| Streptococcus suis type 2 (SS2) and Actinobacillus pleuropneumoniae(APP) are two kinds of critical pathogens in swine infectious disease, and both of them are high incidence and high contagious world wide, which have brought great losses to the Pig industry. SS2 also can cause human's disease or even death, which have great significance for public health, and it is a kind of important zoonosis pathogen; While APP has numerous pathogenic serum type, and the 1,2,3,7 serums type popular in China, so it is very difficult to against it. Many studies show that SS2 and APP have lots of virulence factors. With the development of research, we found some virulence factors can provide immune protection for laboratory animal against the two kind of bacteria, therefore some virulence factors can be used as the subunit vaccine.The gene fragments of muramidase release protein (MRP),suilysin (SLY) and actinbacillus pleuropneumoniae toxin IA(ApxIA) were respectively amplification with the genomic DNA of SS2 (HA9801) and APP (S259) as templates. Then the gene fragments were sub-cloned into plasmid pET-28a (+) vector and obtained restructuring expression plasmid mrp-pET28a,sly-pET28a and ApxIA-pET28a. After enzyme cutting and sequencing, recombinant plasmids were transformed in E. coli BL21 and expressed the 23kDa,26 kDa and 40 kDa fusion protein by IPTG induction respectively. The results of Western blotting showed that three protein can be recognized by rehabilitation serum very well. The mice, immunned by the three kind of fusion proteins mixed with ISA-206 respectively, could produce specific antibodies, the antibody titer goes up to 1:204800 and maintain the high level more than 10 weeks by ELISA monitor. Balb/C mice were immunned by mixture of MRP and SLY, and ICR mice were immunned by ApxIA (50μg each). After the third boostered, the mice were challenged by SS2 HA9801 (10 times LD50, 1.4χ108CFU) and APP1 S259 (5 times LD50,4χ107 CFU).The protection rate of Balb/C mice immunned by mixture of MRP and SLY was 72%, ICR mice immunned by ApxIA was 66.7%.Based on the research of the front, MRP was spliced with ApxIA by a dewatering and flexible polypeptide (GlyGly GlyGlySer)χ2 gene as connector. After constructing ApxIA-linker-mrp fusion gene and sub-cloning in pMD18-T Simple vector, it showed the length of the fragment was 1512 bp by enzyme cutting and sequencing, and the sequence and direction of the inserting fragment was also completely correct. The fusion gene was sub-cloned into plasmid pET-28a(+) vector, obtaining restructuring plasmid ApxIA-mrp-pET28a. Western blot showed the fusion protein ApxIA-MRP could response subtly to the serum from recovery rabbits of the two kinds of bacteria. ICR mice was immunned three times by the fusion protein mixed with ISA-206. It showed that the antibody titer could get up to 1:102400 and last for 11 weeks. The piglets(weaning pigs, aged 30 days) were immunned three times by the vaccine, and the first immunization was lmg each, second was 800μg each, and third was 500μg each. The antibody titer was 1:3200,1:51200,1:204800 at the time of 1 week after each immunization by monitoring respectively. The piglets were injected by 10 times of LD50 of S259 and HA9801 after the third immunization, and the results showed that protection rate of the vaccine was 80%and 100%respectively. The research proofed that the series fusion protein can provide certain protection from the homologous strains, which provide the basis for the research of the genetic engineering subunits vaccine of SS2 and APP... |
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