Study On The Functions Of T3SS Effector Proteins Of Vibrio Alginolyticus

The type 3 secretion system effector proteins of vibrio are with a variety of pathogenic protein function of host cell. So research on the structure and function of effector proteins has great significance to study its pathogenic mechanism and vaccine production. 3 pairs of primers of a cloning of effector proteins va1686, type3hy322 and hopPmaJ were designed according to the sequence number of Vibrio alginolyticus HY9901 in GenBank and obtain the total length of the 3 genes for PCR amplification.Sequence analysis shows that hopPmaJ gene is 345 bp and eneodes a putative protein of 114 amino acids.Using Ex PASy software to analyze the HopPmaJ protein, the results showed that the number of amino acids was 114, the total number of atoms was 1755, the theoretical molecular weight was 12.7742 kD, and the theoretical pI value was 4.45. Signal Server 4.1 software predieted that the HopPmaJ amino acid sequence did not contain a signal peptide or a transmembranous region. The HopPmaJ amino acid sequence contains 2 N- glycosylation sites, acylation sites of 2 protein kinase phosphorylation sites, 2 casein kinase II phosphorylation site, 1 prenyl groups binding sites, 2 micro positioning signal the end of the C-end.The gene va1686 and type3hy322 of V. alginolyticus HY9901 was cloned. Sequence analysis shows that Type3hy322 is a T3 SS transfor protein, the sequence length is 969bp; Va1686 is a a permease protein, length is1164 bp.The va1686 and type3hy322 of V. alginolyticus HY9901 was expressed with E.coli cell. The type3hy322 was expressed excellently in IPTG(0.4mM),37℃, induced for 12 h,the va1686 was expressed excellently in IPTG(0.1mM),37℃, induced for 12 h. Also, the biological information above the two proteins was researched. The Type3hy322 is 322 aa,wt=35.480 KD,Va1686 is 387 aa,wt= 41.342 KD.Using PCR, the amplification above sequence of up-stream and down-stream of hopPmaJ was finished, then, fuse with vector pRE112(Cm+)and pACYC184(Cm+), restriction enzyme digestion, connect with DNA ligase, and transformation, construction of joint bacteria species, homologous recomnination twice times, selection to deficiency bacterial species, after determined by PCR and sequencing, a bacterium lacked of the hopPmaJ gene, deletion in its ORF was constructed, and named ΔhopPmaJ.The biological chracteristics and fishy protectiveness of ΔhopPmaJ was researched. The researching results showed that ΔhopPmaJ is inheritance steadily in 30 continuous from generation to generation,compared with a V. alginolyticus HY9901(Wild-species),the growth rate,biofoam forming, the activity of out-cell enzyme, there are not significant changes, but the swimming capacity, the dead rate to fish is decreased significantly("<0.5, p<0.5). Using 106cfu/m L ΔhopPmaJ to immerse the experimental fish, after 28 d challenge with 5 L 108cfu/mL V. alginolyticus HY9901(Wild-species)to the experimental fish,the fish immersed ΔhopPmaJ gained 35% protection rat,there was no significant(R≥0.5,p≥0.05). Using 105 cfu/m L ΔhopPma J to inject the experimental fish,after 28 d challenging with 5 L108cfu/m L V. alginolyticus HY9901(Wild-species)to the experimental fish, the fish injected ΔhopPmaJ gained 75% protection rat, there was significant(R≤0.5,p≤0.05). The results shows that ΔhopPmaJ decreased its pathopoiesia, and increasing the host immune capacity compared with Wild-species V. alginolyticus.