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Plant oil-body expression vector and process of expressing human acid fibroblast growth factor with plant oil-body

A technology for fibroblasts and growth factors, which is applied in the field of vegetable oil body expression vector and the use of vegetable oil body to express human acidic fibroblast growth factor, and can solve the problems of unsatisfactory product recovery rate and the like

Active Publication Date: 2007-06-27
JILIN AGRICULTURAL UNIV
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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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  • Plant oil-body expression vector and process of expressing human acid fibroblast growth factor with plant oil-body
  • Plant oil-body expression vector and process of expressing human acid fibroblast growth factor with plant oil-body

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Embodiment 1: Preparation and sequence determination of aFGF gene

[0030] Using the haFGF gene as a blueprint, firstly remodel the base sequence of haFGF 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 aFGF gene with plant preference, eleven primers were designed and synthesized, and the length of each primer is about 57-59bp: PA1-PA5 are forward primers, PA6-PA11 are reverse primers, among which PA5 and PA6 is a complementary chimeric primer (with 17 complementary bases); HindIII, KpnI and thrombin cleavage sites, SpeI, EcoRI cleavage sites and protective bases were introduced into primers PA1 and PA11, respectively, so as to clone into Intermediate cloning vectors and plant expression vectors.

[0031] aFGF full-length gene synthesis

[0032] For the fir...

Embodiment 2

[0076] Example 2: Construction of recombinant oil body expression vector

[0077] 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 HindIII restriction sites, and primer 2 introduces XbaI and EcoRI restriction sites. After PCR amplification, about 900 bp The promoter of the 20 KD oleosin gene (PCR reaction conditions: 94°C, 5 minutes; 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute; 72°C extension for 5 minutes after 25 cycles), the PCR product was used After double digestion with HindIII and EcoRI, it was connected to pUC19 to obtain pUCON, and the sequencing results showed that the cloned product was the promoter of rapeseed oleosin gene.

[0078] Primers were designed based on soybean 24kD oil body protein gene sequence: the sequence of primer 1 was: 5′-GAATCT AGA GAT GAT GAT GAT AAG ATG ACC ACA CAA GTA CC, and the sequence of primer 2 was: 5′-GCC GGT ACC ATC AT...

Embodiment 3

[0080] Example 3: Construction of recombinant oil body expression vector containing haFGF gene

[0081] The pUCaFGF plasmid was double-digested with KpnI and SpeI, and the obtained haFGF fragment was connected to the recombinant oil body expression vector p1390ONE to obtain the recombinant plant oil body expression vector p1390ONE-aFGF.

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Abstract

The present invention is gene recombination process, in which human acidic fibroblast growth factors (haFGF) and oil-body protein gene are fused and synthesized to constitute one plant oil-body expressing vector and establish oil-body protein genetically transforming and expressing system. Root nodule agrobacterium is used to transform Arabidopsis thalianuum, safflower, soybean, black bean and rape to obtain transgenic plant. The molecular biology detection on transgenic safflower and rape proves the integration and expression of exogenous gene, and the activity detection proves the bioactivity corresponding to the standard haFGF activity of obtained recombinant protein. The present invention also provides the method of expressing aFGF in plant seed, and the method may be used in producing aFGF at low cost and developing new medicine.

Description

technical field [0001] The invention relates to the biological field, in particular to a plant oil body expression vector and a method for expressing human acidic fibroblast growth factor by using the plant oil body. 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 cen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/62C12N15/09C12N15/29C12N15/12
Inventor 李校堃庞实锋肖业臣柯实张驰
Owner JILIN AGRICULTURAL UNIV
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