Construction Of Live Strain Of Recombinant Mycoplasma Pneumoniae P1 Protein Gene Influenza Vector And Study On Immune Effect

Mycoplasma pneumoniae(MP)infection could causes Mycoplasma pneumoniae pneumonia(MPP).MPP is one of the community acquired pneumonia(CAP),and MP is one of the main pathogen that causes CAP.MP,especially harmful to the elderly and children,usually outbreaks in communities all over the world.However,there is still no effective vaccine for MP.The adherent at one end of the MP plays an important role,which allows the MP to settle on the surface of the host cell.Then,MP fuses with the host cell,which dissolves the cell to cause the death of the host cell.Adhesin,protein P1 is not only the main adhesion protein,but an important immunogen.Therefore,recently,subunit vaccines of P1 protein as the main immunogen have become a research hotspot.Nowadays,many domestic and foreign papers have reported that influenza A virus(IAV)carrying foreign genes in the genome expresses foreign proteins with the growth and replication of IAV.IAV vector has many advantages:IAV vector can carry foreign genes of various pathogens;it can induce the body to produce specific immunity including humoral immunity and cellular immunity;it is safer;it can be used as a bivalent vaccine;Extensive experience in chicken embryo or cell culture.A/Puerto Rico/8/34(PR8)strain,belongs to influenza A virus H1N1.The virus strain,adaptation strain of chicken embryo,is a high-yielding human influenza virus strain,which is often used as the backbone for the production of influenza vaccines.In this study,we first constructed a recombinant plasmid(NS-P1)of the fragment NS gene carrying the P1 protein gene of MP.7 normal plasmids,such as the PB2,PB1,PA,HA,NP,NA,M and NS-P1 recombinant plasmids co-transfected in293T cells,and rescued the recombinant virus carrying the P1 gene of MP(named PR8-NS-P1)by revers genetics technique.Hemagglutination assay(HA)was used to test the PR8-NS-P1 titer,and the degree is 1:128.The extraction of RAN of PR8-NS-P1 was identified and found that it contained the P1 protein gene,which sequencing results showed that the gene sequence was completely consistent.PR8-NS-P1 was expanded and cultured in chicken embryos,and then immunized New Zealand rabbits and BALB/c mice.The experimental animals weight changes and appearance characteristics were observed,and it was found that the weight of the white rabbits gradually increased without any changes in appearance within a week.Intranasal immunization with virus concentrate of mice,the mice weight decreased significantly within a week,while the body weight of mice in the intramuschlar injection group increased.In the 10~6EID50 group mice intranasal immunization,mice weight remained stable.The weight of mice in the other groups was increased.The mice that lost weight showed symptoms such as frowning hair,curling up,and sluggish movement,while the remaining mice had no obvious changes.The sera of the the first immunization and the second immunization mice was collected,and the sera use ELISA to detect whether the serum contains the MP specific antibody.The results show that the maximum OD value of mice exceeds 2.0,while the serum OD value of white rabbits is about 1.0.However,there are significant differences that all experimental group compared with the control group.The immunized rabbit serum was used for growth inhibition experiments to detect the MP antibody titer,and the results showed that the titer of the blank control group was 0,while the experimental group was 1:8.Furthermore,the results of co-cultivation of serum and MP in solid medium plate also showed that serum of the experimental group could significantly inhibit the proliferation of MP.Then,MTT assay was used to detect the effect of immune rabbit serum on MP infected A549 cells in vitro.The results showed that immune rabbit serum could significantly inhibit MP infected A549 cells.Nest,the mice that were given the second immunization were challenged with MP.Mice in the experimental group and control group were then challenged with low dose(106 CCU/ml)and high dose(108 CCU/ml).Four days later,the mice were dissected and their lung tissues were taken.The results of MP load detection of the lung tissues showed that the mice immunized with viral original solution had a lower bacterial load in the lung tissues after receiving the high-dose MP challenge experiment,while the MP clearance rate of the lung tissues was higher after the low-dose MP challenge experiment.It was about 96%in the nasal drip group and lower in the intramuscular injection group.In mice immunized with 10~6EID50 virus,the MP load of lung tissue in the intranasal immunization group was lower,and the lung tissue clearance rate was more than 90%.However,the intramuscular injection group had a higher MP load and a higher clearance rate of 92%,which was somewhat contradictory.Mice immunized with 10~5EID50 virus,intranasal immunization group and intramuscular injection group had higher MP load in lung tissue,and lung tissue clearance rate was about 70%.In summary,the study found that the influenza A virus carrying the P1 gene of MP can cause an immune response in experimental animals,which can induce MP specific antibodies and effectively protect experimental animals from MP infection.The result show that the serum obtained after immunization of the white rabbits and mice was tested to contain MP specific antibodies.The serum significantly inhibited the growth and proliferation of MP in vitro,and also inhibited the infection of MP to the A549 cells model in vitro.This subject provides a theoretical basis for the development of subunit vaccines of Mycoplasma pneumoniae.