Construction And Identification Of The Eukaryotic Expression Vector Of PcDNA3.1⁺-P14 And GST Of Schistosoma Japonicum And Investigation On Their Protective Effects

Objective: To construct the eukaryotic expression vector of pcDNA3.1⁺-P14 and GST of S. japonicum, and to investigate the immune-protective effect of them in mice.Methods: 1. Constuction and Identification of the Eukaryotic Expression Vector of pcDNA3.1⁺-SjP14 and SjGST: Purified mRNA obtained from the total RNA of S. japonicum was used as the template of reverse transcription for cDNA. And then the SjP14 and SjGST encoding genes were amplified from the cDNA by RT-PCR. Both SjP14and SjGST genes were cloned into eukaryotic expression vector pcDNA3.1⁺ and their expression in vitro and in vivo 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 were prepared for immunizing mice. BALB/c mice were randomly divided into 4 groups (n=10), including saline control group, pcDNA3.1⁺ group, pcDNA3.1⁺-SjP14 group and pcDNA3.1⁺-SjP14+pcDNA3.1⁺-SjGST group. Except for the control groups, all mice were immunized intramuscularly three times with a 2-week's intervals. Mice in saline and pcDNA3.1⁺ groups were given 100μg saline and pcDNA3.1⁺ pre mouse respectively. The pcDNA3.1⁺-SjP14 group was immunized with pcDNA3.1⁺-SjP14 alone (100μg/mouse every time). And mice in the synergistic group were injected with pcDNA3.1⁺- SjP14 (100μg/mouse every time) plus pcDNA3.1⁺-SjGST (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: Two DNA fragments (SjP14 and SjGST gene) were amplified from the adult worm of S. japonicum by RT-PCR, and the size were approximately 1070 bp and 670bp respectively. Subsequently, they were cloned into the pcDNA3.1⁺ vector. The pcDNA3.1⁺-SjP14 and pcDNA3.1⁺-SjGST plasmids were digested with BamHâ… and Xhoâ… , and the insert of pcDNA3.1⁺- SjP14 and SjGST were the same in length with the PCR products. The recombinant DNA was identified with completed ORF by DNA sequencing. Then the plasmids were purified and transfected to Hela cells, and screened by Neo⁺ antibiotic resistance. RT-PCR and Western blotting results suggested that they can express in vitro and in vivo. After immune the mice, the results showed that DNA vaccine pcDNA3.1⁺ -SjP14 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%. When combined with DNA vaccine pcDNA3.1⁺-SjGST, the protective effect was significantly strengthened, and both worms and eggs burdens were reduced markedly. 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 synergistic group 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 treatment relieved the pathological changes significantly. Besides, pcDNA3.1⁺-SjP14+ SjGST group reversed the hepatic injury better than pcDNA3.1⁺-SjP14 used alone. 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 SjP14 and SjGST were successfully amplified, cloned and expressed. The pcDNA3.1⁺-SjP14 DNA vaccine has some extent of immuno-protection effect on S. japonicum infection. And the protective effect can be enhanced significantly by combining with pcDNA3.1⁺-SjGST DNA vaccine.