Production of Polyclonal Antibodies to a Recombinant Potato Mop‐top Virus Non‐structural Triple Gene Block Protein 1

Abstract

The gene encoding the triple gene block protein 1 (TGBp1) of Potato mop-top virus (PMTV) was cloned into expression vector pQE32 tagging the protein with 6xHis on the N-terminus. When the gene was enshortened on its 3¢-end by two different restriction digestions, efficient and high yield bacterial expression was achieved in both cases, as shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. One of these two constructs was used for raising rabbit polyclonal antibodies. The obtained sera and antibodies were tested for the detection of PMTV in laboratory host Nicotiana benthamiana and natural host Solanum tuberosum by enzyme-linked immunosorbent assay (ELISA) as well as by Western blots. The obtained antisera are more suitable for Western blot analysis of infected plants than for ELISA.

Molecular characterization of Indian isolate of peanut mottle virus and immunodiagnosis using bacterial expressed core capsid protein

“…The major expressed protein appeared in the insoluble fraction, which could be successfully purified. Similar results of expression of recombinant His-tagged CP in the insoluble fraction have been reported for several plant viruses, pelargonium zonate spot virus [27], faba bean necrotic yellows virus [15], sugarcane yellow leaf virus [13], potato mop-top virus [5,6] and groundnut bud necrosis virus [12]. However, for some plant viruses such as sugarcane streak mosaic virus, cucurbit yellow stunting disorder virus, cymbidium mosaic virus, the His-tagged viral CP was expressed in soluble fraction [10,11,16,23].…”

Preparation of Polyclonal Antibodies to Grapevine fanleaf Virus Coat Protein Expressed in Escherichia coli

“…Proteins from several plant viruses have been produced in E. coli and used for raising virus-specific antibodies for immunodiagnosis (Lee and Chang, 2008;Cerovska et al, 2006;Jain et al, 2005;Abou-Jawdah et al, 2004;Cerovska et al, 2003;Hourani and Abou-Jawdah, 2003;Korimbocus et al, 2002;Kumari et al, 2001). In this study, we report the use of GFLV CP expressed in E. coli as the antigen to produce GFLV-specific polyclonal antibodies, which proved to be efficient, when tested in ELISA and Western blotting.…”

“…Similarly, an optimal duration in expression at a particular IPTG concentration may vary according to the gene of interest or other conditions even though expression for 3-4 h at 37°C has been widely reported as being optimal (Bragard et al, 2000;Jacob and Usha, 2002;Liu et al, 2001;Saini and Vrati, 2003). Like other virus systems such as Faba bean necrotic yellows virus (Kumari et al, 2001), sugarcane yellow leaf virus (Korimbocus et al, 2002), Potato mop-top virus (Cerovska et al, 2003(Cerovska et al, , 2006 and Groundnut bud necrosis virus (Jain et al, 2005) where tagged viral CP was expressed in insoluble fraction, the fusion GFLV CP was detected in the insoluble fraction (pellet) from the transformed E. coli. Expression of recombinant fusion protein in the soluble fraction has also been reported elsewhere (Hema et al, 2003;Hourani and Abou-Jawdah, 2003;Lee et al, 2008;Raikhy et al, 2007), but we detected only a small amount of the fusion protein in the soluble fraction.…”

“…Biotechnology 14 (4): [173][174][175][176][177][178][179][180]2015 which do not detect the related virus in DAS-ELISA (Cerovska et al, 2006: Korimbocus et al, 2002 due to inability of the coated antibodies to react with native viral epitopes even though it was not case in this study.…”

Highly efficient immunodiagnosis of Large cardamom chirke virus using the polyclonal antiserum against Escherichia coli expressed recombinant coat protein

“…Polyclonal antiserum to the purified preparation of LCCV from large cardamom leaf tissues was raised but the quality was not adequate for efficient detection of the virus by enzyme linked immunosorbent assay (ELISA) [10]. Prokaryotic (E. coli) expression of plant viral antigen provides several advantages in antigen preparation over the traditional methods and the recombinant antigen has utilized to develop immunodiagnosis of several plant viruses [2,4,6,12,13,18]. However there is no report of expression of recombinant CP in bacteria and its application in immunodiagnosis of LCCV.…”