Cloning an RBD-T4-LINKER-C5a sequence encoding the SARS-CoV-2 antigen into a plant expression vector

. This study aims to make a plant expression vector with an RBD-T4-Linker-C5a sequence that codes for the SARS-CoV-2 antigen and an A3Dsp signal peptide from the rice 3D amylase gene located before the RBD. Methods of molecular cloning were applied in this study. The plant expression vector pNHL22 harboring the RBD-T4-Linker-C5a sequence was successfully established and conjugated into Agrobacterium tumefaciens LBA4404 by triparental mating. Bacteria A. tumefaciens containing the RBD-T4-Linker-C5a sequence are now ready for genetic transformation into the Nicotiana benthamiana plant for future applications.


Introduction
Coronavirus disease (COVID-19), an infectious disease caused by the SARS-CoV-2 virus, has alerted many countries because of the high fatality rate it has produced [1,2].Due to how quickly it spread across continents, the World Health Organization has called it a pandemic.This led to the urgent hunt for effective vaccines and the development of medicines and quick, affordable testing methods.Several vaccine candidates based on nucleic acids, viral vectors, and subunit vaccines are used and tested in clinical trials [3].
Spike (S) glycoprotein (sometimes also called spike protein) is the largest of the four major structural proteins found in coronaviruses.The spike proteins assemble into trimers that form large structures, called spikes or peplomers, that project from the surface of the virion.There are two components of this protein called the S1 and S2 subunits [4,5].The S1 sequence, which contains the receptor-binding domain (RBD) located between amino acids 319 and 591, plays a decisive role in the viral infection process [4].The These productive systems have many benefits, but they also have drawbacks, such as high production costs [8], which may encourage the use of alternative production environments with significant cost savings.Using plants to make recombinant proteins has many advantages over using conventional platforms.For instance, they are inexpensive and easy to cultivate and scale up production [9,10].Generally, plants can be used as bioreactors to make permanent or temporary pharmaceuticals.The latter is a great way to quickly get heterologous proteins with medical value [11,12].In this study, we cloned the sequence encoding a novel SARS-CoV-2 antigen called RBD-T4-Linker-C5a, fused with rice amylase 3D signal peptide (A3Dsp).This sequence was then transferred into A. tumefaciens LBA4404 to prepare for the antigen's production in N. benthamiana plants.
All of them are synthesized precisely according to the previously mentioned order.
Also, three restriction sites for XbaI, KpnI, and SacI were added to the 5' end, the site between the T4 peptide sequence and the flexible linker region, and the 3' end of the Kozak-A3Dsp-RBD-T4linker-C5a-SEKDEL sequence, respectively (Fig. 1).This full-length sequence was synthesized and cloned into the pJET1.2vector (named pNHL22A) by Nanning GenSys Biotechnology Co., Ltd. (China).

Construction of plant expression binary vector
The Kozak-A3Dsp-RBD-T4-Linker-C5a-SEKDEL sequence from the pNHL22A vector was inserted into the pMYV719 plant expression vector, to form the new vector, pNHL22B.
In the first step, the Kozak-A3Dsp-RBD-T4 sequence was cut out of the pNHL22A vector by KpnI and XbaI enzymes (NEB, UK) and then was inserted into the pMYV719 vector, which had been linearized by the same two enzymes, using T4 DNA ligase (Fig. 2).Next, the Linker-C5a-SEKDEL sequence was digested from the pNHL22A vector using KpnI and SacI (NEB, UK) and put into the vector made above, which had already removed the old SEKDEL fragment in the pMYV719 vector using the same enzymes.The new pNHL22B vector containing the Kozak-A3Dsp-RBD-T4-Linker-C5a-SEKDEL sequence was transformed into E. coli TOP10 using the heatshock method (Bergkessel, 2013).The cloning efficacy was checked by restriction digestion.

Triparental mating
A. tumefaciens LBA4404, E. coli TOP10 with the plant expression vector pNHL22B, and E. coli TOP10 with the helper plasmid pRK2013 were all grown in LB medium with the right antibiotics added.Rifamycin (100 µg/mL) was used for A.
tumefaciens LBA4404 culture, whereas kanamycin (100 µg/mL) was used for 2 strains of E. coli TOP10 culture.Triparental mating was initially conducted by the co-culture of three bacteria in solid LB medium.After 3 days of growth, Agrobacterium with the vector pNHL22B was selected in solid LB medium with 100 µg/mL rifamycin and 100 µg/mL kanamycin.Screening for transformed A. tumefaciens colonies by PCR and digestion with restriction enzymes to confirm the pNHL22B vector had been conjugated successfully [13].

Conclusion
We successfully cloned the Kozak-A3Dsp-RBD-T4-Linker-C5a-SEKDEL sequence into the plant expression vector pNHL22B, which is expected to produce SARS-CoV-2 antigen that binds to the nasal cavity and enhances recipients' mucosal immune response, and are now ready to transfer this expression construction into the N. benthamiana genome.
RBD contains eight cysteines and two Nglycosylation sites (N331 and N343).According to Shin et al. (2021), the addition of glycans in in vitro conditions can significantly affect the folding, dynamics, stability, and solvent accessibility of the RBD [6].The RBD-hACE2 complex comprises the angiotensin-converting enzyme 2 (hACE2), located on the exterior surface of the membrane of several types of human cells.Thus, viral particles can enter the host cells and release their genetic material into the cytoplasm of the infected cells [7].The abovementioned factors have prompted researchers to examine the RBD as a potential protein candidate for a subunit vaccination.Various biotechnological platforms have been used to create medications; however, most pharmaceuticals and recombinant vaccines have been produced by microbial or mammalian cells.

Fig. 4 .
Fig. 4. Verification of the Kozak-A3Dsp-RBD-T4-Linker-C5a-SEKDEL sequence in vector pNHL22B by digestion and PCR amplification.A: Digestion with XbaI and SacI, 1 and 2: Plasmids from two single E. coli colonies.B: PCR amplification with specific primers, 1: PCR product, PC: pNHL22A vector was used as a positive control, NC: non-transformed E. coli was used as a negative control.M: Gene Ruler 1 kb DNA Ladder (Thermo Scientific)