Method for efficiently expressing recombinant human interleukin 12 in eukaryotic cell
A technology for interleukin and eukaryotic cells, which is applied in the field of increasing the expression intensity of recombinant human IL-12 in eukaryotic cells, which can solve the problems of reducing production costs and low expression intensity, and achieve the effect of increasing expression
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Embodiment 1
[0038] Example 1: Amplification of genes
[0039] A. Primer design:
[0040] 1) Amplification primers for P35:
[0041] Forward primer: 5'ctc ctgcag atgtggccccctggtcag-3' (SEQ ID NO: 4), wherein ctc is a protective base, and the underlined part is the cutting point (Pst I) when cloning into the expression vector pMSG (purchased from Pharmacia).
[0042] Reverse primer: 5'-gca gaattc ttaggaagcattcagatagctc-3' (SEQ ID NO: 5), wherein gca is the protective base, and the underlined part is the cutting point (Eco RI) when cloning into the above expression vector.
[0043] 2) Primer design for P40
[0044] Forward primer: 5'-ct gctgcag atgtggccccctggtcag-3' (SEQ ID NO: 6), wherein ctc is a protective base, and the underlined part is the cutting point (Pst I) when cloning into the expression vector pMSG (Pharmacia).
[0045] Reverse primer: 5'-gca gaattc ttaggaagcattcagatagctc-3' (SEQ ID NO: 7), wherein gca is a protective base, and the underlined part is the cutting point ...
Embodiment 2
[0059] Cloning, identification and sequencing of the gene of embodiment 2
[0060] The purified amplification product obtained above was inserted into pGEM-T vector (Promega product). The operating steps were carried out according to the manufacturer's instructions to obtain recombinant DNA.
[0061] The above-mentioned recombinant DNA was transformed into Escherichia coli DH5α strain by conventional methods, and 19 positive clones of P35 and 23 positive clones of P40 were obtained by blue / white screening. After 5 clones were identified by P35 and P40 enzyme digestion, 2 and 3 clones were respectively confirmed to have the target insert.
[0062] Bidirectional sequencing was performed on two positive clones of P35 and P40 identified above on an ABI377 automatic sequencer. The sequencing results are shown as SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing.
Embodiment 3
[0063] Example 3 The relationship between the expression of P35 subunit signal peptide I and II and the expression level of P35
[0064] Western blot studies have proved that the secretion of P40 monomer is much higher than that of P35, and the expression of biologically active IL-12 mainly depends on the expression of P35. Therefore, to increase the expression of IL-12, a feasible way is to increase the expression of P35.
[0065] Since it is believed that P35 cDNA is the same as some cytokine gene sequences, its sequence itself may be a regulatory element that regulates its expression, how to adjust the P35 cDNA sequence to increase the expression of P35 is the key to improving the final IL-12 production.
[0066] The structural characteristics of the P35 structural gene and its translation products were analyzed with the analysis software DNAstar version 3.10, and it was found that the encoded product contained two signal peptides (I and II). The first signal peptide consi...
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