Biocharacteristics Of Terminase Large Subunit Encoded By Streptococcus Suis Phage SMP

Streptococcus suis (S. suis) is a common zoonotic pathogen that leadto sepsis, pneumonia, endocarditis, arthritis and meningitis in piglet ormeningitis and death in human, in which Streptococcus suis type2(SS2)is the most serious and widely distributed and posed a great threat tohuman life and public hygiene. More serious, the increasing prevalence ofantibiotics resistant SS2makes it difficult to prevent and treat the disease.The research based on phage to prevent the disease caused by SS2isbecoming a new hot spot.Terminase is the essential protein that mediates dsDNA packaginginto the viral procapsid. Generally, the large subunit of terminase hasATPase and restriction enzyme activity. SMP is a SS2lytic phageisolated from a mini-pig. Although the whole genome of the phage hasbeen sequenced, the phage terminase is still to be characterized. In thisthesis, The phage terminase large subunit (TlsSMP) was expressed byrecombinant Escherichia coli and the ATPase bioactivity have beentested.The structure and function of the terminase domain was predicted by using SWISS-MODEL, NCBI CDD and PSIPRED. Its homologycompared with G2P (nuclease domain from the bacteriophage SPP1)deposited in GenBank is up to30.5%.To express the TlsSMP, the Tls gene (TlsSMP) derived from thevirulent streptococcus suis phage (SMP) was amplified by PCR and thensubcloned into an expressing vector of pEasy-E1. The recombinantplasmid pEasy-E1-TlsSMP was transformed into E. coli BL21(DE3).The recombinant TlsSMP protein was expressed in supernatant afterinduction with IPTG． The maximized expression was induced by0.4mmol/L IPTG at16℃for15hours. Furthermore, the expressedprotein of about53.26kD was confirmed in the western-blottingexperiment.To characterize the activity, the TlsSMP was purified. The purerecombinant protein could reach the concentration about1-1.5mmol/L.The ATPase function of recombinant protein was found because TlsSMPcould catalyze ATP to ADP and Pi3+, and the bioactivity was detected byphosphate assay kit. The maximum bioactivity could be reached underthe condition of pH7at the temperature of23℃when Mg2+existed inthe buffer. It is reported that terminase of phage also can cut concatemericDNA to generate a headful-size viral genome, but this property need to beconfirmed in TlsSMP. The study of TlsSMP will provide information forfuture uncovering of streptococcus suis phage packaging mechanisms.