Production method of recombinant human follicle-stimulating growth hormone

A growth hormone and follicle-stimulating technology, applied in the field of genetic engineering, can solve the problems of rh-FSH stability and u-FSH activity

Inactive Publication Date: 2008-11-05
SHANGHAI NEWSUMMIT BIOPHARMA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the 20% internal variation in the biological activity of urinary gonadotropin, 751U of u-FSH may

Method used

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  • Production method of recombinant human follicle-stimulating growth hormone
  • Production method of recombinant human follicle-stimulating growth hormone
  • Production method of recombinant human follicle-stimulating growth hormone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The construction of embodiment 1 expression plasmid and the acquisition of high expression engineered cell line

[0054] According to the natural amino acid sequence of human follicle-stimulating hormone, according to the codon preference, under the condition of not changing the amino acid sequence, the target gene sequence (SEQ ID NO: 1) of the recombinant human follicle-stimulating hormone protein is synthesized completely. The optimized The homology of FSH gene sequence and natural FSH gene sequence is 93% (see figure 1 ). The gene was cloned into pET-30a(+) and verified by sequencing.

[0055] For expression vector construction methods, see figure 2 . The target human follicle-stimulating hormone gene was obtained by PCR amplification, and the PCR recovery product of the gene IL-4 and the plasmid pET30a(+) were respectively treated with Nde I and EcoR I double digestion, and the corresponding fragment was recovered after digestion with T4DNA ligase Ligation was...

Embodiment 2

[0057] Embodiment 2 Expression of recombinant human follicle-stimulating hormone and determination of expression form

[0058] Select a well-confirmed expression engineered cell CHO / pET30a(+) / FSH, culture it with α-MEM medium (5% dFBS), detect whether there is a target band in the supernatant by SDS-PAGE electrophoresis, and analyze its expression volume. A part of the cultivated culture fluid was taken and centrifuged, and the supernatant was detected by SDS-PAGE electrophoresis to determine its expression form. Analysis of the supernatant and the precipitate showed that the target protein was mostly in the supernatant, indicating that the target protein was expressed in a secreted form (see image 3 ).

Embodiment 3

[0059] The large-scale cultivation of embodiment 3 recombinant human follicle-stimulating hormone

[0060] Resuscitate 1 tube of frozen cells, inoculate in 1 T-25 square bottle, pass to 1 T-75 square bottle after 2-3 days, pass to 3 T-75 square bottles after 2-3 days, 2- Passage to 9 T-75 square bottles after 3 days, pass to 4 spinner bottles after 2-3 days, inoculate in 2.2L reactor (working volume 1.1L) after 2-3 days, culture in medium containing serum, Monitor the changes in the glucose concentration in the reactor. After the glucose consumption of the cells in the reactor reaches 4g / day, change to a serum-free medium for perfusion culture. Adjust the perfusion speed according to the glucose consumption of the cells. Stop the culture when the cells basically no longer consume glucose. Collect the supernatant.

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Abstract

The invention provides a coded sequence of an rhFSH and a method for producing the rhFSH as well as an expressive carrier and a host cell used for the method. A pET system is adopted to express the FSH for the first time, a cell strain with the expression in a high level is obtained by transfecting CHO engineering cells; the high protein expressive rate is obtained by controlling the culture condition; on the base, a large amount of groping and researching of the purification of the rhFSH of the secretion expression are realized to obtain a set of FSH purifying method. The method is adopted to obtain the rhFSH with high yield and high grade of purity and can satisfy the clinical requirements. The coded sequence and method of the invention can obtain the rhFSH purified product with high efficiency, simplicity and low cost.

Description

technical field [0001] The invention relates to the field of genetic engineering. More specifically, the present invention provides a method for efficiently producing recombinant human follicle-stimulating hormone (FSH), including the construction of related CHO cell lines, the expression and purification process of recombinant human follicle-stimulating hormone. Background technique [0002] Recombinant human follicle-stimulating hormone (FSH) is a polypeptide hormone secreted by pituitary basophils into the blood circulation. Female egg cells control the maturation of egg cells, among which FSH plays a key regulatory role and is an indispensable factor for the development and maturation of follicle cells. [0003] FSH is a polypeptide glycoprotein formed by two chains, including α chain and β chain. The two subchains are connected by non-covalent bonds and are transcribed from different genes. The alpha chain contains 92 amino acids and the beta chain contains 111 amino ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/18C07K14/59
Inventor 黄阳滨孙九如任军游磊朱飞云
Owner SHANGHAI NEWSUMMIT BIOPHARMA
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