Research For Preparation Of NDV HN Gene DNA Vaccine And Its Immunological Effect

DNA vaccine has been widely studied on animal infectious disease since DNA vaccine has been discoverd. Many experiments have shown that the DNA vaccine has a lot of advantages and its requirement is enlarging increasingly, but the cost of the DNA vaccine is still very high, which limit its application, especially on veterinarian fields. In order to estabilish a simple method to produce pure DNA vaccine, in this study we compare some experimental materials and methods in purifing the recombinant plamid DNA under the laboratory conditions. Furthermore, we also observed the immune effect of NDV HN gene DNA vaccine constructed by our laboratotry on experimental animals. Firstly, we optimized the experimental condition of preparation of plasmid DNA. The recombinant plasmids DNA were transformed into E.coli DH5α. After these transformed bacteria were cultured in a volume of 200 mL LB medium containing 100μg/mL ampicillin overnight at 37℃, their growth was observed and cell growth curve was described. The result showed that the density of recombinant bacteria reached the highest level when the incubation for 14 h. Then a large quantity of bacteria were cultured in an optimal cultural time and bacteria were lysed by the alkaline-lysis method under two conditions, namely, ratios of bufferⅠ, bufferⅡand bufferⅢwere 1:1:1 and 2:4:3, respectively. The results demonstrated that the plasmids were two types of congfiguration when the ratio of bufferⅠ, buffer Ⅱand buffer Ⅲ was 1:1:1. The plasmids were three types of congfiguration when bufferⅠ: buffer Ⅱ: buffer Ⅲ was 2:4:3. These indicated the former was better than the latter. Under the optimal conditions the incubated bacteria were lysated. Then the lyset was precipitated with isopropanol (0.6 volumes) and ethanol (2 volumes), respectively. Plasmids and DNA fragments were analyzed by agarose gel electrophoresis (AGE). The result showed that there was no obvious difference between methods with isopropanol and ethanol in the bands on AGE map. The method with isopropanol was simpler and faster than that with ethanol, so in this study the isopropanol was used to precipitate plasmid. Finally plasmid DNA were purified with LiCl + PEG, Q-Sepharos FF, DEAE-cellulose and HA chromatograph, respectively. As a result, plasmid DNA precipated by LiCl + PEG was better than that precipated by either LiCl or PEG,but the production quantity was not large. When Q-Sepharos FF was used, 0.05mol/L NaCl 50mmol/L MOPS(pH8.0)could elute the pure plasmid DNA. No purified plasmids DNA were obtained when DEAE- cellulose was used to purify plasmid DNA for its narrow range of salt concentration. This means that the elution peaks of proteins, RNA, and DNA overlapped extensively with one another and a satisfatory separation could not be obtained. Pure plasmids DNA were aquired by HA chromatography, and the plasmids were eluted by 0.3 mol/L phosphate buffer and the elution peak containing the plasmid DNA was separated well with the peak containing RNA. At last, these methods of purifying plasmid DNA were compared and Q-Sepharose FF chromatograph was chosen. Secondly, Balb/C mice (100μg/dose) and SPF chickens (200μg/dose) were vaccinated with the pure DNA vaccine. The mice were divided into 5 groups. Three groups of mice were vaccinated DNA vaccine twice or three times; One group of mice were inoculated PBS; The last group of mice were vaccinated with plamid DNA vector pcDNA3. The serum was separated from immunized mice and their titres were determinated by ELISA every week after being vaccinated. ELISA results manifested that the IgG antibody could be detected after inoculated for 2 weeks and the antibody titre reached the peak after being vaccinated 6 weeks. SPF chicken serum were separated from 6 chickens chosen at random and the IgG antibody was determinated by ELISA. The ELISA result indicated that the IgG antibody titre reached the summit after being vaccinated 6 weeks. In a word, by comparasion, we chosed Q-Sepharos FF ion exchange chromotograph method to purify plasmid DN...