| Enterococcus faecalis is a normal flora of human or animal intestinal,oral and reproductive tracts.Under normal circumstances,symbiotic enterococci are not pathogenic to the host.But when its ectopic parasitic can cause sepsis,urinary tract infection,suppurative abdominal infection and endocarditis and other diseases,with the increased resistance to Enterococcus faecalis,the infection caused by it more difficult to control,the urgent need to study the new Antibacterial preparations.The emergence of phage for us to provide a new idea for the phage and phage lytic enzyme research also showed great value.Scientists from a variety of infected phage pathogens,including Streptococcus pneumoniae,Bacillus anthracis,Lactobacillus,Staphylococcus aureus and so isolated phages.But with the emergence of antibiotics,people’s attention and transferred to the above antibiotics,thus ignoring the natural enemies of bacteria phage.With the super-strong pathogen bacteria,broad-spectrum drug resistance problems make phage therapy for drug-resistant bacterial infection treatment has become a hot spot.Phage and its cleavage enzymes can specifically cleave bacteria that do not infect eukaryotic cells and do not destroy the body’s normal flora,which is a new concept of antimicrobial substances.In-depth study of phage and its cleavage enzymes will lead to the development of novel antimicrobial agents for the treatment of bacterial diseases.In the previous experiment,we obtained a new phage with high cleavage activity against vancomycin-resistant Enterococcus faecalis from clinical samples.Through the whole genome sequencing and analysis,the phage cleavage enzyme gene was identified and obtained by genetic engineering Lyase LysN10.A total of 36 strains of Enterococcus faecalis were isolated,and 32 strains of LysN10 could be cleaved,and the CHAP fragment of LysN10 and LysGH had some homology to the amino acid sequence.The cleavage enzyme showed broad spectrum and efficient cleavage activity against Enterococcus faecalis,showing the potential of the cleavage enzyme to treat resistant enterococcus faecalis.In this part,the structure of the phage lysate was analyzed by structural analysis.We constructed four recombinant plasmids pET15b-N10,pET15b-NF418,pET15b-NR438,and pETl 5b-NR492,for protein expression,purification and crystal screening.Recombinant proteins were purified by affinity chromatography,ion exchange chromatography and molecular sieve chromatography.Crystallization conditions screening wereproformed on for purified proteins.One condition as follows:PEG4 Suite finally was obtained on NF418.The results of the study show that the use of clinically isolated drug-resistant Enterococcus faecalis phage lysates,construction of recombinant phage lysates,and can be used to treat bacterial infectious diseases can be used for clinical programs.Handmade disease(HFMD)is an infectious disease caused by enteroviruses.Coxsackievirus A group 16(Coxsackievirus A16,CA16)is one of the main members of CV,first in 1951 South Africa was successfully separated.CA16 caused by hand,foot and mouth disease symptoms are relatively light,and with the EV71 caused by the specific symptoms of hand,foot and mouth disease is difficult to distinguish,in addition,children and hidden infection are infected,adult infection and become hidden infection and Asymptomatic carriers do not develop themselves,but can be transmitted as a potential source of infection to children,which is one of the main causes of disease transmission difficult to control.Many viral structural proteins have the ability to assemble automatically into VLPs,which are similar in morphology to natural viral particles and have strong immunogenicity and biological activity.Because VLPs do not contain viral genetic material,they are not infectious,some of which have been successfully used as a vaccine in clinical practice.Because the VLPs surface can repeat and exhibit high levels of exogenous epitopes to elicit a strong immune response,it is an ideal form of vaccine that can be developed for a variety of diseases.Studies have shown that CA16 virus is composed of 60 VP 1,VP2,VP3 and VP4 capsid protein subunits together to form an icosahedral spherical particles.Like the other small RNA viruses,the VP1,VP2,and VP3 proteins each form a subunit of the capsid.The subunit forms a pentamer and the 12 pentamer forms the capsid.CA1 VP1,VP2,VP3 can constitute its VLP.In this part of the experiment we extracted the CA16 genome,through the specific primers using PCR method to retrieve CA16 capsid protein VP 1,VP2,VP3 gene.The recombinant plasmids were constructed with pET28a,PET43.1a,pCOLD-SUMO,pMAL-P2X and pMAL-C2X plasmids respectively.Then the correct recombinant plasmid was expressed in engineering Escherichia coli,and the expression conditions were explored.Finally,the optimal expression conditions were used for prokaryotic expression of VLP.Protein purification was carried out by Ni column affinity chromatography,molecular sieves. |
PDF Full Text Request