| Tuberculosis is one of the fatal infections that harm human health,and it is serious in our country.As the over use of spectrum antibiotics,MDR-TB and XDR-TB have been increasing. Finding a more effective medicine to control the infection,simultaneously reducing the medicine toxicity and preventing the drug resistance has become an important problem to be solved.Bacteriophage lysins has been called "new antimicrobial" because of these reasons:(1) very rapid and potent antibacterial activity both in vitro and in vivo;(2)a completely new mode of action,that is,enzymatic cleavage of peptidoglycan;(3)activity against bacteria regardless of their antibiotic sensitivity;(4) a narrow antibacterial spectrum;(5) low probability of developing resistance;(6) apparent safety;and(7) relatively easy modifications by means of genetic engineering.Mycobacteriophage D29 is a lytic phage,which could infect and destroy M.smegmatis and M.tuberculosis.In this study,we investigate Mycobacteriophage D29 as follows:1.Increasing Mycobacteriophage D29 and analysis of its proteins:M.smegmatis and Mycobacteriophage D29 were cultured in liquid culture medium,and the titer of phage D29 is 3×10~8.After analysis by Blast in NCBI and Interpro in EMBL,the gp10 has 30%～50%identity with lysins and its conserved domain belongs to lysozyme-like superfamily;the gp12 has 28%～42%identity with lysins and cutinases and its conserved domain belongs toα/βhydrolases superfamily.With consulting the literatures,we hypothesize that the gp10 and gp12 has the function of cell lysis.2.Reconstructed the prokaryotic vector and optimized expression target protein:The genome DNA was distilled from Mycobacteriophage D29.The gene10,gene11,gene12 and gene30 were amplified from mycobacteriophage D29 genome DNA by PCR,cloned into pET42a vector after been identified.Then the plasmids were transferred into BL21(DE3),the target proteins expression were induced by IPTG and the expression conditions were optimized.Under the condition of 30℃,0.5 mM IPTG induced 8h,the percentage of gp10 and gp12 of the total protein are 32.3%and 43.1%by the analysis of Bandscan software.3.Purified and renatured gp10 and gp12:The gp10 and gp12 were expressed in the form of inclusion body,and the expressed production can not be soluble by reducing the expression temperature.The gp10 and gp12 were purified by Ni-NTA column after been dissolved by 8M urea,renaturated the inclusion by dilution,dialysis,refolding via purification column,and optimized renaturation rate with 8 kinds of different renaturation solutions.After 1mM EDTA and 0.1%PEG2000 were added into the base renaturation buffer,the soluble renaturation ate of gp10 and gp12 were improved to higher than 30%and 40%.4.Study of the bacteriocidal activity of gp10 and gp12:The gp10 and gp12 were dissolved after had been desiccated,the lysis activity of gp10 and gp12 were evaluated by cylinder-plate method and double dilute method,and the inhibitory effect of gp10,gp12 and streptomycin, kanamycin,rifampicin were compared with.1024μg/mL gp10 formed 2mm transparent zones in the plate by cylinder-plate method and the minimal inhibitory concentration(MIC) is 128μg/mL, the inhibitory effect was lower than streptomycin,kanamycin and rifampicin.The gp12 didn't put up bactedostasis and didn't have assistant effect.In conclusion,we have constructed the prokaryotic expression vectors pET42a-gp10, pET42a-gp11,pET42a-gp12 and pET42a-gp30 and expressed the gp10 and gp12 of Mycobacteriophage D29 successfully.The gp10 and gp12 were purified after had been dissolved by urea.After optimized the renaturation buffer,we got higher soluble renaturation ate.The inhibitory experiments prove that the gp10 have inhibitory effect but the gp12 don't.Our findings will bring our research work to be promising.
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