Extraction method of anti-TNF antibody Fab fragment expressed in periplasmic space of Escherichia coli
A technology of Escherichia coli and periplasmic space, applied in chemical instruments and methods, anti-animal/human immunoglobulin, anti-cytokine/lymphokine/interferon immunoglobulin, etc., can solve problems such as unwell target proteins
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Embodiment 1
[0033] Embodiment 1: the construction of the recombinant Escherichia coli engineering bacterium expressing the Fab fragment of the heavy chain amino acid sequence of SEQ ID No:7 and the light chain amino acid sequence of SEQ ID No:8
[0034] The secretion and expression of Fab antibody in the periplasmic space of Escherichia coli is achieved by introducing the target gene into the empty plasmid expression vector, constructing the recombinant plasmid expression vector, and then transferring it into the Escherichia coli engineering bacteria to construct the recombinant Escherichia coli engineering bacteria.
[0035] 1) Construction and amplification of the target gene
[0036] Before the heavy chain gene sequence of the Fab antibody (the corresponding amino acid sequence is shown in SEQ ID No: 7), a section of OmpA signal peptide gene sequence (the corresponding amino acid sequence is shown in SEQ ID No: 9) is introduced, and then a section containing ribose is inserted The conn...
Embodiment 2
[0054] Example 2: Construction of recombinant Escherichia coli engineering bacterium co-expressing hPDI and the heavy chain amino acid sequence of SEQ ID No:7, the Fab fragment of the light chain amino acid sequence of SEQ ID No:8
[0055] 1) Construction and amplification of the target gene:
[0056] The method for constructing and amplifying the target gene expressing the Fab antibody is the same as in Example 1.
[0057] The amino acid sequence corresponding to the constructed target gene expressing hPDI is shown in SEQ ID No: 11, which already includes a signal peptide sequence.
[0058] Both the target gene expressing Fab antibody and the target gene expressing hPDI were obtained by total gene synthesis, and optimized for the preferred codons of Escherichia coli BL21 (DE3).
[0059] Amplify the cDNA of the target gene expressing hPDI synthesized by PCR. The 5' end primer contains the NdeI restriction site, and the 3' primer contains the XhoI restriction site. These two r...
Embodiment 3
[0077] Embodiment 3: the fermentation of recombinant escherichia coli engineering bacteria
[0078] The fermentation of recombinant Escherichia coli engineering bacteria HL-pET30a / BL21(DE3) and HL-pET30a / hPDI-pCDFduet / BL21(DE3) all adopt the following method:
[0079] The composition of the fermentation medium is as follows: tryptone 16g / L, yeast extract 10g / L, glucose 10g / L, sodium citrate 3g / L, KH 2 PO 4 ·3H 2 O 5.2g / L, (NH 4 ) 2 SO 4 1.5g / L, Na 2 HPO 4 4.05g / L, MgSO 4 ·7H 2 O 1.0g / L, NaH 2 PO 4 2H 2 O 3.0g / L, NH 4 Cl 0.2g / L, FeSO 4 ·7H 2 O 5.45μg / L, ZnSO 4 ·7H 2 O 2.55μg / L, CaCl 2 2H 2 O 3.8μg / L, MnSO 4 ·5H 2 O 1.5μg / L, CuSO 4 ·5H 2 O 0.8μg / L, AlCl 3 ·6H 2 O 0.3μg / L, (NH 4 ) 6 IMo 7 o 24 4H2 O 0.1μg / L, H 3 BO 3 0.5μg / L, Vitamin B 1 2μg / L.
[0080] The composition of feed medium is as follows: glucose 750g / L, MgSO 4 ·7H 2 O 20g / L, citric acid 3g / L, KH 2 PO 4 ·3H 2 O 7.15g / L, (NH 4 ) 2 SO 4 4.8g / L, Na 2 HPO 4 12H 2 O 4g / L.
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