Comparison between natural antibody production and E. Coli system

Antibody Production				E Coli. Production System
VDJ Recombination	Preparation	RSS Sequence	<----------->	Landing Pad Insertion	Module I: Recombination System
		VDJ Recombinase	<----------->	Helper Plasmid Insertion
		VDJ Library	<----------->	Donor Plasmid Construction
	Recombination			DP Insertion and Recombination Induction
Somatic Hypermutation				Junctional Diversity
				ToxR-based Transmembrane Pathway	Module II: ToxR Receptor System
Antigen Selection
				CBD-based Microarray	Module III: Microarray System

Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. Our system is aiming at imitating this recombination process, using E.coli as the gene carrier. It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.

VDJ Recombination vs Lading Pad Recombination

Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.

Our system is aimed to mimic this recombination process, using E.coli as the carrier. Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.

Somatic hyper-mutation vs Junctional mutation

Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.

In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.

Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method

In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.

Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal. CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.

Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.

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