Construction And Immunogenicity Analysis Of The Eukaryotic Expression Plasmid For The Gene Encoding The Outer Membrane Protein P6 Of The Nontypeable Haemophilus Influenzae

Nontypeable Haemophilus influenzae (NTHi), which has a highprevalence rate among the population is frequently associated withmany serious diseases, such as acute otitis media (AOM), sinusitis,conjunctivitis, the repeated outbreak and acute exacerbation of chronicpulmonary diseases (COPD), and it has become a great healthy threat tohuman, especially to the infant and the elderly. Thus, it is high time toresearch a new safe and effective vaccine to prevent NTHi infections.The outer membrane protein P6, which is commonly found in all Histrains (including NTHi), could evoke protective humoral, cellular andmucosal immunity; however, the harvest of P6 is very little and trivial.In recent years, the development of nucleinic acid vaccine has offered anew approach to developing vaccines. In this study, the eukaryoticexpression plasmid for P6 named pcDNA3.1/His A-P6 was constructedand used to immunize BALB/c mice, then the immunizing effects wereobserved to ascertain the applied value of P6 in the vaccine developmentfor NTHi.The P6 gene was amplified from the NTHi chromosomal DNA byPCR；Then the eukaryotic plasmid named pcDNA3.1/His A-P6 wasconstructed and verified by PCR, enzyme digestion analysis and DNAsequencing. The pcDNA3.1/His A-P6 plasmid was also transfected intoHeLa cells to detect its expression by indirect immunofluores cenceassay. A total of 45 8-week-old mice were separated into three cohorts of 15 animals each at random. The PBS group, pcDNA3.1/His A groupand pcDNA3.1/His A-P6 group were respectively immunized byintramuscular injection into the left thigh quadriceps muscle with 100μLPBS, 100μL PBS containing 100μg pcDNA3.1/His A plasmid and100μL PBS containing 100μg pcDNA3.1/His A-P6 plasmids three timeson days 0, 14, 28. 2 weeks after the last immunization, 10 mice fromeach group were killed, the proliferation of spleen lymphocyte wasanalysed by CCK-8 counting; IFN-γand IL-4 in supernatant ofspleenocytes cultured with NTHi in vitro was detected byenzyme-linked immunosorbent assay; 3 weeks after the lastimmunization, live NTHi strains at a dose of 1×108 cfu/head wasintranasally challenged to 15 mice (5 from each group), nasopharyngealwashings (NPWs) were obtained by meticulously washing with 100μLsterile PBS on days 3, 7 after the challenge to determine the clearance ofNTHi; 1 week after NTHi challenge, nasal mucosa histopathology wasanalysed by hematoxylin and eosin staining.The eukaryotic expression plasmid pcDNA3.1/His A-P6 was successfulllyconstructed, and the sequence of the inserted gene was verified tobe P6 by PCR, enzyme digestion analysis and DNA sequencing; Inaddition, the recombinant plasmids could express target protein P6 inHeLa cells. In the animal experiment, the mice spleen lymphocyteimmunized with pcDNA3.1/His A-P6 evoked a higher production ofIFN-γand stimulation index than those of controls which wereimmunized by pcDNA3.1/His A or PBS (P<0.01), while there was nodifference in IL-4 level among them; The nasopharyngeal clearance rateof NTHi immunized with pcDNA3.1/His A-P6 was also apparentlyhigher than those of controls (P<0.01); The H&E staining showed thenasal mucosa immunized with pcDNA3.1/His A-P6 was nearly normal,while the nasal mucosa of controls disordered and fell off.This study has successfully constructed the eukaryotic expression plasmid pcDNA3.1/His A-P6 and this eukaryotic plasmid for P6 inducesobviously protective effects against NTHi in the BALB/c mice model,which displays the recombinant vector for P6 has a potential value forNTHi vaccine research and development .