Part:BBa_K4961004
PEE12.4-33D9-H
The main task of this experiment is to combine the heavy chain variable region and light chain variable region sequences of 33D9 with the constant region sequences (mIgG2a) on pEE12.4 and pEE6.4, respectively, by molecular cloning technology to form an expression vector that can fully express the antibody. To construct our complete antibody expression plasmid, we asked the Gene Synthesis company to synthesize the DNA fragments, then, using the technique of molecular cloning(homologous recombination), we inserted the heavy chain DNA fragment of the antibody into the pEE12.4 vector. After obtaining the recombinant plasmid, we selected the PCR-correct monoclonal colonies and sent them to the company for sequencing. The validation results showed that we obtained the correct recombinant plasmid.
Contribution by Team YangzhouNOFLS 2023
PEE12.4-33D9-H BBa_K4961004
Usage and Biology
Antibody-drug conjugate (ADC) is an emerging class of highly effective drugs that is a perfect combination of chemotherapy and immunotherapy. As of September 2023, 16 ADC drugs have been marketed worldwide. With the development of ADCs, it is crucial to test the stability of ADCs in plasma[1]. An anti-paclitaxel (ptx) antibody (33D9) that binds Antibody-drug conjugate coupled to ptx with high specificity and affinity (52B8hG1ptx).
Construction Design
The part consists of the gene sequence 33D9-H (encoding the antibody light chain/BBa_K4961003) and the vector backbone PEE12.4(BBa_K4961000).
Figure 1. Profile of the vector backbone pEE12.4
The most classical method of antibody production is the hybridoma technique, and through the preliminary research in the laboratory, we have obtained the heavy chain variable region and light chain variable region sequences of 33D9 respectively. The main task of this experiment is to combine the heavy chain variable region sequences of 33D9 with the constant region sequences (mIgG2a) on pEE12.4, respectively, by molecular cloning technology, to form an expression vector that can fully express the antibody. Then, using the transfection technique, the heavy chain expression vector (pEE12.4-33D9H) was packed with polyethyleneimine (PEI) transfection reagent and was transfected into human embryonic kidney epithelial cells (293F) for antibody expression. After 5 days of expression, we could purify the antibody 33D9 from the supernatant of the culture medium of 293F. finally, an enzyme-linked immunosorbent assay (ELISA) was applied to detect the ability of 33D9 to bind ADC (52B8hG1ptx).
Experimental Procedure
For this experiment, we designed the plasmid: antibody heavy chain (33D9H) protein expression plasmid. Among them, the heavy chain variable region sequence of the antibody was obtained from the genomic DNA of hybridoma cells.
In order to construct our complete antibody expression plasmid, we asked Gene Synthesis company to synthesise the DNA fragments, then, using the technique of molecular cloning, we inserted the heavy chain DNA fragment of the antibody into the pEE12.4 vector. We transformed the expression plasmid into E. coli competent cells, and then coated them on LB solid medium plates containing ampicillin and cultured them overnight at 37℃.
Figure 2. Purity verification and colony inoculation A: Purity of the digested vector B: Purity of amplified fragments
After constructing the antibody expression plasmid, it needs to be allowed to amplify inside E. coli, and after the amplified E. coli is lysed, a large amount of antibody expression plasmid will be released, which provides material for the subsequent transfection experiments. We can get the high purity and low endotoxin antibody expression plasmid by using the company's HiPure Plasmid EF Maxi Kit (Megan).
At present, we have obtained a large number of antibody light and heavy chain expression plasmids. The pEE12.4-33D9H was encapsulated with PEI transfection reagent, and then the complex was transfected inside a 293F cell line using a transfection method, and the cell supernatant was collected after 5-7 days. Subsequently, we used protein A affinity purification to obtain the 33D9 antibody from other proteins in the supernatant of the 293F cells.
The size of the 33D9 antibody is 150kD, of which the size of the light chain fragment is 25kD, and the size of the heavy chain fragment is 50kD. The results of polyacrylamide gel electrophoresis (SDS-PAGE) showed that the purity of the protein that we purified from the supernatant of 293F was high. Therefore, the 33D9 antibody was expressed and purified with high quality, which meets the requirements of our subsequent experiments.
Figure 3 Purity of the 33D9 antibody(DetaiBio), the heavy chain fragment is 50kD
Function Testing
Now we have the product we want to develop, the 33D9 antibody, which binds specifically to paclitaxel. With the double antibody sandwich method.We can detect the concentration of ADC coupled to ptx in different media. Before applying it to a real scenario, we have to make a standard curve for our product to determine its upper and lower limits of detection.
The results of the double-antibody sandwich method showed that we successfully made a standard curve that could detect 52B8hG1ptx (Figure 4). By analogy, any drug coupled to paclitaxel, which itself can be directly coupled to fluorescence or indirectly coupled to fluorescence, can be detected in different solutions using our antibody. This provides strong experimental data to support the development of our ELISA kit.
Figure 4 ELISA results：Standard Curve of 52B8G1PTX
Reference:
[1] Kaur, S., Xu, K., Saad, O. M., Dere, R. C. & Carrasco-Triguero, M. Bioanalytical assay strategies for the development of antibody-drug conjugate biotherapeutics. Bioanalysis 5, 201-226, doi:10.4155/bio.12.299 (2013)
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 689
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- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 689
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Illegal PstI site found at 7801 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 689
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Illegal AgeI site found at 4211 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 6973
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