The Preliminary Study Of The PcDNA3.1(+)-SjP14 Nanometer Microballoon-DNA Vaccines

Objective: To extract and identify the eukaryotic expression vector of pcDNA3.1(+)-SjP14 and to investigate and compare the immune- protective effect of its DNA vaccine and nanometer microballoons-DNA vaccine on S. japonicum in mice.Methods: 1. Extraction and Identification of the Eukaryotic Expression Vector of pcDNA3.1(+)-SjP14: The recombinant plasmid, extracted from strains and identified by digested with BamHⅠand XhoⅠ, were transferred into Cos-7 cells. Their expressions in vitro were identified by RT-PCR and Western blotting assay. 2. Preparation of DNA vaccines and their protective effects on S. japonicum : The endotoxin-free DNA vaccines and nanometer microballoons-DNA vaccines were prepared for immunizing mice. BALB/c mice (6-8weeks, weight 18-22g) were randomly divided into 3 groups (n=10), including saline control group, pcDNA3.1(+) control group, pcDNA3.1(+)-SjP14 group and nanometer microballoons-DNA vaccines group. Except for the control groups, all mice were immunized intramuscularly three times with a 2-week's intervals. The pcDNA3.1(+)-SjP14 group was immunized with pcDNA3.1(+)-SjP14 alone (100μg/mouse every time). And mice in the nanometer microballoons-DNA vaccines group were injected with nanometer microballoons vaccines (100μg/mouse every time). Two weeks after the last immune, the cercaria of S. japonicum was infected to mice percutaneously (30±1 per mouse). Six weeks later, all mice were sacrifice by cervical dislocation. The adult worms were collected and the worm reduction rate was calculated as an index to evaluate the protective effect of DNA vaccines. Meanwhile, the liver was removed. Some of them were digested and counted for the egg reduction rate. HE staining method was used to detect the hepatic changes and granuloma.Results: pcDNA3.1(+)-Sj P14 plasmids were extracted successfully and were digested with BamHⅠand XhoⅠ. The insert of pcDNA3.1(+)- SjP14 were the same in length with the PCR products. Then the plasmids were purified and transfected to Hela cells, and screened by Neo+ antibiotic resistance. RT-PCR and Western blotting results indicated that they can express in vitro. After immune the mice, the results showed that pcDNA3.1(+)-SjP14 DNA vaccine enhanced the immune function of mice infected with the cercarie of S. japonicum, the worm reduction rate and egg reduction rate reached to 44.6% and 61.6%, respectively. The effects of nanometer microballoons-DNA vaccines were significantly stronger than the pcDNA3.1(+)-SjP14 DNA vaccines, with the worm reduction rate and egg reduction rate reached to 56.2% and 73.5%, respectively. Gross observation showed that the livers in control groups were dark brown and hard, with miliary nodules of worm eggs diffused in the livers. pcDNA3.1(+)-Sj P14 group has some extent of amelioration, the livers were bright red, relative soft and smooth, with less nodules of worm eggs in livers. The color of livers in nanometer microballoons-DNA vaccine was bright red, with smooth surface and soft texture, and few nodules of worm eggs were found in livers. Hepatic histological section demonstrated that there're generous big granulomas containing worm eggs in the livers of control groups, with extensive infiltration of inflammatory cells and degeneration and necrosis of hepatic cells. pcDNA3.1(+)-SjP14 relieved the pathological changes significantly. Nanometer microballoons-DNA vaccine group reversed the hepatic injury better than pcDNA3.1(+)-SjP14 group. The structure of hepatic lobule was integrated on the whole, and the inflammatory reaction around the worm egg granulomas was light. The area of granulomas was also diminution markedly.Conclusion : The pcDNA3.1(+)-SjP14 nanometer microballoon-DNA vaccines were successfully constructed, and its immuno-protective effects were stronger than that of pcDNA3.1(+)-SjP14 DNA vaccine on S. japonicum infection.