Editing Cloning VH and VL from hybridoma

total RNA purification and first-strand cDNA synthesis, a unique two-step PCR protocol was used to amplify the scFv-encoding gene repertoire. The VH and VL gene pools were amplified from the synthesized cDNA, separately. The scFv genes were assembled randomly by fusing VH and VL fragments using an over-lap PCR. The scFv cDNA and pCDisplay-4 vector were digested by a single, rarely-cutting restriction enzyme SfiI. After ligation, the recombinant constructs were transformed into electro-competent E. coli XLI-BLUE. After overnight culture at 37° C., the amplified recombinant phagemid was extracted from bacteria and kept at −20° C. The library construction was achieved by only one transformation and had a theoretical size of 1.0×107. After that, a standard panning process was carried out. The re-amplified library was incubated with human prominin-1 pre-coated in 96-well plate for 1-2 hours at 37° C. After multiple washes, the binding phage antibody was eluted with a low pH buffer. The retrieved recombinant phages were amplified in E. coli for the next round of panning. After 3 rounds of panning, 30 randomly picked-up clones were checked for their reactivity against prominin-1 by phage ELISA, and nine positive clones were identified. The DNA sequencing results showed that five of them were identical, suggesting that they are all derived from the single hybridoma clone: a hybridoma cell line that secretes anti-prominin-1 MAb. One of them, No. 11 clone, was transformed into E. coli Top10F′ for expression. Soluble expression of scFv was achieved by utilizing E. coli secretion machinery and induced at low temperature (30° C.). The expressed scFv was found mainly in the culture medium and periplasmic space of E. coli. Soluble scFv ELISA was performed to confirm the binding specificity to the target protein. The recombinant No. 11 scFv, expressed both in culture medium and periplasmic space, was specific for human prominin-1 protein, although the binding signal is lower than the positive control, the parent intact bivalent antibody. In conclusion, we were able to successfully clone the scFv gene/cDNA from the selected hybridoma clone.

E. coli XLI-Blue host strain: From Stratagene (La Jolla, Calif.). XLI-Blue is a suppressor strain, in which the amber stop codon would not be recognized and the mouse ScFv antibodies would be fused with phage coat protein III and displayed on the surface of phage virions.

E. coli Top10F′ host strain: From Invitrogen (Carlsbad, Calif.). Top10F′ is a non-suppressor strain, in which the amber stop codon would be recognized and the soluble scFv antibodies would be produced.

VCSM13 helper phage: from Stratagene (La Jolla, Calif.). LB Medium: Per liter: 10 g Bacto-Tryptone, 5 g yeast extract, 5 g NaCl. LB-A Plates: Add 15 g agar to 1 L LB medium, autoclave, when cool, add ampicillin (AMP) to 100 μg/mL. SB Medium: Per liter: 10 g MOPS, 30 g tryptone, 20 g yeast extract. Stir to dissolve, titrate to pH 7.0. Autoclave at 121° C., 20 minutes. Top agar: Add 0.35 g of bacto agar to 50 mL of LB medium. Autoclave. PBS: Per liter: 8 g NaCl, 0.2 g KCl, 1.7 g Na2HPO4, 0.163 g KH2PO4, pH to 7.4 with HCl. Coating Buffer: 0.1M NaHCO3 (pH 8.6) TBS Buffer: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl. Substrate: TMB and H2O2 from Pierce (Rockford, Ill.) Blocking buffer1: TBS (pH 7.4), 1 mg/mLBSA, 0.02% NaN3. Filter sterilized and stored at 4° C. Blocking buffer2: PBS (pH 7.4), 5 mg/mL Non-fat Milk powder, 0.02% NaN3, store at 4° C. HOURP-conjugated anti-M13 antibody: From GE healthcare (Fairfield, Conn.) HOURP-conjugated anti-HA tag antibody: From GenScript Co. (Piscataway, N.J.) HOURP conjugated goat anti-mouse secondary antibody: From Golden Bridge Int., Inc. (Mukilteo, Wash.). Acidic eluting buffer: 0.1M Glycine-HCl (pH 2.2). Phage precipitant (PEG/NaCl): 20% (w/v) polyethylene glycol-6000, 2.5 M NaCl. Autoclave, store at room temperature. Total RNA extraction kit: QIAGEN Co. (Valencia, Calif.). Plasmid purification kit: QIAGEN Co. (Valencia, Calif.). Vector: pCDisplay-4 vector. Agarose gel DNA fragment recovery kit: Takara Co. (Shiga, Japan). Nitrocellulose membrane: Bio-Rad Laboratories, Inc. (Hercules, Calif.).

Mouse VK 5′ primers.









MSCVK-1 GGG CCC AGG CGG CCG AGC TCG AYA TCC AGC TGA CTC AGC C







MSCVK-2 GGG CCC AGG CGG CCG AGC TCG AYA TTG TTC TCW CCC AGT C









MSCVK-3 GGG CCC AGG CGG CCG AGC TCG AYA TTG TGM TMA CTC AGT C









MSCVK-4 GGG CCC AGG CGG CCG AGC TCG AYA TTG TGY TRA CAC AGT C









MSCVK-5 GGG CCC AGG CGG CCG AGC TCG AYA TTG TRA TGA CMC AGT C









MSCVK-6 GGG CCC AGG CGG CCG AGC TCG AYA TTM AGA TRA MCC AGT C









MSCVK-7 GGG CCC AGG CGG CCG AGC TCG AYA TTC AGA TGA YDC AGT C









MSCVK-8 GGG CCC AGG CGG CCG AGC TCG AYA TYC AGA TGA CAC AGA C









MSCVK-9 GGG CCC AGG CGG CCG AGC TCG AYA TTG TTC TCA WCC AGT C









MSCVK-10 GGG CCC AGG CGG CCG AGC TCG AYA TTG WGC TSA CCC AAT C









MSCVK-11 GGG CCC AGG CGG CCG AGC TCG AYA TTS TRA TGA CCC ART C









MSCVK-12 GGG CCC AGG CGG CCG AGC TCG AYR TTK TGA TGA CCC ARA C









MSCVK-13 GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TGA CBC AGK C









MSCVK-14 GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TAA CYC AGG A









MSCVK-15 GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TGA CCC AGW T









MSCVK-16 GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TGA CAC AAC C









MSCVK-17 GGG CCC AGG CGG CCG AGC TCG AYA TTT TGC TGA CTC AGT C

<br/>VK 3′ antisense primers

MSCJK12-BL<br/>GGA AGA TCT AGA GGA ACC ACC CCC ACC ACC GCC CGA GCC ACC GCC ACC AGA GGA TTT KAT TTC CAG YTT GGT CCC<br/>MSCJK4-BL<br/>GGA AGA TCT AGA GGA ACC ACC CCC ACC ACC GCC CGA GCC ACC GCC ACC AGA GGA TTT TAT TTC CAA CTT TGT CCC<br/>MSCJK5-BL<br/>GGA AGA TCT AGA GGA ACC ACC CCC ACC ACC GCC CGA GCC ACC GCC ACC AGA GGA TTT CAG CTC CAG CTT GGT CCC

<br/>VH 5′ sense primers

MSCVH-1<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTR MAG CTT CAG GAG TC<br/>MSCVH-2<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTB CAG CTB CAG CAG TC<br/>MSCVH-3<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG CAG CTG AAG SAS TC<br/>MSCVH-4<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTC CAR CTG CAA CAR TC<br/>MSCVH-5<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTY CAG CTB CAG CAR TC<br/>MSCVH-6<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTY CAR CTG CAG CAG TC<br/>MSCVH-7<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTC CAC GTG AAG CAG TC<br/>MSCVH-8<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG AAS STG GTG GAA TC<br/>MSCVH-9<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG AWG YTG GTG GAG TC<br/>MSCVH-10<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG CAG SKG GTG GAG TC<br/>MSCVH-11<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG CAM CTG GTG GAG TC<br/>MSCVH-12<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG AAG CTG ATG GAR TC<br/>MSCVH-13<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG CAR CTT GTT GAG TC<br/>MSCVH-14<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTR AAG CTT CTC GAG TC<br/>MSCVH-15<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG AAR STT GAG GAG TC<br/>MSCVH-16<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTT ACT CTR AAA GWG TST G<br/>MSCVH-17<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTC CAA CTV CAG CAR CC<br/>MSCVH-18<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG AAC TTG GAA GTG TC<br/>MSCVH-19<br/>GGT GGT TCC TCT AGA TCT TCC CTC GAG GTG AAG GTC ATC GAG TC VH 3′ antisense primers<br/>MSCGlab-B<br/>CCT GGC CGG CCT GGC CAC TAG TGA CAG ATG GGG STG TYG TTT TGG C

Overlap extension primers

RSC-F<br/>GAG GAG GAG GAG GAG GAG GCG GGG CCC AGG CGG CCG AGC TC<br/>RSC-B<br/>GAG GAG GAG GAG GAG GAG CCT GGC CGG CCT GGC CAC TAG TG
[[Category:Antibodies]]