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Expression and Study on Bilogical Activity of Holin by Bacteriophage from Streptococcus Suis

Author: LiNing
Tutor: LuChengPing;SunJianHe
School: Nanjing Agricultural College
Course: Preventive Veterinary Medicine
Keywords: holin (HolSMP) lysin (LySMP) lytic activity anti-infective effect
CLC: S852.611
Type: Master's thesis
Year: 2010
Downloads: 20
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Abstract


Streptococcus suis is an important zoonotic pathogen that causes meningitis, sepsis, arthritis, endocarditis and pneumonia in piglet or human meningitis. It has become an important disease to seriously affect the pig industry. Meanwhile, it is a big threatens to public hygiene and human life. Worse more, the severe antibiotics resistance of bacteria make it difficult to the prevention and treatment of this disease. Bacteriophage is becoming a potential bactericidal agent with high lysis activity based on holin-lysin system. In this work, Purified active holin (HolSMP) encoded by holin gene was obtained by recombinant expression in E. coli. The Bactericidal characteristics of the protein itself and its mixture with lysin (LySMP) encoded by SMP bacteriophage was carried out, and the anti-infective effect of LySMP in vivo was tested by using zebrafish as the infection model of Streptococcus suis.Purified active holin, designated HolSMP, was obtained by recombinant expression in E. coli. The DNA of SMP bacteriophage was extracted before a pair of primers was designed according to the holin gene, after amplification by PCR, the products were then inserted into a prokaryotic expression plasmid, pET-28a (+), to obtain the recombinant plasmid holin-pet28a. It was introduced into E. coli BL21 competent cells and sequenced to ensure a correct expression. After E.coli colones of holin-pet28a induced by IPTG, Recombinant HolSMP protein with 16 kDa was analyzed by SDS-PAGE. The purified HolSMP protein was lmg/mL.To find the biological activity of HolSMP in vitro, the best working conditions and lysis spectrum was analyzed. Mixture of crude products of HolSMP and LySMP lysate exhibited a more extensive lysis spectrum than those of whole phage or LySMP lysate against bacteria investigated. HolSMP can lysis Staphylococcus aureus and Bacillus subtilis. LySMP can lysis four out of seventeen strains of S. suis serotype 2, S. suis serotype 7 and S. suis serotype 9, while thirteen of seventeen strains of S. suis serotype 2 could be lysed with the joint action of HolSMP and LySMP, as well as S. suis serotype 7 and 9, Streptococcus equi, Staphylococcus aureus, Salmonella and Bacillus subtilis. But zooepidemicus and E.coli were not affected. Chromatographically purified HolSMP treated with 0.5% of (3-mercaptoethanol showed higher lytic efficiency. Optimal pH 5.2 and 37℃were investigated for the HolSMP by turbidity reduction assay.Zebra fish was applied as the infection model of Streptococcus suis to evaluate the anti-infective effect of HolSMP or the mixture of LySMP and HolSMP. The purified protein mixed with S. suis were inoculated in peritoneal and the survival rate was calculated. In this work, five strains of S. suis HA9801, SS2-4, SS2-H, S. swuisserotype 7 and S. suis serotype 9 were used respectively. The zebra fish were divided into 8 groups, each group have 30. In group1:Fishes were inoculated with mixture of 50 LD50 S. suis and 2μg HolSMP; in group2:Fishes were inoculated with mixture of 50 LD50 S. suis and 2μg LySMP; in group3: Fishes were inoculated with mixture of 50 LD50 S. suis and 2μg LySMP+2μg HolSMP; in group4:2μg amoxicillin was adminstered post infection with 50 LD50 S. suis. Group 5-8 was the control groups, and the fishes were inoculated only with 50 LD50 S. suis, PBS, LySMP or HolSMP, respectively. The results showed that the survival rate of the fishes treated with mixture of LySMP and HolSMP (group 3) were 53%、87%、70%、67% and 83% when challenging with HA9801, SS2-4, SS2-H, S. suis serotype 7 or S. suis serotype 9, respectively, While the survival rate were 53%、83%、50%、93% and 43% when treated only with LySMP (group 2). The result indicated that HolSMP can increase the anti-infective effect of LySMP, and the mixture of HolSMP and LySMP could be a candidate to control the disease caused by S. suis.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Pathogenic bacteria > Cocci
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