The pComb3HSS vector is the advanced version of the initial pComb vector, signifying substantial improvements and advancements over the first generation. The distinctive feature of the pComb3HSS vector, compared to its predecessor, is its enhanced stability. Moreover, it involves a newly established cloning region that exploits asymmetrical SfiI restriction sites. These restriction sites are used for the directional cloning of full Fab, scFv, peptides, and a variety of other proteins aimed at phage display.
The label 'SS' in pComb3HSS denotes the 'double stuffer,' a unique characteristic of the vector. This inclusion comprises two stuffers: a 1200 bp stuffer present in the Fab light chain cloning area contained within SacI and XbaI restriction sites, and a 300 bp stuffer in the Fab heavy chain cloning region surrounded by XhoI and SpeI restriction sites. An SfiI digest procedure can remove this 1600bp double stuffer (both stuffers plus the leader sequence between the Fab light chain and heavy chain cloning regions). This removal apparatus allows for the cloning of non-Fab genes of interest. One can express soluble Fab by eliminating the gene responsible for the pIII phage fusion protein, a process completed via a SpeI/NheI digest. Apart from this, sufficient soluble protein is intrinsically found in the periplasmic space due to the action of proteolysis.
The use of hybridoma technology to produce the KKU505 mAb for OV infection detection has limitations in terms of stability and genetic changes that affect the quality of the mAb. To solve this problem, preserving antibody sequences in plasmids is a feasible solution to achieve long-term stability. Therefore, the purpose of this study was to construct the pComb3HSS phagemid vector with the KKU505 Fab sequence and then characterize the properties of the KKU505 Fab fragment through experimental studies and molecular simulations.
Briefly, phagemid vectors carrying KKU505 Fab sequences were constructed, and each KKU505 Fab fragment was ligated into the pGEM-T easy vector and then transferred to the pComb3HSS phagemid vector. Phage-displayed KKU505 Fab fragments were successfully generated and verified by a combination of indirect phage ELISA and immunohistochemistry experiments. Furthermore, molecular simulations via antigen-antibody docking were performed to characterize the complex. Molecular simulations have docked KKU505 mAb to the OV myosin head model as an antigen-antibody interaction in Western blot results. The results of this study show that the pComb3HSS phagemid vector carrying anti-OV antibodies was successfully constructed.
Figure 1. Validation of phage display KKU505 Fab fragment by indirect ELISA. (Siripanthong, Sitthinon, et al. 2021)
In the field of research, the pComb3HSS vector plays a crucial role in molecular biology, genomics, and proteomics. Through revealing the interaction between proteins and other molecules, it aids researchers in understanding a vast array of biological processes, modeling of diseases, and discovering potential targets for future treatment. A major use of the vector involves studying protein-protein, protein-peptide, and protein-DNA interactions, vital components of cellular functions.
One significant application of the pComb3HSS vector is in the development of novel therapeutics and production of monoclonal antibodies. Phage display technology using the pComb3HSS vector allows the functional representation of complex protein libraries, essential for designing drugs, identifying novel therapeutic targets, and developing antibody engineering.
The pComb3HSS vector has been implicated in developing high-affinity human antibodies against various antigens, both in the diagnostic and therapeutic field. It can be used to identify antibodies with high specificity to target antigens, enhancing the efficiency of therapeutics.
In biomedical engineering, the pComb3HSS vector can be utilized for tissue engineering and the development of bioactive materials. All these are achieved through the visualization of phage display on the modified gene III and subsequent manipulation of the bacteriophage to display specific biomaterial-binding peptides.
Customer Q&As
What is “SS”?
A: "SS" refers to double stuffing regions, a 1200 bp stuffing region bounded by SacI and XbaI restriction sites in the Fab light chain clone region and a 300 bp stuffing region bounded by XhoI and SpeI restriction sites in the Fab heavy chain cloning region.
Can this "SS" double stuffer be removed?
A: Yes. The "SS" double stuffer region (two stuffers plus the leader sequence between the Fab light and heavy chain cloning regions) can be removed by SfiI digestion so that the non-Fab gene of interest can be cloned.
Ask a Question
Customer Reviews
Worked very well
Using the pComb3HSS vector, I was able to successfully perform the phage display on the modified geneIII.
Highly recommend
It was quite impressive how the second-generation plasmid pComb3HSS showcased a heightened level of efficiency, bringing remarkable ease to my gene manipulation process. I highly recommend this vector to my fellow researchers.
United Kingdom
09/16/2023
Write a Review