Coding gene, eukaryotic host cell and expression method of human interleukin-12
A technique for interleukin and encoding gene, applied in the fields of encoding gene, eukaryotic host cell and expression of human interleukin-12
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Embodiment 1
[0069] Cloning and sequencing of the p35 and p40 subunit gene sequences contained in Example 1 IL-12
[0070] Using the natural amino acid sequences of P35 and P40 in the UniProt database, some codons in the sequence were replaced according to the 10 candidate genes in the scheme design. After gene sequencing, the result was consistent with the expected sequence in Table 4.
Embodiment 2
[0071] Construction and determination of embodiment 2 plasmid
[0072] The gene sequences of 10 groups of p35 and p40 subunits optimized in Table 4 were respectively cloned into pGN-35 and pCDNA3.1 ( figure 1 ) expression plasmid. Considering that the expression level of the p35 subunit is often lower than that of the p40 subunit, the p35 subunit of each pair of genes was cloned into the pGN-35 plasmid using the EcoR1 and Xba1 restriction sites, so as to utilize the accompanying gene of the DHFR gene during MTX pressurized culture The amplified function amplifies the gene of the p35 subunit. The sequence of p40 subunit of each pair of genes was cloned into pCDNA3.1 plasmid using EcoR1 and Xba1 restriction sites, and cells expressing p40 subunit were selected by Neomycin drug resistance gene. Sequence analysis was performed after cloning to confirm the correctness of the sequence.
Embodiment 3
[0073] Example 3 Expression of rhIL-12 protein
[0074] The obtained expression plasmids containing p35 and p40 subunits were transiently expressed to evaluate the effect of optimized sequences. In the transient expression evaluation test, two host cell lines, CHO-DG44 and CHO-3E7, were selected. Among them, CHO-DG44 is a commonly used host cell for the construction of industrial production-grade stable cell lines. High-yield stable cell lines can be obtained after pressurized screening, but when used for transient expression, its expression level is often not high. Therefore, the test also used the transient expression host cell CHO-3E7 as an alternative reference to make up for the lack of CHO-DG44 in the evaluation of transient expression.
[0075] In the transient expression evaluation test, 10 groups of candidate plasmids were transfected into CHO-DG44 cells and CHO-3E7 cells, respectively. The specific operation is as follows: CHO-DG44 and CHO-3E7 cells were respective...
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