Establishment Of DNA Vaccine With Nudix Hydrolase From Trichinella Spiralis And Its Inducing Immune Response

Trichinellosis is a kind of the global distribution of food-borne beast of parasitic diseases, mainly due to raw or half eat pork containing trichinella spiralis larvae and infection. Now to trichinellosis, there is no effective preventive vaccine and control methods. Therefore, countries around the world spend a lot of manpower, material and financial resources used in animal quarantine of trichinella spiralis in meat and veterinary vaccine research every year, hoping to block the spread of trichinellosis from animals to humans.In recent years, the treatment and research of infectious diseases preventive DNA vaccine and tumor DNA vaccine develop quickly. After DNA vaccination, the immune and virus are similar to natural infection process, promoting the occurrence of mucosal immune response, inducting immune response of memory, cross protection response. At the same time, the exogenous gene can express protein antigen constantly in the body and provide immune system stimulation, thus DNA vaccine can stimulate the body to produce more lasting immune response.The adhesion and intrusion of trichinella spiralis into intestinal epithelial cell is the key to its pathogenesis, but its invasion mechanism is unclear. Now it has been speculated that the the invasion of trichinella spiralis into the intestinal epithelial cells may be mediated by related factor or by some secretory protein.Our research group constructed a c DNA library from infective larvae and muscle larvae of trichinella spiralis intestinal curb cuts hybridization in the early time, selecting a molecular weight of 46 k Da and hydrolysis enzyme activity of trichinella spiralis Nudix hydrolase(Ts Nd), this paper adopts the attenuated salmonella as vector of transporting Ts Nd gene into the body, as a result of eukaryotic expression vector carrying plasmid, it has guaranteed the protein expression and the similarity of natural protein. The recombinant attenuated salmonella enters the body through oral pathways, and has invaded the intestinal mucosa. Materials and Methods 1 Trichinella spiralis, carriers, strains, experimental animal and cell lineThe isolate(ISS534) of T. spiralis used in this study was obtained from domestic pigs in Nanyang, Henan Province, China. The Trichinella isolate was maintained by serial passage in Kunming mice every 6–8 months. Speci?c pathogen-free(SPF) female BALB/c mice aged 6–8 weeks were obtained from the Laboratory Animal Center of the Experimental Animal Center of Henan Province. pc DNA3.1 + plasmid vector, e. coli(e. coli) JM109, kept by our laboratory. The cell line BHK- 21(baby hamster kidney cells) was from CDC cell resource center of Henan province. Salmonella typhimurium ⊿ cya SL1344 strains was given by college of animal science and technology, Henan University of Science and Technology. 2 Ts Nd cloning and the construction of eukaryotic expression vectorApplication of bioinformatics and ORF Finder online prediction tools were used to analyze Ts Nd gene ORF, Ts Nd was from 111 to 1358, the total length is 1248 bp, encoding 415 amino acids. PCR primers were designed and amplified the Ts Nd gene fragments, then insert it into the carrier plasmid pc DNA3.1 to form the recombinant plasmid pc DNA3.1-Ts Nd, after enzyme digestion and sequencing to identify it right, the recombinant plasmid will be electricted into attenuated salmonella, and also were kept into the escherichia coli BL21. 3 The construction of recombinant salmonella and transcription and expression in miceIn nucleic acid level in Ts Nd gene was electricted after the PCR identification of salmonella bacteria liquid, after amplification the recombinant salmonella ⊿ cya SL1344 / pc DNA3.1- Ts Nd was build successfully; To test the recombinant bacteria in vivo transcription level, 7 d after primary immune mice’s spleen and mesenteric lymph nodes were tested using RT-PCR, in protein level using immunofluorescence assay(IFA) to observe the Ts Nd expression in the spleen at the same time. 4 Ts Nd recombinant plasmid’s identificationCollecting the trichinella spiralis muscle larva of 35 d after infection.Then extracted trichinella spiralis total RNA using reverse transcription to obtain c DNA. The PCR reaction to confirm Ts Nd gene, Ts Nd gene was cloning into p MDT-19 vector, through Bam HI and Xho I double enzyme and single enzyme digestion, inserting a specific primer PCR identification of positive clones Ts Nd gene fragment size is correct. And then the Ts Nd gene was cloned into eukaryotic expression vector pc DNA3.1, constructing the recombinant plasmid pc DNA3.1-Ts Nd. Sequence determination confirms the correctness of the inserted fragment. In addition, by using recombinant plasmid transfected BHK- 21 cells, the IFA test of Ts Nd in BHK – 21 cells was used. 5 Ts Nd DNA vaccine inducing immune responses in miceTwo ways of immune routes were used with mice tail vein bleeding, ELISA was used to detect resistance expression of Ts Nd antibody Ig G, Ig G1 and Ig G2 a. Collection of immune mice intestinal washings were to detect intestinal secretory Ig A, spleen cells were collected to detect cytokine IFN-γ, IL- 2, IL- 4 and IL- 10. Through IFA test with the immune mice group recognited the specific Ig A of trichinella spiralis body in different developmental stages.At the same time, plasmid muscle injection group by IFA detected Ig G identification of trichinella spiralis body using immune serum in different period. 6 Immune protection induced by Ts Nd DNA vaccine240 BALB/c mice were randomly divided into 6 groups(40 of each group). The mice were respectively orally given and intramuscular injected with DNA vaccine. Empty bacteria and empty plasmid groups, two PBS groups were the control. All groups were immuned 4 times, interval of 2 weeks. At the end of the 7 d after first immunization, the mice were infected with 300 trichinella spiralis muscle larva. After 7 d day and 35 d of infection the mice were killed, collecting trichinella spiralis adult worms and muscle larvaes, observing the number of intestinal adult worms and muscle larvae, calculating the worm reduction rate. Results1. Through Bam HI and Xho I enzyme digestion, PCR identification and sequencing results, it is confirmed that recombinant eukaryotic expression plasmid pc DNA3.1 – Ts Nd was successfully constructed.2. RT-PCR result shows that Ts Nd gene was transcripted in the spleen and mesenteric lymph nodes of immune mice. IFA result shows Ts Nd gene was expressed in the spleen and mesenteric lymph nodes of immune mice.3. The recombinant plasmid pc DNA3.1- Ts Nd transfected the BHK- 21 cells, IFA test founds that Ts Nd gene was expressed in BHK- 21 cells; Western blot analysis showed that Ts Nd expressed in BHK- 21 cells which can be recognited by immune serum.4.Serum antibody testing result shows that the levels of specific Ig G in DNA vaccine orally given and intramuscular injected groups all increased significantly in the 2 and 3 time points after first immunization, and in the last 1 week after immunization reaching the peak, while in the control group and PBS blank control group there is no significant change. 4 and 6 weeks after the first immunization, Ig G1 levels were significantly higher than those of Ig G2 a, Ig G2 a levels were significantly higher before infection after the second immunization, showing that both oral and intramuscular DNA vaccine immunation of mice induced Th1 / Th2 hybrid immune response and mainly Th2 type of immune response predominant.5. Intestinal total Ig A levels were significantly(P <0.05) increased in immunized mice compared with those vaccinated with vector alone or PBS. Specific anti-Ts Nd Ig A was also significantly increased in immunized mice compared with those immunized with vector alone or PBS(P <0.05). And statistical difference was observed in either total Ig A secretion or specific Ig A secretion, especially their ratio in immunized group compared with those vaccinated with vector alone or PBS(P <0.05). At the same time, the immune group produced significantly higher levels of IFN-γ, IL-2, IL-4, and IL-10, compared with those before immunization(P < 0.01). Moreover, the levels of 4 cytokines in immunized mice continued to increase at week 8 after immunization(one week after challenge), showing that both oral and intramuscular DNA vaccine induced the Th1 / Th2 hybrid immune response.6. Oral Ts Nd DNA vaccine immunization group shows adult worm reduction rate(73.32%), and muscle larvae worm reduction rate(49.5%),were obviously higher than empty bacteria in the control group(Fadults = 54.049, Flarvae = 54.049, P < 0.05); Intramuscular immunization group of adult worm reduction rate(40.44%), and muscle larvae worm reduction rate(53.9%) were significantly higher than the empty plasmid as group(Fadults = 43.265, Flarvae = 43.265, P < 0.05). Results show that both orally and intramuscular Ts Nd DNA vaccine immune mice can partly induce immune protection, oral group of adult worm reduction rate is higher than muscle injection(χ2= 22.54, P < 0.01). Conclusions1. The recombinant plasmid pc DNA3.1- Ts Nd was constructed successfully.2. The recombinant salmonella was orally given to mice and recombinant plasmid was intramuscular injected the mice, both inducing the production of specific intestinal secretory Ig A and Th1 / Th2 type hybrid immune response.3. Both Ts Nd DNA vaccines have partly immune protection, and the adult worm reduction rate of oral group was obviously higher than that of muscle injection group, but muscle larvae worm reduction rate difference has no statistical significance.