The Construction of Plant Expression Vector harbouring Carica Papaya L. WRKY Gene in Escherichia coli

Carica papaya is a well-liked and economically important fruit with outstanding nutritional and medicinal values. Its susceptibility to abiotic stress which affects the growth and harvest, causes significant yield loss to farmers. In recent years, significant progress has been made to understand the genes that play critical roles in abiotic stress response, especially some transcription factor (TF) encoding genes. Among all TFs, WRKY TF gene family is one of the best-studied TFs involved in various stress responses. To date, only limited information on functionally characterised WRKY TFs is available for C. papaya. The aim of this study was to produce a recombinant construct harbouring WRKY gene in pGEM®-T Easy cloning vector. The presence of a DNA band of the expected size of 465 bp on agarose gel electrophoresis indicated that WRKY gene was successfully amplified from all treated samples. DNA sequencing analysis revealed that the amplified sequence isolated from the treated samples were closely related to Carica papaya species with 97% similarity. Following transformation, 4 out of 5 colonies that were randomly selected showed the WRKY gene had been successfully inserted into pGEM®-T Easy vector and transformed into E. coli. In future, the WRKY gene from pGEMT-WRKY recombinant construct will be cloned into the plant expression vector pCAMBIA 1304 prior to transformation in the plant. The success of demonstrating the WRKY gene towards the response in abiotic stress will enable us to produce stress tolerant transgenic crops under unfavourable conditions via genetic engineering for sustained growth.

Monoclonal Antibodies B38 and H4 Produced in Nicotiana benthamiana Neutralize SARS-CoV-2 in vitro

The ongoing coronavirus disease 2019 (COVID-19) outbreak caused by novel zoonotic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially reported in Wuhan city, Hubei Province of China, in late December 2019. The rapid global spread of the virus calls for the urgent development of vaccines or therapeutics for human applications to combat the coronavirus infection. Monoclonal antibodies (mAbs) have been utilized as effective therapeutics for treating various infectious diseases. In the present study, we evaluated the feasibility of plant expression system for the rapid production of recently identified therapeutically suitable human anti-SARS-CoV-2 mAbs B38 and H4. Transient co-expression of heavy-chain and light-chain sequences of both the antibodies by using plant expression geminiviral vector resulted in rapid accumulation of assembled mAbs in Nicotiana benthamiana leaves within 4 days post-infiltration. Furthermore, both the mAbs were purified from the plant crude extracts with single-step protein A affinity column chromatography. The expression level of mAb B38 and H4 was estimated to be 4 and 35 μg/g leaf fresh weight, respectively. Both plant-produced mAbs demonstrated specific binding to receptor binding domain (RBD) of SARS-CoV-2 and exhibited efficient virus neutralization activity in vitro. To the best of our knowledge, this is the first report of functional anti-SARS-CoV-2 mAbs produced in plants, which demonstrates the ability of using a plant expression system as a suitable platform for the production of effective, safe, and affordable SARS-CoV-2 mAbs to fight against the spread of this highly infectious pathogen.