Construction Of Hantavirus' Nucleoprotein Gene Vaccine And Primary Study On Its Kinetics Of Gene Expression In Mice

ObjectiveHemorrhagic fever with renal syndrome (HFRS) is a kind of acute viral infectious diseases which is caused by Hantavirus and characterized with the serious symptoms, high case fatality and no effective therapeutic tool until now. So the prevention to Hantavirus seems practically significant. Recent years, as the development of molecular biology, the appearance of DNA vaccinum which is more better than traditional vaccinum and presentative the developing direction of future vaccinum provides a new idea for us with prevention and cure of HFRS. At present, several subtype of Hantavirus DNA vaccinum have been successfully constructed, but more studies are focus on the immune effect rather than kinetics of gene expression in the body. The following experiment constructs the nucleic acid vaccinum including S gene cloned to Hantavirus and makes an initial study to its kinetics of gene expression in the body to approach the application perspective of Hantavirus DNA vaccinums.We carried out the following study: (1) Construct the the recombinant plas-tnid pEGFP/S with pEGFP-C1 and S gene;(2) Observe the expression of pEG-FT/S in eukaryotic cells after tranfected using liposome;( 3 ) Observe the distribution and expression of pEGFP/S in the mice immuned.Method1. The construction of eukaryotic expression recombinant including S geneUsing restrictive enzyme.EcoRI,Sal I to digest pTargeT/S, purify to get S gene, clone to the corresponding site which is likewise digested by EcoRI^Sal I , digest and sequenc to get recombinant pTargeT/S. Purify pTargeT/S with endotoxin - free ultrapure plasmid DNA midi - prep kit, and store at - 20X1.2. Transient transfection of eucaryotic cell with lipoplasmid Artificial palinesthesia cultivate Vero - E6, to make its coverage fraction a-chieve 80% , inoculate to the 10 pore (12 pore cell culture plate) which is already paved with cover glass ( soaked in hydrochloric acid and alcohol ) under the density of 8 xlO4 -2.0 x 105per pore after trypsinization digestion, the cell creep on the glass at 12 hours, recombinant plasmid pEGFP/S(got out from super pure plasmid midi kit) mediated by bangosome transfect Vero — E6 cell ,after 48 hours, in the way of indirect immunofluorescence, drop anti - NP MoAb of rat^rhodamine marked capri — anti — rat di - antibody and DAPI staining solution respectively after fixation and blockage, observe using fluorescence orth - microscope after mounting.3. Amplification and purification of plasmid pEGFP/S to immunity Guiding by < Molecular Cloning > , transfer pEGFP/S into E. coli DH5a,culture it in 30ml LB medium for 16 - 18hours at first, take 25ml culture material into 500ml LB medium for 2. 5 hours, add 2.5ml amphemycin(34mg/ml dissolved in alcohol) ,then continue culturing for 12 - 16h. After alkaline lysis to get out amounts of plasmids from the culture material, use PEG to purify plasmid DNA, measure OD260/28o, calculate the concentration of plasmid DNA, and store at -20t.4. Immunif action of animal30 female BALB/c mice at 6 ~ 8w age are divided into 3 groups randomly. Both experiment group and control group have 14 mice, one group is injected pEGFP/S plasmid, the other is injected PBS, another two mice is blank control group. 0. 75% dolicaine of 50jxl is injected into double side tibial muscle ,anterior of exp. group and control group, next day inject pEGFP/S lOOjxg each (exp. group)at the same site, or PBS lOOjxl each (control group).5. The detection of expression productWhen injected at 3d, 7d, 15d, 30d, 60d, liver, spleen, kidney, lung,brain and partial musculature removed from two from exp. group and two from control group randomly, stay it overnight in alcohol to fix it, then make frozen section 7 pjn thick by freezing microtome, stretched prepare on glass slide treated by polylysine, observe with fluorescence microscope immediately.Results1. Identity of recombinant plasmid pEGFP/STwo fragment of 1. 3 and 4. 7 kb are observed after pEGFP/S positive recombinant plasmid are digested by EcoRI, Sal I , the result is just like anticipate.2. Fluorescence microscope observe the result of transient transfection Transfected at 48 h, green fluorescence protein in blue excitation can be observed by fluorescence microscope;red fluorescence in green excitation can be observed;blue fluorescence in UV excitation can be observed, while nucleus of cells which failed to transfect can give out blue fluorescence only in UV excitation , in negative control group green fluorescence can be observed only in blue excitation, other control group cannot observe fluorescence.3. Tissue distribution and persistence of recombinant plasmid pEGFP/ SBright green fluorescence can be detected in all tissues of liver, lung, kidney brain and muscle of two mice after 3 day, at each time point of the following 3d, 7d, 15d, 30d, 60d, green fluorescence can be detected in liver, lung, kidney, brain and spleen of dead mice, fluorescence can be detected in muscle at 15d and disappear at 30d, the numbers of cell which express fluorescence and fluorescence intensity taper in all tissue as time is going on, any fluorescence cannt be observed in control group.Discussion1. The significance of S and GFP gene to Hantavirus DNA vaccineNP is the main structural protein of Hantavirus, which has strong immuno-gencity and induces the humoral immunity and cell immunity of the body. Plasmid pEGFP-Cll is a kind of eukaryotic expression vector which can highly express in mammalian cells, whose GFP which are safe to cells can be provoked of green fluorescence under a certain wavelength, and no exogeneous matters are needed in this process. The fusion protein carrying GFP can both give out the green fluorescence and remain the physiologic function of target protein, and the expression of exogeneous gene in live cells can be observed directly. This experiment constructs recombinant plasmid pEGFP/S with the physiological nature of GFP to make a more macroscopic observation of the distribution and expression of Hantavirus DNA vaccinum in the body.2. The significance of eukaryotic cell transfected in vivo to Hantavirus DNA vaccineExpression of GFP and NP can be observed under blue light and green light optical excitation in the same eyeshot respectively using fluorescence microscope , which certify indirectly that GFP-NP fusion protein express in successfully transfected cells, which provide scientific basis for our extended experiment of observing the effect of animal immunity.3. Significance of muscle immunization to expression of pEGFP/S DNA vaccine of intramuscular injection has the advantage of large vac, capability , safe injection, convenient. In our study, we get fine immune effect by intramuscular injection, it' s show that intramuscular injection induced by lido-caine is a fine immunal style to pEGFP/S.4. Significance of distribution in tissue after immunization Organism can get long-term persistent expression after immunization, whichmake immune system organism accept long-term antigenic stimulation, so that it can make organism to maintain long-term specific immunity, which show that pEGFP/S is a kind of ideal DNA vaccine.5. Expression of pEGFP/S in brainIt is difficult to the exogenous plasmid expressed in brain, because brain does not contain dendritic cell (DC) and large molecular substance is not easy to pass the brain because of blood - brain barrier. In our study, persistence time of expression in brain is not obvious difference with other tissue, which implies thatthe effect of some nature physiological structure in organism to the pEGFP/S metabolism in the brain tissue is not obvious.Conclusion(1) pEGFP-Cll is a good kind of vector to study Hantavirus'DNA vaccine for us.(2) GFP-NP fusion protein coded by pEGFP/S could be expressed in eu-karyotic cells.(3 ) It show that intramuscular injection induced by lidocaine is a fine im-munal style to pEGFP/S.(4) pEGFP/S intramuscular immuned could be expressed in tissue and have a long - term maintain.( 5 ) It implies that pEGFP/S could not be block by some nature physiological structure in brain .