Evaluation of expression clones for recombinant coat protein of cucumber mosaic virus infecting banana

Abstract

The present study was carried out to evaluate recombinant expression clones for recombinant coat protein of CMV infecting banana, which can be utilized for producing high quality antiserum for the detection of CMV infecting banana. The study was conducted using existing facilities at Banana Research Station, Kannara and College of Agriculture, Vellanikkara during the academic year 2019-2022. The confirmation of recombinant expression clones with pRSET-C and pET28a expression vectors were carried by restriction digestion using Nhe1 and BamH1 restriction enzymes. The insert having 750 bp CMV-CP gene was released from pET28a plasmid. The confirmed pET28a expression clone along with CMV-CP insert was transformed into E. coli DH5α cells to amplify the clone. The recombinant pET28a/CMV-CP plasmid was cloned into E. coli BL21 pLysS cells for over expression of the protein and presence of recombinant clone was confirmed by colony PCR by using specific CMV-CP primers. The E. coli BL21 pLysS cells harbouring pET28a/CMV-CP was grown in LB broth until reached the log phase and induced with 0.3 mM, 0.5 mM and 1 mM IPTG. Induced culture along with uninduced culture were incubated at varying temperature and time. The incubated bacterial culture was lysed by using Tractor buffer (Protein purification kit)/Sonicator. The cell lysate of induced and uninduced culture were loaded on to 12 per cent SDS-PAGE for separation and to understand the protein profile. In the gel, even though slight expression of CMV coat protein was observed, no band was observed in Western blotting which was the confirmation test. As production of recombinant coat protein was not successful in pET28a expression clone, experiments were conducted to develop new expression clone using expression vector pET32a. Infected leaf samples of banana showing typical symptoms, along with healthy control were tested for CMV infection by DAC-ELISA using specific antiserum purchased from the National Research Centre for Banana, Trichy, Tamil Nadu. Isolate namely, NDRNS-4, KANM1 and KAAS-2 showed maximum absorbance at 405 nm and hence selected for molecular detection using reverse transcriptase polymerase chain reaction with CMV- CP specific primer. The PCR product was purified and CMV- CP amplicon of NDRNS-4 isolate was ligated to pGEM- T linear plasmid vector, which was later transformed into E. coli DH5α cells. Positive clones were selected according to blue-white screening. Cloning i.e., E.coli DH5α/pGEMT/CMV- CP was confirmed through colony PCR using T7 and Sp6 primers of that plasmid, followed by sequencing. Coat protein specific forward (5’GGG GAA TTC ATG GAC AAA TCT GAA TCA ACC 3’) and reverse primers(5’CCC CTC GAG AAC TGG GAG CAC CCC AGA TG 3’) were designed along with recognition sites of restriction enzymes EcoR1 and Xho1.The annealing temperature of designed primer was standardized as 55 °C using gradient PCR. The coat protein gene of CMV was amplified at 750 bp using designed primers and phusion DNA polymerase enzyme. Expression vector as well as amplicon were subjected to ligation and the recombination in expression plasmid (pET32a/CMV- CP) was confirmed through colony PCR and followed by sequencing. The recombinant pET32a/CMV-CP plasmid was transformed into E. coli BL21 pLysS cells for overexpression of the protein and presence of recombinant clone was confirmed by colony PCR by using specific CMV-CP primers. The E. coli BL21 pLysS cells harbouring pET32a/CMV-CP was grown in LB broth until reached the log phase and induced with 0.3 mM IPTG. Induced culture along with uninduced culture were incubated at 37 °C for 3 h, 4h, 5h and 20 °C 16 h. The incubated bacterial culture was lysed by using Tractor buffer (Protein purification kit)/Sonicator. The cell lysate of induced and uninduced culture was loaded on to 12 per cent SDS-PAGE for evaluating the over expression of coat protein. Expression was observed on gel at 25 kDa corresponding to the CMV coat protein.