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An expression vector containing human insulin gene and its construction method and application

A human insulin and expression vector technology, applied in the preparation methods of insulin and peptides, genetic engineering and other directions, can solve the problems of difficult to expand reproduction, low plant oil content, small seed volume, etc., to improve transformation efficiency, simplify purification process, The effect of increasing the expression

Inactive Publication Date: 2011-12-07
HEBEI CHEM & PHARMA COLLEGE
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The three giants all use microbial fermentation to produce human insulin products, but the use of microbial fermentation technology has its limitations, such as high cost and difficulty in expanding reproduction
However, the seed volume of the safflower plant is small, and the oil content of the whole plant is low, resulting in a small amount of protein in the final product and high cost

Method used

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  • An expression vector containing human insulin gene and its construction method and application
  • An expression vector containing human insulin gene and its construction method and application
  • An expression vector containing human insulin gene and its construction method and application

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Experimental program
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Effect test

Embodiment 1

[0058] Embodiment 1: Seed-specific plant expression vector

[0059] In the present invention, the rapeseed oil body protein gene promoter (NOP) is first amplified by PCR method, and the promoter is inserted between the HindIII and BamHI restriction sites of pUC19 to obtain pUCN. At the same time, the human insulin gene was designed and synthesized according to the human insulin gene sequence and the preferred codon of oil sunflower, and the synthetic gene was inserted into the 3' end of the peanut oleosin gene (Ole) to obtain the fusion gene of peanut oleosin and human insulin , and a trypsin recognition sequence Klip27 was added between the peanut oleosin gene and the human insulin gene. Then insert the fusion gene between the BamHI and SacI restriction sites of pUCN to obtain pUCNOI, HindIII and SacI double digestion pUCNOI, recover the 1779bp exogenous fragment from agarose gel, and insert the exogenous fragment into the plant for dual expression Between the HindIII and ...

Embodiment 2

[0060] Embodiment 2: Construction of seed-specific plant expression vector pBINOI

[0061] The plant expression vector pBINOI construction process is as follows: figure 2 As shown, the specific steps are as follows:

[0062] Cloning of rapeseed oil body protein gene promoter. Rapeseed is an important oil crop with high oil content (42-45%), and the amount of 20kD oil body protein in rapeseed oil body is 10 times that of 24kD oil body protein. The forward primer pBINOI-1: CCC was designed according to the nucleotide sequence of rapeseed oil body protein promoter (Genbank No.AF134411) AAG CTT TTC AAC GTGGTC GGA TCA TGA CG (SEQ ID NO: 1) and reverse primer pBINOI-2: CGC- GGA TCC GAA TTGAGA GAG ATC GAA GAG (SEQ ID NO: 2), used for PCR amplification of the promoter of rapeseed 20kD oil body protein gene, respectively introduced HindIII and BamHI restriction sites on the primers (the underline represents the restriction site) , using the rapeseed (Brassica campestris) vari...

Embodiment 3

[0072] Embodiment 3: Utilize this carrier to prepare human insulin

[0073] 3.1 The seed-specific expression vector constructed above is introduced into the recipient plant explant;

[0074] 3.1.1 Preparation of Agrobacterium Competent Cells

[0075] (1) Pick a single colony of Agrobacterium tumefaciens LBA4404 in 3 mL of YEB liquid medium (containing streptomycin Sm 125 mg / L), and culture overnight at 28°C with shaking;

[0076] (2) Inoculate 500 μL of the overnight culture solution into 50 mL of YEB (Sm 125 mg / L) liquid medium, shake at 2 g °C until OD 600 is 0.5;

[0077] (3) Centrifuge at 5,000 rpm for 5 minutes;

[0078] (4) Add 10mL 0.15M NaCl to suspend Agrobacterium cells, centrifuge at 5,000rpm for 5min;

[0079] (5) 1mL pre-cooled 20mM CaCl 2 Suspend the cells, put them in an ice bath, and use them within 24 hours, or aliquot them into 200 μL tubes, freeze them in liquid nitrogen for 1 minute, and store them at -70°C for later use.

[0080] 3.1.2 Transformati...

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Abstract

The invention discloses a human insulin gene-containing expression vector and a construction method and application thereof. The plant expression vector pBINOI disclosed by the invention is obtained by inserting a fusion protein expression box which is driven by a rape oil body protein gene promoter and consists of peanut oil body protein genes and human insulin genes between restriction enzyme cutting sites Hind III and Sac I of a plant double expression vector pBI 121. In addition, the invention provides a method for preparing human insulin by converting oil sunflower with the expression vector to construct a plant bioreactor. By adopting the method, the yield of the human insulin can be improved, and the production cost can be greatly reduced; and the method is suitable for industrial production.

Description

technical field [0001] The present invention relates to an expression vector containing human insulin gene and its construction method and application, in particular to an expression vector containing human insulin gene and its construction method and a method for preparing human insulin in sunflower oil using the vector. Background technique [0002] In recent years, the incidence of diabetes has increased year by year, presenting an epidemic trend. Diabetes has become a common and frequently-occurring disease in many countries in the world. Recent figures from the World Health Organization point out that about 3.2 million people worldwide die from diabetes every year, that is, 6 people every minute. [0003] In diabetes medication, insulin is one of the most effective therapeutic drugs, and it is also the only therapeutic drug for patients with type I diabetes. In addition, 30% to 40% of patients with type II diabetes will eventually need to use insulin. At present, the...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/62C07K14/62C07K1/20
CPCC12N15/8257C07K14/62
Inventor 安胜军柴锡庆邵铁梅焦展温昕李雪刘培卢海刚王崑声胡良元许海民于成钢
Owner HEBEI CHEM & PHARMA COLLEGE
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