Construction Of Eukaryotic Expression Vectors Harbouring HN And F Gene Of Paramyxovirus, Tianjin Strain And Their Immunization On Infant Mice

Parainfluenza virus has been known to be pathogen in a series of respiratory tract infections, among which croup is a typical sign of hPIV1 infection in children, if the infants and toddlers suffering from the hPIV1 infections can not be hospitalized and treated timely, not only will the infants have severe symptoms of respiratory tract infections, even endanger their lives, but the morbidity also appears to be significantly increased. In addition, hPIV1 infection also involved in adult patients. Sendai virus, also known as murine PIV1, a (-)ss RNA virus, gives rise to respiratory tract infections in many rodents, even results in fatal pneumonia among mice. The first step in the infection process is mediated by two kinds of envelope glycoproteins HN and F of the virion, which are main protective antigens as well. A study showed attenuated Sendai virus can protect Africa green monkey from challenge with hPIV1, it provides with experimental evidence that antigenic components of Sendai virus could protect infants human being from hPIV1 infections. However the research on immunization of F glycoprotein of hPIV1 or SeV and of combination of HN and F glycoproteins of Sendai virus was poorly understood, a strain of virus possessing high HA titer was isolated from common cotton-eared marmosets suffering from acute respiratory tract infection, it was verified primarily as a strain of Sendai virus so that Sendai virus, tianjin strain was then designated at the first place and Paramyxovirus, Tianjin strain was renamed later, current research shows the virus could be a new genotype of Sendai virus. therefore, eukaryotic expression vectors harbouring HN and F gene of the Sendai virus were constructed and given solely and jointly to infant mice, its immune response and immunological protection against subsequent infection were observed in order to evaluate these constructs and their administration approaches. HN coding region of the viral genome was amplified using RT-PCR, after the PCR product was digested by both HindⅢand Xba I, it was inserted unilaterally into downstream of Pcmv of pcDNA3 and eukaryotic expression vector harbouring HN gene of Paramyxovirus, Tianjin strain was then constructed. On the other hand, F coding region of the genome was amplified by PCR using its cDNA as template, and then F PCR products and T vector was ligated together to construct transitional plasmid which makes it easier for the plasmid to be digested by both EcoR I and Not I, the enzyme-digested fragment was later inserted to pcDNA3 as it did the case of HN. Both constructs were confirmed by such protocols as PCR, RE digestion and sequencing.Successfully-constructed eukaryotic expression vectors were designated as pcDNA.HN and pcDNA.F respectively, both constructs transfected'COS7 cell for instant expression with the aid of cation liposome after being de-endotoxinated, their transcription and translation were then checked by protocols such as RT-PCR, IFA and ELISA. RT-PCR results showed both pcDNA.HN and pcDNA.F can transcript mRNA in COS7 cell. In addition, IFA showed that pale greenyellow fluorescence substance was seen in the construct-transfected cells whereas no fluorescence stain in the pcDNA3-transfected cells. Furthermore, both ELISA in situ and ELISA of cell lysate produced similar results that both construct can trigger translation in COS7 cell.De-endotoxinated recombinant pcDNA.HN and pcDNA.F were administrated solely and jointly via intramuscular route in 4-6 week old infant ICR mice after pre-treated the mice with bupivacaine, their immunization was monitored on a regular weekly basis. The immunological protection test was carried out by challenging the mice population with the same strain of the virus after the mice had been inoculated three times for 4 weeks, and its protective outcomes were examined by variables such as survival rate, target organ responses and immune response, that is, variables such as survival rates, spleen index, thymus index, pathology of lung, sera HI titer, sera HLI titer, SI of lymphocyte B&T, sera IgM&IgG level, sera IFN-γ, IL-2 and IL-4 level were employed for immunological evaluation, compared to inactivated Paramyxovirus, Tianjin strain as positive control, and to both pcDNA3 and NS as negative control. the experiment results showed combination of pcDNA.HN and pcDNA.F can efficiently induce humoral immunity and cellular mediated immunity, so that these recombinants can protect the mice against subsequent challenge with the same strain of the virus. At the same time, many variables including SI of lymphocyte B&T, sera IgM&IgG level manifested the immune response induced by pcDNA.HN seemed to be better than that of pcDNA.F, most variables such as survival rates, pathology of lung, sera HI and HLI titer, SI of lymphocyte B&T, sera IgG level, sera IFN-y and IL-2 level indicated that combination approach seemed to be superior to that of sole one. Variables such as survival rate, pathology of lung, SI of lymphocyte B&T, sera IgG level, sera IFN-y and IL-2 level support an idea that immunization induced by the inactivated'virus appeared to be more effective than that of pcDNA.HN or pcDNA.F, nevertheless, the immunization compared between the construct combination and the inactivated virus needs to be further confirmed.In summary, combination approach of pcDNA.HN and pcDNA.F had satisfactory performance on preventing infant mice from respiratory tract infections by Paramyxovirus, Tianjin strain, it laid experimental foundation upon which the viral envelope glycoproteins HN and F prevent infant from hPIV1-related infections. Under such circumstance that current approach has no effective preventive intervention against hPIV1 infections in infant, should the main protective antigens HN and F's DNA immunization be considered as a alternative to prevent hPIV1 infections.