Part:BBa_K2865001:Design
AR185-T2A-EGFP, nanobody inhibiting RyR2 phosphorylation
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 155
Illegal NgoMIV site found at 302 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
After utilizing RyR2 to stimulate camels’ immune system and isolating the mRNA coding for heavy-chain antibodies, we got a gene library of camelids-S2808 nanobodies containing several million clones by reverse transcription and polymerase chain reaction. As for the screening techniques we chose phage display, a technique inserting the gene encoding S2808’s nanobody into a phage coat protein gene, causing the phage to "display" the nanobody on its outside while containing the gene for the protein on its inside. We transfected the phages into engineering bacteria and gained amplification products. Followed with multiple rounds of antigen affinity adsorption-elution-amplification, we finally got the S2808’s specific nanobody clones. Camel antibody library construction To construct the camel library, peripheral blood mononuclear cells (PBMCs) were isolated from a total of 300ml blood sample. Total mRNA was extracted from PBMCs for cDNA synthesis. VHH genes were cloned by nested PCR from cDNA library as described previously. The final PCR products (~ 400bp) were cloned into the phagemid vector pCANTAB5E and transformed into electro-competent E. coli TG1 cells for the preparation of phages. Phage Display and Biopanning Phages displaying the VHH proteins on its surface were prepared as described previously. For biopanning, we coated 96-well plates with RyR2. The phages were added to each well to allow bingding. After 1 hour of incubation at room temperature, the unbound and nonspecifically bound phages were removed using 5 washes. The specifically bound phage was then eluted and used to infect freshly prepared E. coli TG1 cells. After four rounds of panning, 300 randomly picked clones were analyzed for RyR2 binding by phage ELISA. Among these clones, 276 antibody fragments specifically bound to RyR2. One antibody fragment which did not bind to RyR2 was choose as a negative control, termed as VHH-AR117.
ELISA
To obtain antibodies that functionally inhibit of RyR2 phosphorylation, each of the antibody fragments was tested for its effect in an ELSA based RyR2 phosphorylation assay. 4 antibody fragments were potent inhibitors of RyR2 phosphorylation. The complementary determining regions (CDRs) were confirmed by sequence analysis and the result revealed that there was only one unique clone in this panel of antibody fragments, termed as VHH-AR185.
