| BackgroundsSince the antibody technology has appeared, mice are usually immunized with protein. These proteins are usually purified from the tissue cell or expressed through the genetic engineering, which is tedious and complicated, and its experiment cycle is long; Some protein's conformational epitope are very easy to change. Substitutes for the protein with the synthesis peptide, the price is expensive, the output is small.SARS, the bird flu, hand-foot-mouth disease and other Infectious disease emergencies unceasingly remind us: Infectious disease emergencies may bring disaster to the mankind at any time. These pathogens always have many subtypes besides gene mutation, reorganization and the rearrangement. Serological diagnosis in early time is important to prevent and to control its dissemination. Serological diagnosis' premise is obtaining the specificity antibody. The conventional method to preparing antibody is too slow.The theory of genetic immunization is that plasmid DNA replaces protein antigen and is transfered into the cell. The protein are expressed in vivo stimulate immune response. The gene immunity may prepare the monoclonal antibody to aim directly at the natural epitope and save 2-3 months. It is a very promising methods to preparation antibody .At present, transgene expression level is quite low and limits in the injection spot as a result of this kind of partial injection. Although conventional routes intravenous injection plasmid DNA may cause DNA to distribute in many organs, after plasmid DNA rapidly degrades by the blood and the organization surface's nuclease, and eliminates very quickly from the blood circulation.In order to overcome these questions, in 1999 Liu and Zhang injected large volume plasmid DNA solution (to account for mouse body weight 8% - 12%) through mouse tail vein (3—8s) fast, discovered that in a short time many kinds of organizations have gene expressions, expresses the highest level in the liver organization. this method is called the hydrodynamics-based procedure. This method can raise the plasmid DNA transfection efficiency significantly, and raised the expression of protein.Since hydrodynamics-based procedure has appeared, it has been obtained the widespread application in the gene therapy aspect, but has no the report on the preparation antibody by gene immunity.DR5 (death receptor 5) was a key receptor of TRAIL receptors and played a important role in TRAIL-induced apoptosis. TRAIL have been tested on a number of different tumor cell lines, and results showed that a high proportion of human tumor cells was sensitive to TRAIL-mediated apoptosis, at the same time many experiments showed TRAIL had minimal cytotoxic to most normal cells and tissues. TRAIL was supposed to be the potential drug target for cancer therapy.AimsTo provides a new way for preparation monoclonal antibody by genetic immunization via Hydrodynamics-based procedure.MethodsTotal RNA was prepared from Jurkat cells by Trizol. DR5 cDNA was cloned into pcDNA3.0 vector, and was confirmed by sequence analysis. Mice were immunized with pcDNA3.0/DR5 4 times(delivered on weeks 0, 2, 4, 6) by Hydrodynamics-based procedure and intramuscular injection. The sera were collected. The titers were identified by ELISA. Specific of antibody was identified by Western blot and immunohistochemical method.MAb was obtained by hybridoma technology. Non-competition ELISA method to analyze affinity constant, Ig sub-class and specificity of mAb was analyzed by ELISA , FACS identified the capacity of antibody binding DR5 in Jurkat cell surface .ResultsThe plasmid pcDNA3.0/DR5 which express DR5 in eukaryotic cells was successfully constructed. Then immunity mice via hydrodynamics-based procedure obtained the higher level polyclonal antibody serum. ELISA measurements showed that the antibody titer of group hydrodynamics-based procedure and of group intramuscular injection can be 1∶102 400 and 1∶25 600. Western blot indicated that the antibody of group hydrodynamics-based procedure could obtain more obvious result. The native DR5 protein in the cellular nuclear membrane of esophageal cancer cells can be recognized with immuno-histochemistry.We had a hybridoma cell (A9) by hybridoma technique. It is IgM hypotype. Its affinity constant is 1.64×10~9L /mol. FACS identified the antibody can specific bind DR5 in Jurkat cell surface .ConclusionsFirst, we gain the pcDNA3.0/DR5 .Second, Genetic immunization by hydrodynamics-based procedure can offer higher titer of mouse anti-DR5 polyclonal antibody than by intramuscular injection.Third, Genetic immunization by hydrodynamics-based procedure can prepare monoclonal antibody.
|
PDF Full Text Request