Anti-His tag antibody

We describe scalable and cost-efficient production of full length, His-tagged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein trimer by Chinese hamster ovary (CHO) cells that can be used to detect SARS-CoV-2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both human embryonic kidney (HEK) and CHO cells mediated by polyethyleneimine was increased significantly (up to 10.9-fold) by a reduction in culture temperature to 32 degrees C to permit extended duration cultures. Based on these data GS-CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9-fold to 53 mg/L. Purification of recombinant spike by Ni-chelate affinity chromatography initially yielded a variety of co-eluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in enzyme-linked immunosorbent assay format to detect immunoglobulin G antibodies against SARS-CoV-2 in sera from patient cohorts previously tested for viral infection by polymerase chain reaction, including those who had displayed coronavirus disease 2019 (COVID-19) symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS-CoV-2 trimer which can be used as antigen for mass serological testing.

Galactose-binding lectins (GBLs) are a subdivision of jacalin-related lectins and known to perform an essential role in plant protective mechanisms against plant pathogens, fungi, and viruses. A cDNA sequence encoding GBL was cloned and the gene has an open reading frame (ORF) of 657 bp, encoding a protein of 219 amino acids. The protein is predicted to have a molecular weight and isoelectric point (pl) of 23.76 kDa and 6.29, respectively. GBL has a jacalin-type lectin domain and fits into the JRL superfamily. The evolutionary relationship of GBL revealed that the GBL from Morus multicaulis was firmly related to that of Morus rotundibila, Morus alba, Morus notabilis, Artocarpus integer, Artocarpus nitidus and Champedak (cgb). qRT-PCR analysis showed that the gene was expressed in all the tissues tested, leaf, and bud displaying the highest transcript levels. There was a general decrease in the mRNA transcript level under cold and salt stress as compared to the mRNA transcript level of the control. SDS-PAGE and western blot results showed that His-tagged fusion GBL protein was positively expressed in E. coli. In total, our findings disclosed the molecular basis for the signal transduction mechanisms when abiotic stress is induced in a mulberry tree.