| Summary: | 碩士 === 朝陽科技大學 === 生化科技研究所碩士班 === 100 === Banana bunchy top virus (BBTV) is taxonomically belonging to the genera of Babuvirus of Nanoviridae family. It infects banana systemically and induces symptoms of stunting, shortening of internodes, small and upward leaves and witches’ broom like of apical stem. Dark greenish lines can be observed on the diseased leaves, midribs, and petioles and the leaf margins turn frequently from yellowing into necrotic that weaken significantly the vigor of the infected plants. Besides aphid transmission, the virus can also be easily transmitted by vegetatively propagated suckers. Nowadays, since industrialized production of banana depends mainly on tissue cultured plantlets as growing materials, the virus indexing of mother plants before tissue culture cloning becomes highly important. Traditionally, detection of BBTV can be approached by both serological and molecular based techniques. However, production of antibody against BBTV is always confined by the difficulty of purifying virions from infected banana tissues. Our laboratory once tested the possibility of producing recombinant coat protein (CP) of BBTV by bacterial expressing technology, but the result was not satisfactory due to low expression of its CP in pET system. In a separate study, we found that a construction of pET-28b(+) expression cassette, namely CyOrN, using a 177 bp sequence encoding 59 amino acid residues from the N-terminal region of coat protein (CP) gene of Cymbidium mosaic virus (CymMV) as upstream leader sequence followed by partial sequences of Odontoglossum ringspot virus (ORSV), could generate significant quantity of expressed recombinant viral proteins. In this study, we took the advantage of this unique expression cassette, CyOrN, by maintaining the sequence of CymMV but replacing the following ORSV sequences with the complete or partial CP gene sequences of BBTV and tested the differential accumulation of different recombinant BBTV coat proteins. The CP gene of BBTV was obtaine by amplification and cloning in pGEM-T Easy vector using primer pairs designed in this study. To facilitate later directional cloning into expressing cassette of CyOrN for replacement of ORSV sequences, we designed the primers by creating a Pst1 site at the 5’-terminus and Xho1 site at the 3’-terminus of the amplified BBTV CP genes. After amplification using the designed primers, the BBTV amplicons were then digested with Pst 1 and Xho 1 restriction enzymes and directionally cloned into CyOrN expression plasmid separately. Three constructions of pET-28b expression plasmids, namely CyBTFCP, CyBTNCP and CyBTCCP, expressing full length (541 bp), N-terminal (304 bp), and C-terminal (198 bp) of BBTV CP gene sequences were made and tested, respectively. These plasmids were then separately transformed into host bacteria, E. coli strain Rosetta, and analyzed its protein expression by induction the bacteria cultures with IPTG. In agarose gel electrophoresis, recombinant proteins with expected sizes of 26 kDa, 17 kDa and 13 kDa were located in the bacterial lysates containing expression plasmids of CyBTFCP, CyBTNCP and CyBTCCP constructs, respectively. These proteins were confirmed by Western blotting tests all reacting positively with antisera against CymMV. The expression levels of these three recombinants were all significantly higher than the control clone of pET-28b expressing only BBTV CP sequence. Two of the expression constructs (CyBTFCP and CyBTNCP) were selected for mass production of their recombinant proteins in IPTG induced bacteria cultures. After purification the recombinant proteins by preparative PAGE, they were used as immunogens to produce their respective antisera (anti-CyBTFCP and anti-CyBTNCP) in rabbits. Both antisera were shown in SDS-immunodiffusion and Western blotting tests to react strongly and specifically with their homologous recombinant proteins and to CymMV CP, indicating the recombinant proteins had indeed raised antibodies against epitopes from CymMV CP sequences. Therefore, these recombinant proteins should theoretically be able to induce antibodies against BBTV CP due to the co-expression of BBTV and CymMV CP sequences in the same protein. This was later confirmed by the revelation of reactivity of both antisera with bacteria expressed BBTV CP in Western blotting test. In the later experiments, we tested the reactivities of both antisera in indirect ELISA with BBTV-infected bananas and CymMV-infected orchids and found that the antiserum against full length BBTV CP (anti-CyBTFCP) could only strongly react with CymMV-infected orchids but not with BBTV-infected bananas. However, antiserum against N-terminal sequences of BBTV CP (anti-CyBTNCP) could give positive reactions against both CymMV-infected and BBTV-infected banana tissues, although the reactivities with BBTV were not as high as those against CymMV. Altogether, we have shown in this study that by the use of CymMV leader sequence in the same pET-28b expression vector, different size of BBTV CP can be expressed and the expression level was significantly higher than the control pET-28b plasmid expressing only BBTV CP sequence. We also showed that the expressed recombinant proteins of CymMV and BBTV can induce both antibodies against CymMV and BBTV. Furthermore, at least one of the antiserum against N-terminal sequence of BBTV CP can be used in indirect ELISA and positively detects BBTV infection in bananas.
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