| Newcastle disease (ND),caused by Newcastle disease virus (NDV) , is a kind of highly contagious disease. NDV can infect many kinds of poultry such as chicken, turkeys, ducks, pigeons, wild parrots and enormously make economic losses, so the World Organization for Animal Health (OIE) determines ND as class A of animal infectious disease. It is well known that F fusion protein located on the membrane of NDV plays an important role in the course of NDV infection, such as its penetration into host cells, the fusion of viral and cellular membranes and the transference of viral genetic material in the cells. F protein is also a kind of viral protective antigen and crucially virulent structure. Therefore, it is considered as the most predominant antigen in the vaccine including genetic engineering vaccine and DNA vaccine. Interleukin-2 (IL-2) is one of important cytokines produced by activated T lymphocyte, which can significantly enhance the differentiation and activation of T cells, development of B cells, as well as increase the stimulation of natural killer (NK) cells. Based on its key role in immune, recently IL-2 is a focus of attention to increasing effect on immune response, especially as an adjuvant to improve the immune process of DNA vaccine.In order to further research DNA vaccine of ND and improve its efficiency in immune response, we cloned NDV F gene and chicken IL-2 gene with the technology of gene manipulation and constructed recombinant plasmid containing them. These genes were well expressed in eukaryotic expression and prokaryotic expression cells.Firstly, according to F gene sequence of F48E9 strain and IL-2 gene sequence of chicken registered in GenBank and multi-clone restriction enzyme sites of eukaryotic expression plasmid pcDNA3, we designed two pairs of special primers for F gene, namely PFA: 5' -CCCAAGCTT-ATGGGCCCCAAATCTTCTACC-3',PFBS:5'-CGGGATCC-T CAGATTCTTGTAGTGGCCCTC-3' and PFBF:5'-CGGGATCC-GATTCTTGTAGTGGC CCTC-3'. We designed also two pairs of special primers for IL-2 gene, namely PIAS:5'CGGGATCC-CTGCCATGATGTGCAAAGTAC-3', PIAF:5'-CGGGATCC-GGTGGC GGACTGCCATGATGTGCAAAG-3' and PIB:5'-GCTCTAGA-CTTATTTTGCAGATAT CTCAC-3'. To manupulate the cloning and expression of genes, corresponding endonuclesae sites, protective sites, linkers, inititation and termination codons were designed at the 5' terminal of the PCR primers. By reverse transcriptase-polymerase chain reaction (RT-PCR), one DNA fragment about 1660bp was amplified from the allantioc fluid propagated NDV F48E9 strain; while the other fragment about 440bp was amplified from chicken spleen cells that were stimulated with ConA for 8 hours, and they were similar to the length of NDV F cDNA and chicken IL-2 cDNA registered in GenBank. The results of endonuclesae digestion of RT-PCR products showed that the two genes had the same eddonuclease specific sites as NDV F and chicken IL-2 gene fragments known. Furthermore, the two fragments were inserted into pcDNA3 vector respectively and identified with PCR and restriction enzyme digestion, the positive recombinant clones (pcDNA3-IL-2 and pcDNA3-F) were sequenced and analysed. The result indicated that the open reading frame (ORF) of NDV F gene consisted of 1662bp, and encoded a protein of 553 amino acids; the ORF of chicken IL-2 gene consisted of 432bp, and encoded a protein of 153 amino acids, there were 99% and 98% homology of nucleic acid sequences with NDV F and chicken IL-2 gene's published respectively. These results suggested that NDV F gene and chicken IL-2 gene were cloned successfully. Under this condition, we linked F gene fragment with IL-2 fragmnet by the linker as a F-IL-2 fusion gene and inserted it into HindIII/XbaI sites of pcDNA3 vector. The further results of PCR, restriction enzyme digestion and sequencing showed that a recombinant plasmid pcDNA3-F-IL-2 was constructed.Secondly, COS-7 and p815 cell strains were transfected with pcDNA3 vector and the recombinant eukaryotic expression vector pcDNA3-F by lipofectamine respectively. To examine whethe...
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