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High-efficiency expression method of human interleukin-10 (hIL-10)

A technology for expressing vectors and exogenous proteins, applied in the field of bioengineering, can solve the problems of poor results, difficult to achieve, low high-copy strains, etc., achieve good stability, avoid endoplasmic reticulum pressure, and prevent high-efficiency expression clones. The effect of losing

Inactive Publication Date: 2013-07-10
UNIV OF SCI & TECH OF CHINA
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Problems solved by technology

Because these conventional Pichia expression vectors have a low probability of obtaining high-copy strains, pAO815 vectors (Jing Shenrong, Zou Quanming, etc. Construction of multi-copy expression cassettes of interleukin-10 gene and its establishment in Pichia pastoris Expression. Although the method of Chinese Journal of Biological Products, 2007, 20 (2)) can also obtain high-copy strains, this method is very time-consuming and laborious, and the results are poor and not easy to achieve
At the same time, conventional screening of high-efficiency expression strains is carried out at a temperature of 28-30°C, ignoring the possible adverse effects of the yeast secretion mechanism on the expression of recombinant proteins, resulting in the retention of exogenously expressed proteins in the cells and causing yeast cell viability influences
Therefore, high expression strains of hIL-10 could not be obtained by conventional screening methods

Method used

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  • High-efficiency expression method of human interleukin-10 (hIL-10)
  • High-efficiency expression method of human interleukin-10 (hIL-10)
  • High-efficiency expression method of human interleukin-10 (hIL-10)

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Embodiment 1

[0045] Embodiment 1, the construction of Pichia pastoris high-efficiency expression vector pUCZ9K, the specific process includes the following steps:

[0046] 1.1. Construction of pUCZ plasmid

[0047] The multiple cloning site (MCS) was amplified by PCR using the pUC19 plasmid (purchased from Bao Bioengineering) as a template, and one BglII site was introduced at each of its two ends; the pPICZαA plasmid was digested with BamHI and BglII to recover TEF1- The EM7-Zeocin-CYC1TT-pUC ori fragment was ligated with the recovered MCS fragment after digestion with BglII, and the ligated product was transformed into competent Escherichia coli DH5α (purchased from Treasure Bioengineering), and positive recombinants were screened. The positive recombinant plasmid was sequenced by Yingjun Company. The results showed that the MCS fragment was correctly connected with the TEF1-EM7-Zeocin-CYC1TT-pUCori sequence and the sequence was correct, and it was named pUCZ. The construction process is...

Embodiment 2

[0143] Example 2, Construction of IL-10-His / pUCZ9K Expression Vector and Screening of Yeast High Expression Strain

[0144] 2.1. Construction of hIL-10-His / pUCZ9K recombinant expression plasmid

[0145] 2.1.1. Amplification of hIL-10 gene

[0146] TRIzol (purchased from Invitrogen Company, Cat. No. 15596-018) was used to extract the total RNA of mononuclear cells from isolated healthy human peripheral blood (purchased from Hefei Blood Bank) according to its instructions, and the total RNA was extracted in Turbo Reverse Transcriptase (purchased from Beijing Yuanpinghao Co., Ltd.). , Cat. No. PC108), oligo dT (purchased from Shanghai Sangong, Cat. No. B0181) was used as a primer to synthesize human cDNA, using the synthesized cDNA as a template, the forward primer 5'-ATGCACAGCTCAGCACTGCTCTG-3' and the reverse primer 5 PCR was performed on '-GTTTCGTATCTTCATTGTCATG-3', and the PCR product was inserted into the TA vector pMD18-T (purchased from Treasure Bioengineering). The plasmi...

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Abstract

The invention relates to a high-efficiency expression method of hIL-10, and concretely provides a method for expressing a foreign protein in yeast. The method comprises the following steps: 1, constructing an expression vector, wherein the expression vector includes an rDNA non-coding region, an antibiotic resistance gene, and a coding sequence secreting a signal peptide and the foreign protein; 2, gradually increasing the concentration of an antibiotic to amplify the copy number of the expression vector and construct a clone database containing different copy numbers; and 3, selecting expression cloning, expressing, and recovering the foreign protein, wherein the foreign protein is hIL-10 preferably. The invention also relates to the expression vector used for the method, and uses of the expression vector and yeast strains, wherein the expression vector includes yeast strains of the expression vector. High-efficiency strains of hIL-10, having industrialized values, can be easily obtained through the method, so hIL-10 is expressed and recovered.

Description

technical field [0001] The invention belongs to the technical field of bioengineering. Specifically, the present invention relates to a method for preparing human interleukin-10 (hIL-10) by biotechnology, in particular to a method for constructing a vector for highly expressing hIL-10 in Pichia pastoris and expressing hIL-10 efficiently Screening and establishment of strains. Background technique [0002] hIL-10 can inhibit the immune inflammatory response mediated by T cells and participate in the regulation of humoral immunity by stimulating the B lymphocyte response, so it has a good clinical application prospect. At present, it has been applied to the clinical experimental research of various immune diseases including type I diabetes, psoriasis, rheumatoid arthritis, multiple sclerosis, and hepatitis C. Natural hIL-10 is mainly produced by macrophages with a molecular weight of 18.5KD and no glycosyl modification. The active form is a homologous oligodimer formed by no...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C12R1/84
Inventor 肖卫华钟永军田志刚郭雨刚李光伟沈翼张国英
Owner UNIV OF SCI & TECH OF CHINA
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