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Production process of recombinant human horny cell growth factor-2

A keratinocyte and growth factor technology, applied in the field of genetic engineering, can solve the problems of limited expression and low efficiency of human keratinocyte growth factor-2

Inactive Publication Date: 2009-05-13
浙江三万药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method optimizes the selected expression vector, so that the expression amount can be improved (about 200ug / ml), and optimizes the separation and purification process, yet the hKGF-2 coding sequence used is still a natural human hKGF-2 sequence, Therefore, the improvement of expression is still very limited
[0007] However, so far, the efficiency of producing hKGF-2 with E. coli is still low

Method used

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  • Production process of recombinant human horny cell growth factor-2
  • Production process of recombinant human horny cell growth factor-2
  • Production process of recombinant human horny cell growth factor-2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Construction of engineering bacteria expressing rhKGF-2

[0066] 1. Synthesis of target gene

[0067] According to the known natural amino acid sequence of hKGF-2, according to E. coli codon preference and considering the elimination of hairpin structure and other unfavorable secondary structures for expression, the coding sequence of hKGF-2 is designed without changing the amino acid sequence (As shown in SEQ ID NO:1). Entrusted Shanghai Shenggong Biological Engineering Technology Co., Ltd. to synthesize the optimized sequence of hKGF-2 designed.

[0068] When artificially synthesizing the full sequence of the recombinant human keratinocyte growth factor-2 (rhKGF-2) gene, the NdeI site is introduced at the 5'end of the gene and the Sal I site is introduced at the 3'end.

[0069] 2. Construction of expression plasmid-pET30a(+) / rhKGF-2 and transformation of host bacteria

[0070] The expression plasmid is the pET30a(+) expression vector purchased from Invitrogen Company (the...

Embodiment 2

[0077] Selection of suitable expression vector

[0078]According to the method similar to Example 1, insert SEQ ID NO:1 into several different expression vectors, such as pTrc / HisA (purchased from invitrogen company), pBAD / HisA (purchased from invitrogen company), pSE380 (purchased from invitrogen company) ) At the same restriction sites (NdeI / SalI) to obtain plasmids pTrc / HisA / rhKGF-2, pBAD / HisA / rhKGF-2, pSE380 / rhKGF-2.

[0079] The plasmids pTrc / HisA / rhKGF-2, pBAD / HisA / rhKGF-2, pSE380 / rhKGF-2 were respectively transformed into corresponding host bacteria, and resistant strains were selected. A shake flask test was carried out with the engineered bacteria containing plasmid pET30a(+) / rhKGF-2. After 2 hours of induction by IPTG (or arabinose), the target protein expressed by the engineered bacteria containing plasmid pET30a(+) / rhKGF-2 was expressed The amount of the target protein expressed by the engineered bacteria containing plasmids pTrc / HisA / rhKGF-2, pBAD / HisA / rhKGF-2, pSE380...

Embodiment 3

[0082] Choose the best medium

[0083] Pick the E. coli BL21(DE3) (hereinafter referred to as "engineered bacteria") monoclonal that was transformed into pET30a(+) / rhKGF-2 prepared in Example 1, and inoculate it into the LB first-level seed solution, and cultivate for 17-20hr; Two-stage inoculation in a 250ml LB 1L Erlenmeyer flask at a ratio of 1:30, incubate for about 2~3hr, wait for OD 600 It can be fermented in the tank to reach 0.5~1 (M9 medium, modified M9-2 medium, or modified M9-4 medium), pH 6.8~7.2, temperature 37℃, DO>50%, wait for OD 600 After reaching 3~4, add 1mM IPTG to start induction. After the pH rises (the carbon source is exhausted), add 10% carbon source (glucose) and nitrogen source feed to maintain the pH at 6.8~7.2 after the pH rises (the carbon source is exhausted). . The samples were detected by SDS-PAGE (and scanned) to determine the protein content.

[0084] The protein content determination results are as Figure 4 Shown. The results show that both M9 ...

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Abstract

The present invention is the coding sequence and production process of optimized human horny cell growth factor-2 (rhKGF-2), and expression vector and host cell for the production process. The optimized rhKGF-2 series of the present invention has high expression amount and simple separating and purifying process. The present invention also provides corresponding expression vector and engineering bacterium.

Description

Technical field [0001] The present invention relates to the field of genetic engineering, and more specifically to an optimized human keratinocyte growth factor-2 (rhKGF-2) coding sequence, a method for producing recombinant human keratinocyte growth factor-2, and an expression vector used in the method And host cells. Background technique [0002] Recombinant Human Keratinocyte GrowthFactor-2 (rhKGF-2) is a country that treats intractable and chronic wounds such as diabetic venous ulcers, ulcerative colitis, mucositis caused by high-dose chemotherapy, burns and scalds. Class 1 new drugs for therapeutic biological products. It can specifically stimulate the proliferation of epithelial cells, promote the growth of epithelial cells and the formation of granulation tissue, degrade excess collagen, and reduce the production of scars. [0003] rhKGF-2 was originally listed as a member of the FGF family through sequence homology analysis, also known as FGF-10. This family is a large fa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/63C12N15/70C12P21/02C07K1/14
Inventor 任军黄阳滨孙九如丰涛杜碧金
Owner 浙江三万药业有限公司
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