| AIDS stands for Acquired Immunodeficiency Syndrome, it is caused by HIV(HumanImmunodeficiency Virus).This disease is very contagious and has high fatality rate. Morethan2million people died of AIDS each year. The HIV virus poses one of the biggest viralthreats to human today.HIV DNA vaccine is considered one of the noval ways that we canused to delvelop potential new vaccines.It could be delivered into host cells directly andsynthesizes antigen protein in vivo. Although HIV DNA vaccines has a number ofadvantages over conventional vaccines, including the ability to induce a wider range ofimmune response types, many trials have ended in failure, in part due to its poorimmunogenicity.The current problem of how to improve the immunogenicity of HIVDNA vaccine should be urgently solved.In recent years, many researches show thatco-adminstration of plasmid adjuvants can significantly enhance immune responses. Ourresearch topic is using HIV-1gp120DNA vaccine with different adjuvants immunizeBALB/c mice and evaluating the immunogenicity of different DNA vaccine-adjuvantcombinations.China CDC provided pENVPOL (EP) and pGAGTNR (GT) plasmids which areexpected to very pure for animal experiments, so it is urgent problem to be solved in ourlaboratory.Our first task is to find a simple,economical and fast method for plasmid DNAextraction.Many methods of isolating plasimd DNA that have been published in chinese andforeign literatures. We try most of these methods which are all based on the alkaline lysismethod and eventually get the most effective method.After a standard lysis procedure,plasmid DNA releases from the host cells,then we use calcium chloride to selectivelyprecipitate RNA and PEG8000to selectively precipitate the plasmid DNA.The chemicalmethod can be used to remove contaminating RNA,protein,genomic DNA and otherimpurities,then the total crude is purified away from endotoxin by strong anion exchangechromatography(Q Sepharose F.F.).Finally,we prepare approximately7mg of high qualityand endotoxin free plasmid DNA（endotoxin less than0.05EU/μg,concentration of2μg/μl）.The final product basically meet defined quality and can be applied to experimental animals,we think that the simple, cost effective method will have wide application in thelaboratories.Many experiment results demonstrate that adjuvant can enhance the immunogenicity ofDNA vaccine.Based on the existing adjuvants in our laboratory, we choose MF59,CpGODN,WFR as adjuvant to combine with plasmid DNA and immunized the BALB/cmice.The mice were divided into four groups (EP+GT;MF59+EP+GT;CpGODN+MF59+EP+GT;WFR+EP+GT) and were immunized intramuscularly(i.m.),three timesat two-week interval.After56days of first immunization, we measured splenic lymphocyteproliferative responses by MTT assay in immunized mice(EP+GT;WFR+EP+GT). Theresult showed that1μg/ml gp120peptide can stimulate more splenic lymphocyteproliferation in vitro than10μg/ml gp120peptide.We detected the specific antibody responseof gp120peptide by ELISA at different time points.These results clearly demonstrate that thegroup which use WFR oil adjuvant can induce significant humoral immune response thanapplication of other adjuvants or plasimid DNA alone.Then we use another oil adjuvant PFRto immunize BALB/c mice.The mice were divided into two groups (EP+GT; EP+GT+PFR),we took the same way to immunize mice.We also detected the specific antibodyresponse of gp120peptide by ELISA at different time points. These results showed that thehumoral immune response to DNA immunization can also be enhanced by using PFR oiladjuvant.In our experimental study, we explore different methods for DNA vaccine manufactureand finally found a simple and cost effecitve method for the preparation DNA vaccine whichcan be applied to animal experiments. In animal experiments, we found that the humoralimmune response can be dramatically enhanced and accelerated by WFR and PFR.Thisresults can provide a basis for the further exploration of HIV-1gp120DNA vaccine andadjuvant. |
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