VHH antibodies against new coronavirus, SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1 complete genome provided by the National Center for Biotechnology Information (NC_045512.2) was analyzed and the gene of the receptor binding domain (RBD) of spike protein was synthesized.

RBD binds to ACE2 (angiotensin converting enzyme 2), which is a receptor for SARS-CoV-2 that is expressed on the human cell surface. The RBD gene was ligated to pET22b E. coli protein expression vector and expressed as 6xHis-tag fusion protein.

Affinity purification using Ni-NTA was performed and used as a target antigen for VHH antibody screening. As a control, 6xHis fusion proteins on the N-terminal region and the C-terminal region of the S1 subunit were also prepared.

*Click on the image to enlarge.

Based on a VHH antibody-displaying phage library having a diversity of 20 billion, VHH-displaying phages having binding properties were concentrated by a three-round panning method, and then cloned.

The FR3-CDR3-FR4 gene of the obtained VHH antibody clone was amplified by PCR, ligated with the FR1-CDR1-FR2-CDR2 gene having diversity of 1 billion, inserted into a phage display vector, and transformed into E. coli. As the result, we created a SARS-CoV-2 specific CDR3 preservation library with about 100 million diversity.

From the SARS-CoV-2-specific CDR3- preserved library, VHH-displaying phages that had binding properties were concentrated by two rounds of panning and then cloned. As a result, clones having 19 different amino acid sequences were obtained.

Five clones were selected from these, VHH-Fc (Fc is derived from mouse IgG1) expression constructs were prepared, and HEK293T cells were transiently transduced, and ELISA was performed using the culture supernatant.

As a result of examining the binding to the full length (S1-Full), RBD, and C-terminal side, it was confirmed that the all VHH clones specifically bind to RBD and S1-Full having RBD.

It was also confirmed that those also bind to the S1 subunit protein and RBD that were transiently expressed in HEK293T cells.

*Click on the image to enlarge.

As a result of verifying whether the obtained VHH clone can be used in Western blotting, it was confirmed that it could be used.

(the display data used 2-mercaptoethanol-treated antigen, but it can be detected without it)

*Click on the image to enlarge.

Development period information

*It shows the period of development as an emergency response when the company's business was relatively light. Therefore, it is not always possible to produce other antibodies at the same time.

*The above period does not include the waiting time for the ordered reagents to arrive and the period spent for other tasks.

Providing method

We will provide the acquired VHH antibody gene sequence data in PDF and VHH antibody protein expression / purification protocol in E. coli system for NON-commercial research.

If you would like to apply, please agree to the following provisions and fill in the necessary items in the application form below.

Those who apply will be deemed to have agreed to the provision terms, so please carefully check the contents and apply only if you can agree.

Provision clause

1.

The provided materials and data shall be used for non-commercial research purposes only, and shall not be used for commercial or commercial purposes, or for human treatment, diagnosis, food and drink, etc.

2.

The provided materials and data are used only by me and the research groups to which I belong and are not provided to third parties.

3.

The provided materials and data have experimental and research properties that were created during research, and RePHAGEN does not guarantee the provided materials and data, whether express or implied, and caused by use, maintenance, and storage. I agree that RePHAGEN will not be liable for any result and that RePHAGEN will not be liable for any damages, compensation claims, lawsuits or other losses, whether direct or indirect.

4.

When handling the provided materials and data, I will comply with applicable laws and regulations.

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