Dissertation > Excellent graduate degree dissertation topics show

Transgenic Study on the Pseudorabies Virus Entry Receptor Gene Nectin-1 Animal

Author: ShangZuo
Tutor: LiuZhengFei
School: Huazhong Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Pseudorabies Virus nectin-1 Eukaryotic expression Lentiviral
CLC: S852.65
Type: Master's thesis
Year: 2011
Downloads: 33
Quote: 0
Read: Download Dissertation

Abstract

Pseudorabies virus (Pseudorabies virus, PRV) can cause pseudo-rabies in wildlife and a variety of livestock, especially swine pseudorabies, causing huge economic losses to the pig industry of China and the world. Other α-herpes virus subfamily members, pseudorabies neural tropism and latent infection characteristics, is an important research significance of animal viral diseases. Currently, the main measures for the prevention and control of the disease is still immunization vaccine. Although it can reduce the establishment of latent infection, but can not completely prevent the virus infection and Sandu, can not prevent the establishment of latent infection. To be able to proceed from the animal's own point of view, bred transgenic animals with anti-pseudorabies, no doubt for the aquaculture industry, is an epoch-making progress. PRV genome encoding a membrane protein similar to other herpesviruses, GD gene plasmid as an immunogen inducing a cytotoxic lymphocyte reaction, a glycoprotein that is necessary for invasion of target cells of the pseudo-rabies virus. Nectin-1 gene is the alpha herpes virus receptor mediated pseudo-rabies virus into the pig epithelial cells and nerve cells. Nectin-1, have an N-terminal immunoglobulin-like domain, this domain is combined with the pseudo-rabies virus glycoprotein gene gD, leading to the viral envelope with the cell membrane fusion, nucleocapsid enter cells, incidence of infection caused by the body. Constructed a transgenic animal model is the use of this feature of nectin-1, first, the transgenic animal expressing nectin-1 gene extracellular region of a soluble fragment, in the pseudo-rabies virus-infected cells before the first competitive binding with the gD gene, and barrier off gD gene and nectin-1 gene in the cells of the receptor combination, so as to achieve the purpose of prevent the pseudo-rabies virus infected cells, and enhance the ability of the transgenic animal against pseudorabies. Given the above background, the present test were obtained by the method of transfection and transduction expression of nectin-1 gene in a soluble fragment of the extracellular region of the PK-15 cell line, to lay the foundation for building the model of transgenic pigs having anti pseudorabies. The main work includes: 1.PRV receptor nectin-1 gene cloning and molecular characterization of the test based on the the GenBank logged pigs nectin-1 gene sequence (accession number AF308632) to design a pair of primers P1 P2, this test first pig nectin-1 gene cloned from pig liver tissue using RT-PCR method, a comparative analysis of its nucleotide sequence and the deduced amino acid sequence and bioinformatics software. Pig nectin-1 gene with dogs, people, rhesus monkeys, chimpanzees, mice, cattle, horses nectin-1 gene nucleotide sequence homology of 92%, 92%, 91%, 91%, 89% , 93% and 93%; homology of the deduced amino acid sequence is 100%, 95%, 67%, 100%, 100%, 100% and 100%, respectively. Having a highly conserved, sequence analysis of the three N-glycosylation sites in the extracellular region of the executed function and 7 cysteine ??residues are located in the extracellular region, and also between different species have highly conserved, which partly explains the PRV infected pigs, sheep, cattle, cats, dogs and other 35 kinds of vertebrates, has a wide host range. Also provides a theoretical basis for later use nectin-1 extracellular region of transgenic animals to produce anti-pseudorabies virus. 2. Pig nectin-1 extracellular domain outside the District gene cloning and original expression of this trial also use Prime 5 software design one pairs of primers P3, P4, to extract total RNA from pig liver tissue as template, RT-PCR amplification of pig nectin- complete coding sequence of the extracellular region, and cloned into the pGEX-6p-1 prokaryotic expression vector pGEX-6p-1-nectin-1-ecto plasmid was constructed successfully, IPTG 37 ℃ under the conditions of induced expression after 3h, E. coli BL21 expression system, expressing the nectin-1 extracellular region of the protein gene of the pig. SDS-PAGE and detection can be a size of about 62 kDa GST-tag fusion expression band, and mainly exists in the form of insoluble inclusion bodies. An anti-monoclonal antibody anti-six histidine specificity of protein expression, the expression product immunized with three New Zealand white rabbits were prepared nectin-1-ecto polyclonal antibody was confirmed by Western blot analysis . 40 days after the arterial blood collecting a small amount of serum, the titer was measured by ELISA tests 1:640 3. Construction of the PK-15 cell line was constructed expressing porcine nectin-1 extracellular region gene expression of porcine nectin-1 extracellular region genes eukaryotic expression plasmid pCA-nectin-1-ecto PK-15 cells transfected using liposome method, and in three weeks with G418 to select stable expression of pig nectin-1 extracellular region of PK-15 cell line. And flow cytometry, indirect immunofluorescence and Western blot confirmed swine nectin-1 extracellular domain gene expressed in cell lines constructed from the protein level. PK-15 cell line inhibition of the PRV virus proliferation test, compared with the normal PK-15 cells and filtered by the PK-15 cell line has a significant inhibition of the proliferative capacity of the PRV virus. 4 lentivirus transduced PK-15 cell line Construction of in many retroviral vector, lentiviral vector as the use of this test HIV vector construct a transgenic animal. 293FT cells simultaneously as the packaging cell prepared lentivirus particles. This test in transfected of 293ft packaging cells, while transfection addition to carrying the other outside of the the foreign gene pLenti-7.3CA plasmid the three helper plasmid pLP1, pLP2, and pLP / VSVG to express four H1V lentiviral structural gene. After assembly by packaging cell secreted into the cell supernatant, the cells were collected supernatant transcend the HIV lentiviral particles. Exceed accordance with 5 × 108pfu/ml away lentiviral viral particles with low-speed centrifugation method transduction PK-15 cells after 72 hours by inverted fluorescence microscopy positive cell clones were picked expanding culture to obtain lentiviral transduction capable of stably expressing of swine pLenti7.3CA-Nectin-1-ecto PK-15 cell line. Both cell lines to inhibition of virus proliferation experiments comparing this test by inoculation test three aspects of the one-step growth curve test and the Western blot method to verify that the two cell lines to inhibition of virus proliferation assay. Inoculation trials, both PK-15 cell lines compared with normal PK-15 cell line, can inhibit the proliferation of the virus. The comparison between the two cell lines, the difference is not obvious. In one-step growth curve experiments, normal PK-15 cells and successfully constructed using the above two ways PK-15 cell line by the same amount each culture bottle at 7 bottles of T-25 cells, 24h after inoculation 5MOI measured at the The virus concentration of 1 × 108 the PRV-Ea strains of the virus, according to Oh, 3h, 6h, 12h, 18h, 24h, 7 point in time to collect the supernatant of the three cells. Titer was measured and found by lentiviral transduction method to obtain the PK-15 cell line is valid in the first 9h inhibit the proliferation of the virus, and the cell lines obtained by transfection of PK-15 began in the first 12h proliferation of the PRV inhibition. PK-15 cells by Western blot to verify transfection and transduction empty vector and the above two ways to build a successful PK-15 cell line protein amount. The test results showed that: at 36 kDa, the supernatant of both cell lines expression of the target protein and a small amount of total protein of the cell itself nectin-1 expression at 57 kDa, and transfection and transduction empty vector the PK-15 cells, only a small amount of nectin-1 protein expression at 57 kDa. These results one step closer description of the two cell lines. Antiviral effect, and no significant differences between the two. 6. Lentiviral vector in a transgenic animal test This test uses lentiviral infection Law, select Hubei white pigs, pregnant sows autologous transplantation, the above obtained lentiviral virus particles in accordance with a 6 × 108pfu/ml lentiviral concentration The ,4-5pL / embryo dose, injected into the fertilized egg of egg week gap microinjection method, a total of successful injection of 153 embryos and transplanted to six conceive pigs. After 2 months, the sow all abortion, failed to obtain the survival of transgenic pigs. Take the six aborted fetal tissue samples in the DNA, PCR revealed no positive transgenic pigs.

Related Dissertations

CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
© 2012 www.DissertationTopic.Net Mobile