Construction of recombinant SAG22 Bacillus subtilis and its effect on immune protection of coccidia

Abstract

Avian coccidiosis causes huge economic losses to the global poultry industry. Vaccine is an important means to prevent and control coccidiosis. In this study, Bacillus subtilis was selected as the expression host strain to express anti Eimeria tenella surface protein SAG22. The synthesized surface protein SAG22 gene fragment of E. tenella was ligated with Escherichia coli-bacillus shuttle vector GJ148 to construct the recombinant vector SAG22-GJ148. And then the recombinant Bacillus strain SAG22-DH61 was obtained by electrotransfer. The results of SDS-PAGE and Western Blot showed that the recombinant protein SAG22 was successfully expressed intracellularly. The immunoprotective effect of recombinant Bacillus strain SAG22-DH61 on broiler chickens was evaluated in 3 identically designed animal experiments. The birds were infected with E. tenella on d 14, 21, and 28, respectively. Each batch of experiments was divided into 4 groups: blank control group (NC), blank control group + infected E. tenella (CON), addition of recombinant SAG22-DH61 strain + infected with E. tenella (SAG22-DH61), addition of recombinant empty vector GJ148-DH61 strain + infected with E. tenella (GJ148-DH61). The animal experiments results showed that the average weight gain of the SAG22-DH61 group was higher than that of the infected control group, and the difference was significant in the d 14 and 28 attack tests (P < 0.05); the oocyst reduction rate of the SAG22-DH61 group was much higher than that of the GJ148-DH61 group (P < 0.05); the intestinal lesion count score of the SAG22-DH61 group was much lower than that of the GJ148-DH61 group (P < 0.05). In addition, the SAG22-DH61 group achieved highly effective coccidia resistance in the d 14 attack test and moderately effective coccidia resistance in both the d 21 and 28 attack tests. In summary, recombinant SAG22 B. subtilis has the potential to become one of the technological reserves in the prevention and control of coccidiosis in chickens in production.

Keywords: Bacillus subtilis; E. tenella; SAG22 protein; immunological effect.

Figure 1

The PCR amplification of gene SAG22. M:DL2000 marker;1: gene SAG22.

Figure 2

The PCR amplification of GJ148-SAG22.M;DL2000 marker;1-12: Recombinant plasmid GJ148-SAG22 Identification product.

Figure 3

Enzyme digestion of GJ148-SAG22.M;DL5000 DNA marker;1: EcoRI/BamHI double digestion products;2: Negative control.

Figure 4

Identification of recombinant SAG22 protein by SDS-PAGE.M.26616 marker;1:Recombinant strain lysis supernatant;2:Recombinant strain lysis bacteriophage precipitation;3:Vaccum lysis supernatant;4:Lysis of bacteriophage precipitated by unloaded bacteria.

Figure 5

Identification of recombinant SAG22 protein by Western. 1:Maker;2:Recombinant strain lysis supernatant;3:Recombinant strain lysis bacteriophage precipitation;4:Vaccum lysis supernatant;5:Lysis of bacteriophage precipitated by unloaded bacteria.

Figure 6

Weight gain results in broilers infected with E. tenella at different ages. All data were analyzed with ANOVA: **P < 0.01, and *P < 0.05. Abbreviations: CON, blank control group + infected E. tenella; GJ148-DH61, addition of recombinant empty vector GJ148-DH61 strain + infected with E. tenella; NC, blank control group; SAG22-DH61, addition of recombinant SAG22-DH61 strain + infected with E. tenella.

Figure 7

Oocyst output results in broilers infected with E. tenella at different ages. Among them, d 14 broiler infect test is A, d 21 broiler infect test is B, d 28 broiler infect test is C. All data were analyzed with ANOVA: **P < 0.01, and *P < 0.05. Abbreviations: CON, blank control group + infected E. tenella; GJ148-DH61, addition of recombinant empty vector GJ148-DH61 strain + infected with E. tenella; NC, blank control group; SAG22-DH61, addition of recombinant SAG22-DH61 strain + infected with E. tenella.

Figure 8

Mean lesion scores results in broilers infected with E. tenella at different ages. All data were analyzed with ANOVA: **P < 0.01, and *P < 0.05. Abbreviations: CON, blank control group + infected E. tenella; GJ148-DH61, addition of recombinant empty vector GJ148-DH61 strain + infected with E. tenella; NC, blank control group; SAG22-DH61, addition of recombinant SAG22-DH61 strain + infected with E. tenella.

Figure 9

Anticoccidial index results in broilers infected with E. tenella at different ages. Abbreviations: CON, blank control group + infected E. tenella; GJ148-DH61, addition of recombinant empty vector GJ148-DH61 strain + infected with E. tenella; NC, blank control group; SAG22-DH61, addition of recombinant SAG22-DH61 strain + infected with E. tenella.