Nanobody for cytoskeletal protein beta-actin and coding sequence thereof
A nanobody and cytoskeleton technology, applied in the direction of immunoglobulin, anti-animal/human immunoglobulin, biochemical equipment and methods, etc., can solve the problem that it is difficult to obtain antiserum
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Embodiment 1
[0025] The preparation process of β-actin recombinant protein with FLAG tag at the carboxyl terminal:
[0026] (1) According to the coding gene sequence of β-actin in NCBI (NCBI Reference Sequence: NM_001101.4), and the FLAG tag sequence (Sigma, USA), the 5' end and 3' end are artificially synthesized with Nco1 and HindⅢ restriction enzymes respectively The gene expressing β-actin-FLAG recombinant protein at the site (Suzhou Jinweizhi Biotechnology Co., Ltd.), the DNA sequence is shown in SEQ ID NO:9, and the encoded amino acid sequence is shown in SEQ ID NO:10.
[0027] (2) Use Nco1 and HindⅢ double enzymes (purchased from New England Biolabs, UK) to obtain the digested β-actin-FLAG DNA fragment and pET28a DNA fragment, and β-actin-FLAG DNA fragment with T4 ligase (purchased from British New England Biolabs) was connected to the pET28a expression vector, and after gene sequence determination, the correct pET28a-β-actin-FLAG expression vector was finally obtained. (See figur...
Embodiment 2
[0031] Nanobody screening process against β-actin:
[0032] (1) Antigen coating: According to the instructions of PureCube NHS Activated MagBeads (cube bioteach, Germany), magnetic beads coated with β-actin-FLAG protein and FALG polypeptide were prepared respectively.
[0033] (2) Pretreatment of the nanobody library: get 500 μl containing 2% (g / ml) skimmed milk powder PBS buffer solution (pH7.4), add non-immune American camel (llama) nanobody phage display gene library (nanobody phage display References for gene bank construction:
[0034]Goldman E R, Anderson G P, Liu J L, et al.Facile generation of heat-stable antiviral and antitoxin single domain antibodies from a semisyntheticllama library.[J].Analytical Chemistry,2006,78(24):8245-55.), making The amount of nanobodies in the solution reaches 1x 10 11 pfu, and add Tween-20 (final volume ratio is 0.1%). After mixing, add 20 μl of magnetic beads coated with FLAG polypeptide, and then rotate and incubate at room temperatur...
Embodiment 3
[0042] Preparation of nanobodies
[0043] In Example 2, after the third round of screening for phage infection, Escherichia coli SS320 (Lucigen, USA) was plated, and 100 single clones containing phage plasmids were picked for sequencing. According to the sequencing results, the single clone with the highest repetition rate (marked as NB001) was selected, the sequence shown is shown in SEQ ID NO:8, and the encoded amino acid sequence is shown in SEQ ID NO:7. The selected nucleotide sequence encoding the Nanobody was connected to the expression vector pET28a by PCR method and T4 ligase ligation method, and the specific process was as follows:
[0044] (1) Design PCR primers for encoding the nucleotide sequence of the Nanobody according to the above sequencing results:
[0045] upstream primer GATC CCATGG GC CAA GGT GTC CAG GCT GAG GTG CAG CTC (SEQ ID NO: 11)
[0046] downstream primer GATC CTCGAG GTC TTC GCT GTG GTG CGC TGA GGA G (SEQ ID NO: 12)
[0047] (2) Introduce the...
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