Cloning, Expression, and Antigenesity Analysis of the PCV2 Coat Protein Gene (ORF2)
| Autor: | Yu-San Chen, 陳裕森 |
|---|---|
| Rok vydání: | 2006 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 94 Porcine circovirus type 2 belongs to the Circoviridae family. PCV2 is a small, nonenveloped virus with a single-stranded circular DNA genome of about 1.76 kb. The open reading frame ORF2 of the PCV2 genome encodes the immunogenic structural capsid protein. The expression for this immunogenic protein using full length ORF2 DNA was difficult to obtain in large quantity in prokaryotic expression system. Therefore, an alternative approach was to divide ORF2 DNA into three segments for expression, F1, F2, and F3. It was discovered that expression in E.coli was obtained in segments other than F1, which contained the N-terminal sequences. When F1+F2 (named F6) and F2+F3 (named F5) fragments were cloned and expressed, F6, similar to F1, was not expressed due to the N-terminal sequences. On the other hand, F5 was still expressed in large quantity. When dividing the full length ORF2 into half, F4 with C-terminal sequences was again expressed in large quantity as predicted. Analysis on PCV2 ORF2 full length sequence showed that within F1, there were 21 arginine codons, far more than the 9 arginine codons in F2 and F3 combined. Therefore, five additional constructs with the first 9, 21, 36, 48, 111 nucleotides missing of ORF2 (named F10, F22, F37, F49, and F112 respectively) were cloned and subsequently expressed. It was discovered that PCV2 ORF2 recombinant protein expression increased as arginine codons decreased. When immunizing rat with purified F5 recombinant protein, high antibody titer against PCV2 was induced, as confirmed by the IPMA method in PCV2 infected PK-15 cells. Currently, since PCV2 vaccine is not commercially available, this research project was also aimed for the development of a PCV2 subunit vaccine. Rats were used as an experimental animal model to evaluate the various recombinant proteins, F2, F3, F5, and F49, for their abilities to induce antibodies against PCV2. In addition, F5 and F49 recombinant proteins, in conjunction with ORF1 and/or ORF2 recombinant eukaryotic expression plasmids, were injected in rats to evaluate their abilities to induce antibodies against PCV2. Other than using IPMA for the detection of neutralizing antibodies, I used ELISA to measure the levels of antibodies induced in the collected rat serum of different experimental groups. The result of ELISA indicated that F2 recombinant protein induced the highest level of IgG antibodies in rat. As for neutralizing antibodies, neither the recombinant proteins nor when used in conjunction with recombinant eukaryotic expression plasmids were induced when using rat as an animal model. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
| Externí odkaz: |
|