Human SP-A1 expression in pichia pastoris

An SP-A1 and expression method technology, applied in recombinant DNA technology, biochemical equipment and methods, applications, etc., can solve problems such as loss of water-soluble SP-A, limited quantity and ethics, failure, etc., and achieve easy induction regulation. , high transcription efficiency, not easy to lose the effect

Inactive Publication Date: 2005-02-09
珠江医院
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AI Technical Summary

Problems solved by technology

[0004] The SP-A used clinically is the extract of animal lung tissue or human amniotic fluid. Due to the imperfect extraction process, a large amount of water-soluble SP-A is lost, which restricts the function of PS and the application of SP-A.
On the other hand, due to the immunogenicity of proteins, SP-A drugs cannot be extracted from animal lung tissue like PS phospholipid components, and the extraction from human lung tissue and amniotic fluid is also obviously limited in quantity and ethics
[0005] So far, all efforts to express active SP-A in prokaryotic cell expression systems have failed, and although there have been successful reports on the expression of non-human SP-A in mammalian cell expression systems, there is no genetic engineering of SP-A. product report

Method used

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  • Human SP-A1 expression in pichia pastoris
  • Human SP-A1 expression in pichia pastoris
  • Human SP-A1 expression in pichia pastoris

Examples

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Embodiment Construction

[0028] The plasmid pPIC9K and yeast strains Pichia postoris GS115, KM71, SMD1168, GS115 Albumin, and GS115 β-Gal involved in this example were purchased from Invitrogen.

[0029] The PCR primers used in this example for amplifying the target gene SP-A and identifying transformants were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0030] The main enzymes and reagents involved in this example include DNA restriction endonuclease, T4DNA ligase, and Taq enzyme were purchased from Dalian Bao Biological Engineering Co., Ltd. and Huamei Biological Co., Ltd. Yeast lyase, G418, and glass beads were purchased from Sigma company.

[0031] The operation steps of this embodiment are as follows:

[0032] 1. Medium

[0033] The reagents required for YPD, MGY, MGYH, RD, RDH, RDB, RDHB, MD, MDH, MM, MMH, BMG, BMM, BMGY, and BMMY were all commercially available, and their concentrations were prepared according to the instructions.

[0034] 2. Construction of SP-A1 gene expression...

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Abstract

The present invention discloses the expression of human SP-Al in Pichia pastoris. Said method is characterized by that it clones human SP-Al gene into expression vector, and utilizes electric shock transformation technique to transfer in into pichia pastoris, and screens out multicopy transformant from transformed strain, and utilizes high-expression methanol oxidase 1 (AOXI) to gene promotor, and uses methanol as unique carbon source to make induction expression of recombinant P. pastoris, in the described inductive expression the concentration of methanol is 0.1-1.0% (g/ml). Said ivnention can produce lots of high-activity human SP-Al, its maximum expression amount can be up to 150 mg/L,l and the expressed object protein can possess opsonic action for making macrophage to kill pathogen.

Description

technical field [0001] The invention relates to a DNA recombination technology, in particular to the expression of human lung surfactant-related protein human SP-A1 in Pichia pastoris. Background technique [0002] Pulmonary surfactant (PS) is a lipoprotein complex synthesized and secreted by lung type II cells. Its main function is to reduce alveolar surface tension, maintain alveolar expansion, prevent lung collapse, and maintain normal respiratory physiological activities. The complex is composed of phospholipids and proteins, of which 90% are phospholipids, mainly dipalmitoyl lecithin (DPPC) and phosphatidylglycerol (PG), and 8-10% are pulmonary surfactant-associated protein (surfactant-associated protein). , SP), divided into SP-A, SP-B, SP-C and SP-D, etc. SP-A is the most abundant, accounting for about 50% of the total protein. SP-A is a multifunctional glycoprotein that plays a very important role in regulating the metabolism of pulmonary surfactant, SP gene express...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/81
Inventor 封志纯兰和魁
Owner 珠江医院
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