Construction And Identification Of Recombinant Adenovirus Containing Chimeric Gene Of Hantavirus And Antigen Presenting Molecules

Hemorrhagic fever with renal syndrome (HFRS) is an acute infectious disease characterized by fever, hemorrhage and nephritis, which is caused by Hantavirus. In China about 20,000–50,000 people per year were infected by Hantavirus with the severe symptoms and high mortality. There are still no effective therapeutical drugs directed to HFRS until now. Recently, it has become a serious worldwide health threat.At present, China has successfully developed three types of HFRS inactivated vaccine: the suckling mice brain inactivated vaccine (HTN- type), inactivated vaccine of cell cultivation (inactivated vaccine of sand rat kidney cell cultivation HTN type, inactivated vaccine of hamster kidney cell cultivation SEO type) and bivalent vaccine (HTN type and SEO type). Although the vaccines have effectively prevented the occurrence and spread of HFRS, there are still many problems: the main thing is that it can not stimulate cellular immune responses effectively, in addition, its ability to induce neutralizing antibody is weak, and the antibody titer is low.Hantaviruses belong to the Bunyaviridae family of viruses. Hantaviruses are enveloped viruses with a genome that consists of three single-stranded, negative sense RNA segments designated L (large), M (medium), and S (small). The L segment encodes the L protein, which functions as the viral transcriptase/ replicase. The M segment encodes a polyprotein that is cotranslationally cleaved to yield the envelope glycoproteins G1 and G2. The S segment encodes the nucleocapsid protein (NP).It is indicated that the glycoprotein (GP), which was encoded by M segment, could elicit organism to produce neutralizing antibody. Moreover, the neutralization sites of GP mainly exist in G2. But the immunogenicity of GP is weak. The antibody elicited by GP is produced later and the titer is low. NP has been proved to be the structural protein with highest immunogenicity and may also evoke protective immunity.We have fused M segment and 0.7 kb fragment of S segment, and then immunized mice with the chimeric gene. The results suggested that the chimeric gene could directly elicit specific anti-HTV humoral and cellular immune response and can be used as a candidate component for HFRS genetic vaccine. But we also found some problems, although a variety of systems are suitable for express the fusion protein, but the amount of protein expression is still low. In addition, athough many of the expression system can stimulate humoral and cellular immune responses in mice, and the adenovirus expression system is superior to other systems, but the overall level of immune response is still low. Therefore, there are some problems need to be resolved in the research of the HFRS genetic vaccine: select a suitable expression vector, optimize expression systems to enhance the expression of chimeric gene; enhance the ability of antigen-presenting to stimulate the immune response more effectively.Is this study we cloned M, S chimeric gene to adenovirus expression system, in order to optimize of the structure of transcriptional regulatory ,at last improve the level of chimeric gene expression. In previous studies, we used CAG promoter and the WPRE post-transcriptional element to optimize the transcriptional regulation of adenovirus expression system. It was found that after using CAG promoter in the adenovirus vector, chimeric gene expression level is higher than using WPRE. On the basis of the optimized structure of transcriptional regulation, we use HSP70C (heat shock protein 70 gene encoding N-terminal fragment) and ubiquitin, to promote the antigen processing and presenting, further enhance the humoral and cellular immune response.1. Constructing recombinant pShuttlePCR primers were synthesized according to GenBank.The fragments HSP70C and Ub were obtained by PCR and then cloned into the pShuttle-G1S0.7-CAG and pShuttle-G2S0.7-CAG. Recombinant pShuttle, plasmid DNA were digested with restriction enzyme, and then verify digestion by analyzing sample on agarose gel. Positive plasmids were named as pShuttle-G1S0.7-CAG- HSP70C,pShuttle-G2S0.7- HSP70C,pShuttle-G1S0.7- CAG-Ub,pShuttle-G1S0.7-CAG-ub.2. Constructing recombinant Adenoviral DNAExcise expression cassettes from recombinant pShuttle plasmid DNA by digesting with I-CeuⅠand PI- SceⅠ. Subsequently ligate expression cassettes to Adeno-XTM Viral DNA. Isolate putative recombinant adenoviral DNA and using PCR to confirm that it contains inserted genes. 3. Propagate and identify recombinant adenovirusTransforming E. coli with recombinant adenoviral DNA, then purify recombinant adenoviral DNA using the large-scale procedure. Digest recombinant adenoviral DNA with PacⅠ.Transfect low passage HEK 293 cells with PacⅠ-digested recombinant adenoviral DNA using standard transfection techniques. Periodically thereafter, check for cytopathic effect (CPE). Virus is obtained by manually lysing cells with a series of freeze-thaw cycles and collect cell lysate. Infect a fresh culture by adding cell lysate,CPE could be evident within one week, collect cell lysate as before, name this stock"Primary Amplification". The presences of recombinant constructs were verified by PCR.4. Plaque purification and determining viral titerUse agarose overlay medium to overlay the infected cell monolayer to prevent virus progeny from spreading to neighboring plaques. Isolate virus deriving from a single plaque on a monolayer of 293 cells. The viral titer is a quantitative measurement of the biological activity of recombinant virus and is expressed as plaque forming units (pfu) per ml. Determine viral titer using the End-Point Dilution Assay, check each well for cytopathic effect, count the number of wells having CPE, then use the formula to determine the titer (pfu/ml) of viral stock.After careful calculation, the titers of recombinant adenoviruses were 109 pfu/mL.5. Analyzing recombinant adenovirus Expression in Infected CellsInfecting target cells at a multiplicity of 100 pfu/cell. Detectable levels of gene product could be evident 24–48 h after infection. HEK293 cells transfected with recombinant adenoviruses were detected positive by immunofluorescence assay using a specific mAb 1A8 against Hantavirus nucleoprotein. Cells infected with recombinant adenoviruses Ad-G1S0.7-CAG-HSP70C and Ad-G2S0.7- CAG-HSP70C were detected positive by a specific antibody against HSP70C. Cells infected with recombinant adenoviruses Ad-G1S0.7-CAG-Ub and Ad-G2S0.7-CAG-Ub were detected positive by a specific antibody against Ub.