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Gene sequence for expressing soluble recombinant human hyaluronidase PH20 in CHO (Chinese hamster ovary) cell

A technology of human hyaluronidase and gene sequence, applied in genetic engineering, plant gene improvement, recombinant DNA technology, etc., can solve the problems of high production cost and low level, and achieve the effect of improving the expression level

Active Publication Date: 2013-06-26
广州白云山拜迪生物医药有限公司
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Problems solved by technology

[0006] In our research, we found that the engineered cell lines constructed by transfecting genetically engineered CHO cell lines (specifically DG44 cell lines) with vectors containing soluble rHuPH20 native gene sequences expressed low levels of PH20, resulting in high production costs

Method used

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  • Gene sequence for expressing soluble recombinant human hyaluronidase PH20 in CHO (Chinese hamster ovary) cell
  • Gene sequence for expressing soluble recombinant human hyaluronidase PH20 in CHO (Chinese hamster ovary) cell
  • Gene sequence for expressing soluble recombinant human hyaluronidase PH20 in CHO (Chinese hamster ovary) cell

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Experimental program
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Embodiment

[0044] 1. Optimization of the gene sequence of soluble human hyaluronidase PH20

[0045]On the basis of analyzing the natural gene sequence of human hyaluronidase PH20 (the nucleotide sequence is shown in SEQ ID NO.1), eliminate rare codons, remove unstable motifs, remove RNA stem-loop structures and utilize Chinese hamster ovary Optimal codon redesign for cells. The optimized gene sequence is shown in SEQ ID NO.2. The nblast homology comparison between the optimized gene sequence and the natural sequence did not give the result of homology between the two; while the homology comparison with tblastx showed that the amino acid sequences encoded by the two were 100% homologous ( See figure 1 ).

[0046] In order to improve the expression efficiency of the gene sequence in eukaryotic hosts, a Kozak consensus sequence GCCACC was added before the codon corresponding to amino acid position 1 (methionine) encoded in the gene sequence. The optimized gene sequence was synthesized b...

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Abstract

The invention discloses a gene sequence for expressing soluble recombinant human hyaluronidase PH20 by a CHO (Chinese hamster ovary) cell. The gene sequence has a nucleotide sequence shown in SEQ ID NO.2; after a kozak consensus sequence is added to 5' terminal of the optimized gene sequence, the optimized gene sequence is connected to pOptiVEC-TOPO carrier, and stably transferred to the Chinese hamster ovary cell improved by genetic engineering, so as to achieve high expression of the soluble recombinant human hyaluronidase PH20 in a mammalian cell.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a gene sequence for recombinantly expressing soluble hyaluronidase in Chinese hamster ovary cells (CHO cells) and an engineering cell strain containing the gene. Background technique [0002] Hyaluronidases are a family of enzymes that degrade hyaluronic acid, an essential component of the extracellular matrix and a major component of the interstitial barrier. Hyaluronidase can reduce the molecular weight of hyaluronic acid, thereby reducing the activity and viscosity of hyaluronic acid, and improving the liquid permeability in tissues. [0003] The hyaluronidase family includes bacterial hyaluronidases, hyaluronidases from parasites and crustaceans and hyaluronidases from mammals. There are six hyaluronidase-like genes in the human genome: HYAL1, HYAL2, HYAL3, HYAL4, HYALP1 and PH20 / SPAM1. Among them, HYAL3 and HYAL4 show no or little activity of degrading hyaluronic a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/63C12N15/85C12N5/10C12N9/26
Inventor 李润明丘力功黄明陈建农韦剑蔡小杰孙希海
Owner 广州白云山拜迪生物医药有限公司
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