Macrophage-Specific Expression Of Antimicrobial Peptide PR39 Protects Against Intracellular Bacterial Infection

Background:Infectious diseases, especially chronic infection are global diseases threatening animal and human health, of which many refractory and serious infections such as TB, virus hepatitis are involed in intracellular infection. The most common intracellular infectious bacteria include Salmonella Typhi, Mycobecterium Tuberculosis, and Legionella Pneumophila, et al. Infected the body, these bacteria can usually escape the killing of macrophage, live and proliferate in macrophages and escape from the human immunity and antibiotic therapy, so that macrophages become theirs shelter.Antimicrobial peptide (AMP) as a kind of peptide with antibacterial activity plays an important role in the innate immunity of organisms. It is a kind of defensive peptide with broad-spectrum antibacterial activity, nearly having no cross-resistance with other kinds of antibiotic and can hardly inducing drug-resistance. Antimicrobial peptide PR39 is a proline-rich AMP extracted from pig intestine firstly, which is a member of the cathelicidin family. PR39 has many additional functions such as neutralizing endotoxin, anti-inflammatory effection, immune induction, tissue repairing and inhibiting apoptosis, exepting its broad-spectrum and efficient antibacterial activity to bacteria including Salmonella Typhi and Mycobecterium Tuberculosis. Compared with traditional antibiotics, PR39 has multi-aspect advantage for the therapy of bacterial infection, and it is promising to be a new antibiotic against intracellular bacterial infection.However, AMPs are facing several handicaps during its clinical application:â‘ It is hard to extract and purify, which cause its high cost not endured by most patients.â‘¡As a peptide, AMP would be digested easily in gastrointestinal tract when orally administrated, and could induce allergic reaction when administratded intravenously.â‘¢Needing continious administration because of it is easy degradation.â‘£As many other antibiotics, AMP cannot enrich in cells easily, so it is difficult to kill intracellular bacteria. However, it becomes an important problem to find a new efficient, safe and low-costed method to apply AMP in the therapy of intracellular bacteria infection.Objective:To construct a recombinant adenovirus vector encoding antimicrobial peptide PR39 gene regulated by a macrophage-specific promoter, and express PR39 effiently and macrophage-specifically in vitro and in vivo. To detect the expression of PR39 gene transcriptionally and translationally in vitro and in vivo, and detect it is macrophage-specifitiy. To furtherly study the protective effects of the recombinant adenovirus encoding PR39 against intracellular bacterial infection in vitro and in vivo. To establish a foundation for the gene therapy of intracellular bacterial infection with antimicrobial peptides.Methods:1. RT-PCR method was used to synthesize PR39 gene, which was cloned into pIRES2-GFP vector after digested by NheI and SalI. The recombinant vector was transfected into mouse macrophage RAW264.7 cell, and cells stably expressing PR39 was screened out with G418. The expressions of PR39 in cells were detected with RT-PCR and immunocytochemisty. The capacity of PR39-expressing RAW264.7 cells to kill intracellular bacteria was also investgated.2. Preparation of PR39 rabbit polyclonal antibody: the epitope including 1-19 amino acids of PR39 was choosened with software DNASTAR and synthesized by chemical method. The synthesized peptide was subjected to immunize rabbit after linked to KLH protein. The antiserum from rabbit was purified with ammonium sulfate salting, and its puritiy and titer were detected with SDS-PAGE and ELISA.3. Antimicrobial peptide PR39 sequence which was designed according to its sequence in Genbank and the condon preferences of E.coli was synthesized by RT-PCR, and cloned to prokaryotic vector pET-32a (+), pET28a, pQE30, pET31b and so on. The expression of PR39 gene was induced by IPTG.4. Mrophage-specific promoter sequence, which was designed according to its gene sequence in Genbank, was synthesized by splicing PCR and cloned to eukaryotic vector pEGFP-N1 to substitute its CMV promoter. The recombinant vector pSP-GFP was equimolarly mixed with pERFP-N1 and transfected mouse macrophage cell line RAW264.7, human colorectal cancer cell Lovo, human breast cancer cell line ZR-75-30, human hepatocellular carcinoma cell line HepG2 and african green monkey kidney cell line Cos-7. 48 hours after transfection, the GFP and RFP were observed with fluorescence microscopy.5. Shuttle vectors encoding PR39 regulated by marophage-specific promoter were constructed and adenovirus backbone plasmids were transformed into E. coli BJ5183 to get AdEasier cell. Recombinant shuttle vectors were then transformed into competent AdEasier cell to construct the recombinant adenovirus plasmid Ad-SP-PR39 through homologous recombination. Recombinant adenovirus plasmids were transfected into packaging cell line 293 to generate adenovirus, which were amplified and then purified further. The recombinant adenovirus was used to infect mouse macrophage RAW264.7 and other non-macrophage cells. The expression of PR39 gene in RAW264.7 was identified by RT-PCR and immunocytochemisty, and the macrophage-specifity was also detected.6. Recombinant adenovirus Ad-SP-PR39 was used to infect mouse macrophage RAW264.7 in vitro and its antibacterial ability to kill salmonella typhi and Mycobacterium bovis BCG was detected by colony-counting methods. C57BL/6 mice were infected by recombinant adenovirus, fluorescentmicroscopy was used to study the orientation and survival of adenovirus, and RT-PCR and immunofluorescence was used to assay the expression of PR39 gene. C57BL/6 mice were infected by Mycobacterium bovis Bacille Calmette-Gue`rin before they were infected with recombinant adenoviru intratracheally and the antibacterial activity of Ad-SP-PR39 against Mycobacterium was investgated.Results:1. PR39 gene was amplified and the recombinant eukaryotic expression vector pIRES2-PR39 was constructed, the vector was transfected into macrophage RAW264.7 to get the PR39-stably-expressing cell line, in which the expression of PR39 gene could be detected both in mRNA and protein levels. Macrophage RAW264.7 expressing PR39 has the capacity to kill intracellular bacteria.2. Rabbit anti-PR39 polyclonal antiboby was successfully prepared and purified. SDS-PAGE and ELISA showed that it has a high puritiy and titer (1:10000).3. Some prokaryotic expressing vector encoding PR39 was constructed and the induced expression of PR39 gene was also tried many times, but PR39 gene could hardly be expressed in E.coli. The reasons may be that the expressed PR39 is high toxic to the host, which inhibited its further expression.4. Macrophage-specific promoter was synthesized by splicing PCR, and pSP-GFP vector regulated by the promoter was then constructed. GFP gene regulated by macrophage-specific promoter was expressed in macrophage cell line RAW264.7, but nearly not in various non-macrophage cell lines, in contrast, the RFP gene regulated by CMV promoter can be expressed in both macrophage cells and non-macrophage cells.5. Recombiant adenovirus Ad-SP-PR39 encoding PR39 regulated by macrophage-specific promoter was constructed. Mouse macrophage RAW264.7 cell infected by these recombinant adnovirus could express PR39 gene effiently, and recombiant adenovirus Ad-SP-PR39 could specifically express PR39 gene in macrophage cells.6. Mouse macrophage RAW264.7 infected with recombinant adenovirus Ad-SP-PR39 could express antimicrobial peptide PR39, which enhanced its ability to kill salmonella typhi and Mycobacterium bovis Bacille Calmette-Gue`rin in vitro. Recombinant adenovirus Ad-SP-PR39 could infect C57BL/6 mice efficiently and express antimicrobial peptide PR39. PR39 gene expressed in C57BL/6 mice lung, which were intratracheally infected by recombinant adenovirus Ad-SP-PR39, could kill Mycobacterium bovis Bacille Calmette-Gue`rin given intratracheally.Conclusions:1. PR39-gene-transfected mouse macrophage RAW264.7 could express antimicrobial peptide PR39 successfully, which enhances the intracellular antibacterial activity of macrophage cells.2. Rabbit anti-PR39 polyclonal antibody was successfully prepared.3. Antimicrobial peptide PR39 could hardly be expressed in prokaryotic host E.coli because of its antibacterial action and toxicity to host cells.4. GFP gene regulated by macrophage-specific promoter could be expressed in macrophage cells specifically, which suggested the macrophage-specific expression of PR39 gene.5. Recombinant adenovirus Ad-SP-PR39 could infect mouse macrophage and express PR39 gene efficiently. Ad-SP-PR39 could specifically express PR39 gene in macrophage cells.6. Recombinant adenovirus Ad-SP-PR39 could enhance the antibacterial ability of macrophage to salmonella typhi in vitro and to Mycobacterium bovis Bacille Calmette-Gue`rin in vitro and in vivo.