| [Objective] Construct the eukaryotic expressing plasmids which can express the hemagglutinin (HA) and neucleoprotein (NP) gene of H5N1 influenza virus and the live trager vaccines(CAV-2-HA and CAV-2-HP) of expressing the HA and NP gene recombinant Canine Adenovirus Type 2. Then use the eukaryotic expressing plasmid and the live trager vaccines to immunize the mice, the humoral immunity and cell-mediated immunity response of the immunized mice are detected and the protective rates of the eukaryotic expressing plasmid is analyzed. The work done here provides a valuable theoretical and technical bases for the nucleic acid vaccine and the recombinant live vector vaccine development against mammalian H5N1 subtype influenza virus.[Method] HA and NP gene of A/Tiger/Harbin/132/2007 (H5N1) from the chick embryo allantoic fluid were amplificated through PCR by the contrivable synthetic primer of HA and NP gene,then cloned and sequencing analyzed. Ordinary molecular methods were used to construct eukaryotic expressing vectors, pVAX-HA and pVAX-NP expressing HA and NP gene of H5N1 subtype influenza virus isolated from tiger. Dual-expressing vector pIRES2-IL-18-HA expressing HA gene of H5N1 subtype influenza virus and IL-18 gene of feline was also constructed. The protein expressed by eukaryotic vectors in BHK-21 cell was detected by indirect immunofluorescence assay and indirect ELISA.Then the expression cassette containing NP/HA gene (CMV+NP/HA+PolyA) was cloned into the Ssp I enzyme losses division of pVAXE3,then obtaining shuttle vector (pVAXΔE3-NP/HA) containing NP expression cassette. Using Sal I+Nru I double digestion for pVAXΔE3-NP/HA and pPoly-2-CAV2. The fragment containing NP/HA expression cassette was directionally cloned into pPoly2-CAV2 and obtained recombinant plasmid (pCAV-2-NP/HA) of NP/HA expression cassette inserted into the E3 lacking area. After Releasing CAV-2-NP/HA recombination genome and transfecting DK cells by liposomes mediate, the CAV-2-NP/HA recombination virus which typically made the DK cells adenovirus-like cytopathic. From morphology, genomics level, NP/HA genes transcription, NP/HA protein expression and the growth characteristics of recombinant virus, etc, we indentified the recombinant virus.The mice immunization experiment and conteracting toxic substances and protection experiment were taken with four eukaryotic expressing plasmid and two recombinant viruses respectively. Design steps of eukaryotic expressing plasmid immunization experiment as follow.7 immune groups were used:immunized with pVAX-HA, pVAX-NP, pVAX-Ha and pVAX-NP, pIRES2-IL-18-HA, IRES2-IL-18-NP, pIRES2-IL-18-HA and pIRES2-IL-18-NP, PBS, respectively.,11 mice in each group. Mice were immunized 3 times with a interval of 15 days by intramuscular injection. 15 days after inoculation the antibody titers of the mice in each group against AIV were detected by indirect ELISA, MTT method was used to detect the cell-mediated immunity response. The mice in each group were inoculated with highly pathogenic H5N1 subtype influenza virus, in order to measure the protective rates.Design steps of the immunization experiment of recombinant virus as follow.3 immune groups were used: immunized with CAV-2-HA,CAV-2, combined immunized with CAV-2-HA and CAV-2-NP. There were 13 mice in each group. Mice were immunized 2 times with a interval of 14 days by drip into the nose (50μL) and subcutaneous injection(250μL).14 days after inoculation of each time, two mice were killed in each group to collect serum and took conteracting toxic substances in 3 mice of each group to detect lung titer four days later. The remaining was used to detect the protective rate after conteracting toxic substances.[Result] The total NP and HA gene of A/Tiger/Harbin/132/2007 strain are cloned successfully, the lengths are 1704bp and 1479bp, each of them encodes 568 and 493 amine acids respectively. Four eukaryotic expressing plasmids (pVAX-HA, pVAX-NP, pIRES2-IL-18-HA and pIRES2-IL-18-NP)of expressing HA and NP gene of H5N1 subtype influenza virus isolated from tiger and two recombinant viruses(CAV-2-NP and CAV-2-HA) are constructed successfully. These four eukaryotic expressing vectors all have the ability to express HA and NP protein with biological activity in the mammalian cell, BHK-21. Mice in each immunized group are inoculated high titer of highly pathogenic virus (10000 MLD50/each), the protective rates of pVAX-HA, pVAX-NP, pVAX-HA and pVAX-NP, pIRES2-IL-18-HA, pIRES2-IL-18-NP, pIRES2-IL-18-HA and pIRES2-IL-18-NP, PBS immunized group are 60%,20%,60%,20%,0%,20%,0%, respectively.CAV-2-NP/HA has the typical morphological characteristics of CAV-2, the fragment of H5N1 NP/HA expression cassette is not missing or resort in the process of reproduction of CAV-2-NP/HA, and can transcribes the mRNA of H5N1 NP/HA, ELISA and immunofluorescence analysis show that the product of recombinant expression can be identified by the NP/HA monoclonal antibody of flu virus, has a good in vitro genetic stability. The animal immunization experiment shows that the two recombinant vaccines can induce mice to create specific immunereaction and CAV-2-HA is better than the other one, its conteracting toxic substances protective rate to mice is 83.3%, the protective rate of combined immunization of CAV-2-HA and CAV-2-NP is 16.7%, control group(CAV-2) is 0%.[Conclusion] The total NP and HA gene of A/Tiger/Harbin/132/2007 strain is cloned successfully; Four nucleic acid vaccines and two recombinant live vector vaccines of expressing HA and NP gene of H5N1 subtype influenza virus isolated from tiger are constructed successfully, they all can introduce mice to create the immunoprotection effect. This research provides a valuable theoretical and technical bases for the nucleic acid vaccine and recombinant live vector vaccines development against mammalian H5N1 subtype influenza virus.
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