| Pseudorabies virus(PRV)can cause pseudorabies in a variety of wild animals and livestocks. Especially the swine pseudorabies, it has caused tremendous economic losses in the swine industry all over the world. Like other members of a-herpes virus subfamily, nerve tropism and latent infection are important research significance and animal viral diseases characteristics. At present, vaccine is still the main method to prevent and control this disease. Although it can reduce the establishment of latent infection, it can not completely prevent the virus infecting and diffusing, and it also can not prevent establishment of latent infection. From the animal's own point of view, undoubtedly, anti-pseudorabies transgenic animal is an epoch-making progress to the breeding industry.Similar to other herpes viruses, PRV genome encodes membrane proteins. As an immunogen, gD plasmid may induce cytotoxic lymphocytes, which is a necessary glycoprotein for PRV to invade its target cells. Nectin-1 gene is a a herpesvirus receptor which mediates porcine pseudorabies virus into epithelial cells and nerve cells. Nectin-1 has a similar N-terminal immunoglobulin domain, which can bind to pseudo-rabies virus glycoprotein gD, leading to the fusion of viral envelope and cell membrane nucleocapsid and causing the body infected by the disease. Using this feature of nectin-1, the transgenic animal models can be constructed, Firstly, transgenic animals express the soluble extracellular fragment of nectin-1 gene, which could competitively bind to the pseudorabies virus gD gene before the virus infected cells. Consequently it can prevent pseudorabies virus infecting cells, enhancing the resistance ability of transgenic animals.Giving the above background, this experiment have obtained PK-15 cell lines which can express extracellular soluble fragment of Nectin-1 gene using transduction and transfection respectively. This research established the basis for the construction of transgenic pig model with the ability of resist pseudorabies virus. The detailed research were as following:1. The cloning and molecular characteristics of PRV receptor nectin-1 geneAccording to the GenBank pig nectin-1 gene sequence (Accession No. AF308632), we designed one pair primers P1, P2.To further understand the pig nectin-1 gene structure and function, this study combined with bioinformatics methods and RT-PCR to cloned nectin-1 gene in liver tissue from pig, and analyzed its nucleotide sequence and deduced amino acid sequence. Homologies between porcine nectin-1 gene and the dog, human, monkey, chimpanzee, mouse, cow, horse nectin-1 gene nucleotide sequences were 92%,92%,91%, 91%,89%,93% and 93%; deduced amino acid sequences'homologies were 100%,95%, 67%,100%,100%,100% and 100%. In the extracellular domain of executive function are highly conserved. Three N-glycosylation sites and seven cysteine residues are located in the extracellular region, and in different species have very high conservation, which to some extent explains the reason why the pseudorabies virus could infect pigs, cattle, sheep, dogs, cats and other 35 kinds of vertebrates, of a wide host range.2. The cloning and prokaryotic expression of procine nectin-1 extracellular regionAccording to the GenBank pig nectin-1 gene sequence (Accession No. AF308632), we designed one pair primers P3, P4, and extracted total RNA from pig liver as template. Complete coding sequence of porcine nectin-1 cells was amplified by RT-PCR, and cloned into pGEX-6p-1 prokaryotic expression vector to construct the plasmid pGEX-6p-1-nectin-1-ecto. Induced by IPTG at 37℃after 3h in E.coli/BL21, The protein of porcine nectin-1 gene extracellular regoin was expressed. SDS-PAGE electrophores showed a size of about 62kDa fusion protein with GST tag band, and which was mainly in the form of insoluble inclusion bodies. Further, we detected and confirmed the specificity of protein by Western blot with anti-six histidine mAb as the first antibody. Then we produced polyclonal antibody to this protein by immunizing New Zealand rabbits rabbit. A small amount of arterial blood was collected after 40 days, the serum was measured by ELISA and test titer was 1:640.3.Constructed PK-15 Cell Line expressing porcine nectin-1 gene extracellular regionWe constructed a eukaryotic expression vector pCA-nectin-1-ecto expressing porcine nectin-1 ectodomain by using liposome, and screened stable expression of porcine nectin-1 extracellular domain of PK-15 cell lines with G418 within 3 weeks. Then we confirmed that pig nectin-1 gene ectodomain could be expressed in this constructed cell lines which were decked by flow cytometry, indirect immuno-fluorescence and Western blot. In the test of inhibition PRV multiplication, the screening of PK-15 cells inhibited the proliferation of PRV were compared with the normal PK-15 cells.4. Constructed PK-15 Cell Line by Lentiviral transductionAmong retroviral vectors, we chose the HIV lentiviral vector as a vehicle for constructing transgenic animals. At the same time, we used 293ft cells as the packaging cells for preparing lentivirus particles. In this experiment, transfected 293ft packaging cell with carrying the gene pLenti-7.3CA plasmid and the other three plasmid pLPl, pLP2 and pLP/VSVG, they expressed four HIV lentiviral structural gene. Assembled in the packaging cells and secreted into the culture supernatant, collecting supernatant, getting the HIV virus particles by hyperspeed centrifugation. lentivirus obtained from a certain concentration of virus particles in accordance with the low-speed centrifugation transduced PK-15 cells, after 72h observed by inverted fluorescence microscope, positive clones were picked and then expand cultivation to obtain PK-15 cell lines of lentiviral transduction which can stabilizedly express the nectin-1-ecto gene.5. Comparison of inhibition of virus proliferation experiments within two cell linesIn this experiment, we used one-step growth curve and Western blot methods to verify the two cell lines inhibit viral proliferation of static tests. In the static point inoculation, both PK-15 cell lines obviously inhibited virus proliferation comparing to normal PK-15 cells. The comparison between the two cell lines, the difference is not obvious. In the one-step growth curve experiment, the normal PK-15 cells and the successfully constructed two PK-15 cells which were cultured in the same volume of each bottle in the 7 T-25 cellbottles and then inoculated with 5MOI measured PRV-Ea strain of the virus after 24h, at Oh,3h,6h,9h,12h,18h,24h total of 7 time points, we collected the supernatant of the three kinds of cells, we founded the PK-15 cells lines obtained by lentivirus transduction started to effectively inhibit the proliferation of the virus after affected 9 hours, and the PK-15 cell lines obtained by transfection started to effectively inhibit the proliferation of the virus after affected 12 hours. The results of Western blot showed that the two kinds of cell lines expressed a lot of 36 kDa protein in the supernatant.6. Testing of utilizing lentiviral vector in transgenic animalsWe used the lentivirus infection test method, selected Hubei White pigs and then used autologous manner, collected virus particles by using the above methods, which injected into the gap of fertilized eggs by ultra-microinjection. Finally we successfully injected a total of 153 embryos and transplanted into six pregnant pigs.2 months later, sow were all aborted, we failed to obtain the survival of transgenic pigs. Aborted fetal tissue extraction of six samples of DNA, we analysised by using PCR methed.The result showed that there were no positive transgenic pigs. |
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