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Construction and transform expression method of human alkaline fibroblast growth factor plant expression vector

A plant expression vector and fibroblast technology, applied in the biological field, can solve the problems of unsatisfactory product recovery rate and the like, and achieve the effects of overcoming correct folding and glycosylation, facilitating purification and improving purification yield.

Active Publication Date: 2012-09-05
吉林农大生物反应器工程有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process is generally done on a trial-and-error basis, and often unsatisfactory product recoveries are not obtained

Method used

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  • Construction and transform expression method of human alkaline fibroblast growth factor plant expression vector
  • Construction and transform expression method of human alkaline fibroblast growth factor plant expression vector
  • Construction and transform expression method of human alkaline fibroblast growth factor plant expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: Preparation and sequence determination of bFGF gene

[0026] Using the hbFGF gene as a blueprint, first remodel the base sequence of hbFGF according to the codons preferred by plants, and eliminate the enzyme cutting sites used in this experiment according to the degeneracy of the codons, and use the online software Primerfinder and DNASTAR to design primers , in order to synthesize the full-length bFGF gene with plant preference, twelve primers were designed and synthesized, each primer length is about 57-59bp: P1F-P6F are forward primers, P7R-P12R are reverse primers, among which P6F ​​and P7R is a complementary chimeric primer (with 19 complementary bases); BamHI and thrombin cleavage sites, SpeI, EcoRI cleavage sites and protective bases were introduced into primers P1F and P12R, respectively, so as to clone into intermediate clones In the vector and plant expression vector, the primer sequence is as follows:

[0027] p1F: CGGGATCCATTGAGGGAAGACCAGCTTT...

Embodiment 2

[0056] Example 2: Construction of recombinant oil body expression vector

[0057] Using the total DNA of Brassica napus as a template, primers were designed based on the sequence of the promoter of the oleosin gene. Primer 1 contains a HindIII restriction site, and primer 2 introduces a PstI restriction site. The oleosin gene of about 900 bp was obtained by PCR amplification (PCR reaction conditions: 94°C, 5 minutes; 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute; after 25 cycles, 72°C for 5 minutes), the PCR product was treated with HindIII and PstI double enzymes After cleavage, it was connected to pUC19 to obtain pUCON, and the sequencing result showed that the cloned product was the promoter of rapeseed oil body protein gene.

[0058] Design primers based on soybean 24kD oleosin gene sequence:

[0059] The sequence of primer 1 is: 5′-aactgcagtcaaccatgaccacagtgccaccac

[0060] Primer 2 sequence is: 5′-cgggatcctgcggttgcggttgttgctgtcactg

[0061] Primer 1 cont...

Embodiment 3

[0063] Example 3: Construction of recombinant oil body expression vector containing hbFGF gene

[0064] After the pUCbFGF plasmid was double-digested with BamHI and SpeI, the obtained hbFGF fragment was connected with the recombinant oil body expression vector p1390ONE to obtain the recombinant plant oil body expression vector p1390ONE-bFGF.

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Abstract

The invention provides an expression vector used for expressing human basic fibroblast growth factors (hbFGF for short). The method adopts a method of gene recombination in vitro, namely, by the fusion of the hbFGF gene and oleosin gene, to construct a plant oleosin expression vector. Arabidopsis thaliana and safflower are transformed by Agrobacterium tume faciens to obtain transgenic plants. Molecular biology proves the integration and expression of a foreign gene. By virtue of activity detection, the obtained recombinant protein has biological activities which are equal to that of a standard plasmid of the hbFGF. The invention also provides a method for expressing the growth factor of a basic fibroblast in a plant seed, which can economically produce the growth factor of the basic fibroblast, thus providing a new way for the drug development of the bFGF.

Description

technical field [0001] The invention relates to the field of biology, in particular to a plant oil body expression carrier and its application in expressing human basic fibroblast growth factor. Background technique [0002] The use of transgenic plants to produce medical proteins has attracted more and more attention in recent years. To express medicinal proteins in plant oil bodies, the target gene is inserted into the N-terminus or C-terminus of the oil body protein gene to construct a fusion protein gene. Since the oil body protein is embedded on the surface of the oil body, the constructed fusion protein gene can be specifically expressed in the oil body of the recipient plant seed, so that the expression of the foreign protein in the plant tissue can be greatly improved, and the oil body can be utilized Lipophilic and hydrophobic properties, after the seeds are crushed, the oil phase can be recovered through steps such as liquid extraction and centrifugation, and the f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/62C12P21/02C12N15/29C12N15/12
Inventor 李校堃李海燕田海山杨晶庞实锋刘秀明王艳芳董园园
Owner 吉林农大生物反应器工程有限公司
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