The Research Of New Materials With Anti-FMD Virus Transgenic Pig

Foot-and-mouth disease (FMD) is an acute and highly contagious diseaseoccurring in cloven-hoofed animals, whose etiological agent is foot-and-mouthdisease virus (FMDV). FMDV has7serotypes and more than70different subtypes,so there are many difficulties of prevention and control, which require us to explorethe new methods and way to anti-FMDV. In recent years, with the rapid developmentof biological technology, RNA interference and transgenic technology were widelyused, so using this technology to foster the transgenic animal with anti-virus willbecome possible. In this study, we obtained the new breeding material of anti-FMDV(O subtype) by using RNA interference and transgenic technology, which will lay thefoundations of anti-disease breeding and mass production of transgenic pigs.We chosed the conservative sequences of the VP1and3D gene of FMDV-Oserotype, and selected the period of21nt long nucleic acid sequences. Then the single-strand DNA was synthesised according to the design principles of the sequence,annealing synthetic double-stranded vector RNAi-Ready pSIREN-Retro Q-ZsGreenmixed connection. Interference connections were transformed into competent E. coliDH5α cells, then the positive coloies were be screened and sequecing. The plasmid ofpositive colonies was extracted, and transfected into BHK-21cells of different groupsaccording to the instruction of Lipofectamine2000Reagent Reagent manual. After48h, the virus virulence was measured by TCID50, then the2×103TCID50/hole ofFMDV infected, and observed the CPE changes at10h,20h,40h,80h respectivelyafter inoculation and detected that whether the difference existed between the testgroup and controls. The RNA was extracted from the cells at different periods, andwas reversed transcript into cDNA. The clone of VP1,3D plasmid was used template,the standard curves of target gene and β-actin housekeeping gene was drawn.Thesample of target gene and housekeeping gene repeated3times respectively, taking theaverage of the Ct value, the average Ct value of the sample standard curve divided bythe average number of beta-actin Ct value into the β-actin standard curve. We foundout the best anti-FMDV efficiency from the expression level of the sample, and theninterference segment with the strongest effectiveness of anti-FMDV were selected andconnected to the PXL-EGFP-NEO. The embryonic capsule, the fetal organs, head, tail, spine, limbs and bones wereremoved firstly when the pig fetal fibroblast cells were isolated from the embryo ofpig, the rest were washed with PBS and cut into1mm3size of the block, then15mLdigestive juice of cell were added. The cells were digested fully at39℃,5%CO2incubator, then digestive juice was removed by centrifugation，the culture mediumwith20%fetal bovine serum was added to incubation. The part cells were collectedand frozen storage, and the rest cell was expanding cultured. The plasmid wasextracted from the cells, and was digested into linearization with ApaLI. The plasmidwas transfected into the fibroblast cells of pig embryo after which was precipitationby ethanol. After48h, the positive colonies were selected with350μg/mL of G418antibiotics and cultured mature. The some cells used for identification, the rest cells asdonor cells by as donor cells.Sow ovaries were collected from the city slaughterhouse, then the follicles wasisolated with20mL syringe. The occytes was washed3times under the microscope inthe egg wash liquid put38.5℃, then washed3times again in the mature medium ofmaturation. The occytes was cultured at5%CO2incubator. After mutation, theoccytes was be removed the nucleus use blind suction method under the microscopicoperation instrument, then the fibroblast cells was extracted with Note nuclear needlefrom positive pig fetus which was injected into the transparent zone of perivitelline,then was continually incubated after electrofusion. After the success of cultivation,the appropriate rutting sow was selected, surgical site was exposed after which washad been hocused and bound, then ovarian was found out when we opened theenterocoelia, then the reconstructed embryos was injected into the fallopian tube. Wesutured the enterocoelia after the exposed viscera and the surgical site were had beenapplied the antibiotic. The sow was isolated and continuously used antibiotics6d, andmade the observation of conception. The tail tissue of transgenic cloned pig was taken,the fibroblast cell was isolated and cultivated, and we observed whether it took thegreen fluorescence. Ear tissue of transgenic cloned piglets was used the template forPCR and sequencing, to identify whether there existed402bp band. The isolatedfibroblasts were infected with FMDV, then RNA was extracted from the cells andreversed transcript into cDNA. Then the Real-time PCR identification of transgeniccloning was carried out to identify whether which had the efficacy of inhibit FMDV. In this study,16pairs of shRNA，siRNA16pairs of annealed synthetic double-stranded were designed for VP1and3D gene of FMDV-O serotypes and16interferecarriers were constructed.16interference vectors were confirmed correctly bysequencing. The TCID50of the original virus was calculated using the Reed-Muenchformula, the result indicated that it was0.1mL of10-3.95diluted virus solution, that is,the original virus diluted8913fold which was a1TCID50. Interference vectorsuccessfully transfected BHK-21cells, the extracted RNA was reversed transcriptaseinto cDNA. The standard curves correlation coefficient was0.998of the target geneand β-actin gene, and which had a better linear relationship and higher accuracy. Theinhibition efficiency of pSiRe-Zs-VP1-3and pSiRe-Zs-3D-6were96.8%and87%,respectively. Test results of PXL-EGFP-NEO-VP1-3and PXL-EGFP-NEO-3D-6vectors were confirmed correctly by restriction endonuclease and the sequencing. Wesuccessfully isolated34days pig fetal fibroblast cells with enzyme digestion, and thelinearized plasmid (PXL-EGFP-NEO-VP1-3and PXL-EGFP-NEO-3D-6) wassuccessfully transfected into pig fetal fibroblasts as well as the positive cloning wasformed. Under the PCR detection, positive cells contained target gene segments. Ourlab successfully cultivated porcine oocytes and built the reconstructed embryoscontaining fragments of the anti-FMDV replication interference. In this study, weobtained a total of54cloned transgenic pigs, and12transgenic cloned piglets ofreceptor sows was obtained by natural parturition after114days. The tail fibroblastsof the12transgenic cloned piglets were detected,5out of12five fibroblasts carriedwith green fluorescent. The genomic DNA was extracted from ear tissue of piglets asthe PCR template, the402bp band was amplificated from the f5piglets, the results ofReal-time PCR results showed that the fibroblast cells of transgenic cloning piglethave the efficacy to inhibit FMDV-O serotype replication.The result showed that the transgenic cloning of pig could successfully inhibitthe reproduction of FMDV-O serotype, which can control the FMD disease to someextent in the cell level. This can establish scientific foundation for obtaining anti-FMD resistant breeding material.