 95% (38/40), and the protection rate of the inactivated vaccine immunization group was 97.5% (39/40). With 2 MLD challenge, the protection rate of the inclusion body immunization group was 90% (36/40), and the protection rate of the inactivated vaccine immunization group was 95% (38/40).
Two sows immunized with crude inclusion body produced 25 piglets and the other two sows immunized with engineered strain inactivated vaccines produced 24 piglets. As a control group, the sow produced 12 piglets. All piglets were healthy and were challenged. After the newborn piglets were challenged, 23 piglets immunized with crude inclusion body survived and the protection rate was 92% (23/25) and 21 piglets immunized with the inactivated vaccinecrude survived and the protection rate was 87.5% (21/24). In the control group, 11 of the 12 piglets died, and the mortality rate was 91.67% (11/12).
Neutralization Test of intragastric administration in the suckling mouse
Different doses (10, 12, 15, 17, 20 μL) of STa enterotoxin were administrated intragastrically to suckling rats, and the G/C values were 0.072, 0.081, 0.093, 0.108, 0.124, respectively. Therefore, it was determined that the STa enterotoxin of 1 murine unit was 15μL.
The STa enterotoxin of 1 murine unit (15μL) was added with 15 μL of immune rabbit serum and diluted with normal saline to 0.1 mL. After induction at 37 ℃ for 1 hour, the test of intragastric administration in the suckling mouse was carried out.The result showed that the G/C value of the control group(STa+Normal Saline,STa+Sera of healthy rabbit) is not less than 0.09, while the G/C value of the test group(STa+Sera of immunized rabbit) is close to the G/C value of sera of healthy rabbit and LB broth. The G/C value of the test group is not higher than 0.083. This indicates that the serum antibodies induced by the inclusion bodies of the K88ac-3STa-LT2 fusion protein in rabbits can neutralize the STa enterotoxin produced by pathogenic bacteria (Table 1).
Effect of different media on the growth of strains
The three configured mediums had a greater impact on the growth of bacteria. Among them, the number of bacteria in the modified LB medium compared with the LB medium increased significantly (P<0.01), and the number of bacteria in the normal broth medium compared with the LB medium increased significantly (P< 0.01), there was little difference in the number of bacteria between the normal broth medium and the modified LB medium (P>0.05). From the perspective of the stability of the strain culture environment and the reduction of industrial production costs, the modified LB medium was the most suitable medium (Table 2).
Effect of different inducers on the growth of strains
The final concentrations of 1 mmol/L IPTG and 100 mmol/L lactose were used to induce expression, respectively. SDS-PAGE and gel imaging analysis showed that the expression levels of the two were 29.43% and 33.24%, respectively. Considering the production cost, lactose should be used as an inducer(Fig.3).
Effect of different induction time on the growth of strains
As the induction time increased, the pH value decreased first and then increased, and the total bacterial count and protein expression also increased accordingly. When the induction time was 6h, the protein expression reached the maximum value. SDS-PAGE and gel imaging analysis showed that the protein expression level was 30.02% (Fig.4).
Influence of different aeration on the growth of strains
The BL21(DE3)(pxK88ac3STaLT2) strain was aerated and cultivated with modified LB medium. The ventilation volume is 50 L/min, 100 L/min, 500 L/min. Among them, the number of bacteria under the culture condition of 500 L/min was the highest, and there was no significant difference in the expression of the target protein in the three cases .
Formaldehyde solutions of three different concentrations (0.4%, 0.6%, 0.8%) were added separately to the cultured bacteria for inactivation. The results showed that 0.4% formaldehyde solution for 48h can achieve a good inactivation effect.
Passive protection test of newborn piglets during the susceptible period
The healthy piglets produced by the sows were selected for a challenge test. Through this test, it was determined that the protection efficiency of piglets could reach over 80% on the 1st day after suckling and the protective effect was 90% on the 7th after suckling, which was the highest in the efficacy test(Table3).
Determination of the minimum immune dose in mice
After the 1MLD challenge, the immune protection rates of the group1, group3 and group5 were 63.3%(19/30), 86.7%(26/30) and 90%(27/30) in immunized mice. However, all mice in the control group died. The results showed that the minimum immune dose of the vaccine to mice was 0.2mL/mouse (Table 4).
Determination of the minimum immune dose of pregnant sows
After the 1MLD challenge, the immune protection rates of the Group1 and group3 were 84.5%(49/58) and 91.7%(55/60) in immunized piglets. However, all piglets in the control group died. The results showed that the minimum immunization dose for pregnant sows was 2.5 mL/sow(Table 5).