| Recently, an ever-increasing prevalence of antibiotic-resistant tuberculosis, especially multidrug-resistant tuberculosis(MDR-TB),results in the control of tuberculosis infection disease facing serious challenge. Mycobacteriophage can lyse Mycobacterium tuberculosis(MTB) and has the potential to treat TB.Studies indicated that phage delivery route was an important influencing factor on the phage therapy. The purpose of the study was to construct oral-nasal exposure model used for delivery of phage D29 aerosol, to discuss the phage aerosol delivery efficiency, to figure out the metabolic characteristics in targeted organs, and to evaluate the immunogenicity and safety of phage D29 in various delivery routes.The related report of mycobacteriophage lysin therapy was not reported yet. Only one of the mycobacteriophages existing lysin was identified.The purpose of this study was to identify the lysis gene of phage D29 and establish a therapy method for drug-resistant tuberculosis in the future.Contents:1. To construct an oral-nasal exposure model used for delivery of phage D29 aerosol to guinea pig;2. Intranasal delivery was used as control and the delivered efficiency of targeted phage D29 to lung was evaluated between treatment and control;3. To investigate the decay characteristics of phage D29 in the lung of guinea pig;4. To investigate the immunogenicity and safety of Phage D29 by different delivery route.5. To identify the lysis gene of phage D29;6. To construct the E.Coli. carrier to express the lysis gene of phage D29.Methods:1. Construction of the animal mouth/nose exposure system for delivery of aerosolized phage D29: put guinea pigs grouped by gender and weight in barrels of up layer and low layer of exposure equipment. Phage D29 aerosol (6×109PFU/ml) was produced by DV40,and the distribution of aerosol particle in the system was measured by TSI3321 aerodynamic particle sizer spectrometer.AGI-10 liquid impingers were used to sample the aerosol of phage D29 in the exposure equipment.Anderson-2 sampler located outer of the exposure system in the aerosol exposure tank sampled the air in the tank to measure whether the exposure system was leaked or not.Guinea pigs were exposed 1h to phage aerosol in the exposure equipment.The counts of phage D29 in the sampling solution of AGI-10 were measured by double-layer agar plate count method and the real-time PCR at the same time.The phage aerosol concentration in the exposure system was calculated from the phage concentration values in sampling solution.2. Guinea pigs exposed in control and treatment group were executed at 1h,8h,and 24h after exposure.The picked-off whole lung and trachea were used to prepare the lung homogenate and marination respectively after anatomization.Double-layer agar plate count method and the real-time PCR were used to measure the phage concentration of the preparations.Phage delivery efficiency were evaluated between aerosol and intranasal delivery methods by the intake value of phage D29.The survival and total phage D29 in lung at different time points were analyzed statistically and the metabolic characteristics were summarized.3. Phage D29 was delivered to guinea pigs through aerosol(concentration of generating liquid 109PFU/ml),nasal(108PFU/50μl;108PFU/200μl),and hypodermic (108PFU/200μl) routes and aerosolized 7H9 medium,nose delivered 7H9 medium,aerosolized normal saline,and nose delivered normal saline were used as controls.Exposure was performed twice at the first week,then once a week and blood was taken at the last day of every week.Guinea pigs were executed and anatomized after five weeks.Following studies were carried out:(1)observation of animal physiological and behavioral state;(2)observation of organs at a gross and histological level;(3) measurement of phage D29 neutralizing antibodies in blood serum through plaque reduction neutralizing test;(4)measurement of serum IL-2,IL-4,and IFN-γby ELISA test.4. Gene10 was found to be highly homological to lysins, based on the analysis of ORFs encoded by Mycobacteriophage D29.This gene was cloned to E.coli-Mycobacterium shuttle plasmid pUV15tetORm, electroporated into M. smegmatis mc2155. Expressed product was examined by SDS-PAGE. The lytic activity was identified by culture turbidity measurement and transmission electron microscopy (TEM). Then recombinant plasmids pET28-gene10 without tag and pQE80-gene10 with His-tag were constructed, respectively. 5. TMHMM analysis indicates that gene11 encodes a holin-like protein. This gene obtained by PCR amplification, was cloned into pQE80L and expressed in E.coli M15 and the lytic activity was identified by culture turbidity measurement.Results:1. Phage D29 suspension(6×109PFU/ml) was nebulized by a DV40 generator and the mean aerodynamic diameter of the total aerosol particles in the exposure equipment was 0.860±0.017μm.No plaque was detected by the double-layer agar plaque test from Anderson-2 sampler plates after 1h sampling,which indicated that the system was air-sealed and no phage aerosol was leaked.The phage mean plaque forming unit in the sampling solution was 1.52×108±5.41×107PFU measured by double-layer agar plaque test and 4.33×108±4.27×107PFU measured by real-time PCR.The difference was not significant between the two test methods(P>0.05).The concentration of phage D29 aerosol in the exposure barrels was 6.19×109PFU/m3 which was calculated from the phage counts in sampling solution and the theoretical inhalation dose of phage D29 per guinea pig was 2.38×105PFU.2. Data from guinea pigs exposed to phage D29 aerosol at the first hour was analyzed statistically.The total inhalation dose of lung and trachea was not significant difference from the exposure dose(P>0.05).The inhalation dose of lung did not differ from the exposure dose significantly(P>0.05),while it was higher than the inhalation dose of trachea(P<0.05).However,the intake dose of guinea pig through nasal delivery was lower than the exposure dose(P<0.01)and the intake dose of lung higher than the trachea intake dose(P<0.05).3. The detection results of double-layer agar plaque test and real-time PCR test on the phage D29 in guinea pigs at 1h,8h,and 24h after exposure were analyzed by paired t-tests.There was no difference between two methods at 1h(P>0.05),which indicated that the phages inhaled still alive and no obvious death appearance,but the culture method is significant lower than the real-time PCR at 8h and 24 hou(rP<0.05),which indicated the total and alive phage counts were significantly different.The inactivation rate and clearance rate of phage D29 in lung at different time points were caculated.The inactivation rate and clearance rate of phage D29 in lung at 8h was 99.19% and 27.08% and at 24h was 99.99% and 95.19%.4. The outcome of repeated exposure experiments indicated that the death rates of guinea pig from all the groups including control were high(20%-100%).Summarizing the results of weight,pathologic reaction of gross organs and the histological findings,the lung of guinea pig occurred pathological changes which influenced the respiratory function and death occurred.All the indexes between the groups were not significantly different(P>0.05).5. The PRNT(plaque reduction neutralizing test) result revealed that the neutralizing antibody didn't appear in guinea pigs serum of control groups(media 7H9 and physiological saline).Low-titer antibody appeared in phage D29 aerosol treatment and intranasal drop treatment groups 7ds later, declined during next week and disappeared after three weeks. Steady neutralizing antibody appeared in subcutaneous injection group. Two weeks later antibody titer declined, and then increased by weeks. ELISA test result indicated that the lever of Interleukin-2,4 and interferon-γin serum of all groups had no significant difference with the control groups.6. A recombinant shuttle plasmid pUV-gene10 was successfully constructed, and gene10 was successfully expressed in Mycobacterium. The expressed product could effectively reduce the turbidity of host strain culture by hydrolyzing its cell wall. Hydrolization resulted in the mass pieces of cell wall and vacuoles, which was observed by Transmission electron microscopy.7. Recombinant plasmids pET28-gene10,pQE80-gene10 were constructed successfully.8. Constructed recombinant plasmid pQE80-gene11 and electroporated into E.coli M15. The absorption measurement result indicated that the expressed product of gene11 could lyse host obviously.Conclusions:1.The aerosol delivery route presented high efficiency. Most of the phage D29 aerosol could be delivered into the lung which was targeted organ of TB and could keep alive at least within 1 hour;2. The aerosol delivery route and nasal delivery route were obviously weeker to the hypodermic route in terms of causing neutralizing antidody in hosts;3. The therapy effect and the safety of aerosol delivery route should be further investigated;4. Gene10 and gene11 are lysin and holin genes of phage D29, respectively;5. Recombinant plasmids pET28-gene10 and pQE80-gene10 were constructed successfully. Phage D29 aerosol delivery route was worthy of our expectancy because of its high delivery efficiency, low phage activity damnification and causing low immunogenic reaction. The identification of phage D29 lysin and holin was a groundword for phage D29 lysis regulation mechanism research and phage D29 lysin therapy research. It hadn't been reported neither the phage D29 aerosol delivery route nor the phage D29 lysis genes identification, this research was of certain innovation.
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