Production of Squash Mosaic Virus-Specific Polyclonal Antibody Using the Virus Coat Protein Produced from Synthesized Gene as An Antigen

Abstract

Squash Mosaic Virus (SqMV) is one of the causal agents of virus diseases infecting cucurbits causing damage to their products. It is transmitted primarily by some beetles and also reported that the virus can spread through melon seeds. The occurrence of this disease has not been reported in Thailand yet. Nevertheless, Thailand shares a big market of imported and exported cucurbit seed therefore SqMV may become a threat on cucurbit production. This research aimed to produce SqMV recombinant coat protein and used it as an antigen to produce a specific antibody for further development of an effective detection assay. Large and small coat protein genes were synthesized based on the nucleotide sequences of the melon strain (Group 1) of SqMV (GenBank database: Accession number NC_003800.1). The synthesized gene was 1,677 nucleotides in length and the deduced amino acid sequences was 570 residues encoding 62.77 kDa of the SqMV recombinant coat proteins (rSqMV–CP). This protein was tested to confirm their native epitopes by reacting with a commercial SqMV antibody before use as an antigen for polyclonal antibody production in rabbit. The antiserum had been collected for 11 weeks and the highest titer was present in week 11 at 102,800 determined by indirect platetrapped antigen enzyme-linked immunosorbent assay. The diluted antiserum at 1:1,000 was used to react with commercially available SqMV positive controls and other viruses. High specificity to SqMV without cross reaction with 21 species of other viruses brought into examination was observed which included Cucumber leaf curl virus, Cowpea mosaic virus, Cucumber mosaic virus I, Cucumber mosaic virus II, Chili veinal mottle virus, Papaya ringspot virus, Potato virus Y, Tobacco etch virus, Zucchini yellow mosaic virus, Cucumber green mottle mosaic virus, Pepper mild mottle virus, Tobacco mosaic virus, Capsicum chlorosis virus, Groundnut bud necrosis virus, Groundnut ringspot virus, Impatiens necrotic spot virus, Iris yellow spot virus, Melon yellow spot virus, Tomato chlorotic spot virus, Tomato spotted wilt virus, Watermelon silver mottle virus.