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Fusion protein hfbi-rgd gene and protein

A technology of fusion protein and gene, applied in the field of protein genetic engineering, to achieve the effect of high yield, good solubility and solving the problem of dispersion

Active Publication Date: 2020-07-28
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it has many important theoretical and practical values ​​for hydrophobin, there are still deficiencies in its application in molecular diagnosis, drug delivery and targeting carriers.

Method used

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  • Fusion protein hfbi-rgd gene and protein
  • Fusion protein hfbi-rgd gene and protein
  • Fusion protein hfbi-rgd gene and protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Construction of pET-28a-HFBI-RGD:

[0051] With the HFBI nucleotide sequence shown in SEQ ID NO.3 in Trichoderma reesei (Trichoderma reesei) as template, with F1 as forward primer, with R1 as reverse primer, carry out the first round of PCR, obtain the first PCR product; take the first PCR product as the template, use F1 as the forward primer, and use R2 as the reverse primer to carry out the second round of PCR to obtain the first sequence containing the fusion protein HFBI-RGD gene. T4 DNA ligase was ligated, and the fusion protein HFBI-RGD gene was constructed into the PET-28a vector to obtain the PET-28a-HFBI-RGD plasmid. Specific steps are as follows:

[0052] a) Enzyme digestion system

[0053]

[0054] b) Conditions

[0055] i. The target gene was digested for 1 h at 37°C.

[0056] ii. The plasmid was digested for 1 h at 37°C.

[0057] iii. Inactivation at 80°C for 5min after digestion.

[0058] c) Using the HFBI nucleotide sequence shown in SE...

Embodiment 2

[0071] Example 2 Construction of pPIC9k-HFBI-RGD:

[0072] 1) The selection vector is pPIC9k, the restriction sites are NotI and EcoRI, and primers are designed for PCR. The PCR system is shown in Table 3. Primers were designed as F2, R3. Using the plasmid PET-28a-HFBI-RGD constructed in the above Example 1 as a template, PCR can obtain the second sequence containing the fusion protein HFBI-RGD gene with the restriction sites of NotI and EcoRI.

[0073] 2) The above PCR product and the vector pPIC9k were respectively double digested with the restriction enzymes NotI and EcoRI to make the target gene fragment and the vector appear sticky ends. The specific enzyme digestion system and method are as follows:

[0074]

[0075] 3) The target gene fragment with sticky ends and the vector pPIC9k are ligated using T4 DNA ligase, and the ligation system is shown in Table 6 above.

[0076] 4) After transformation and double-enzyme digestion verification, the obtained positive resu...

Embodiment 3

[0077] Example 3 Screening, expression and purification of fusion protein HFBI-RGD positive clones:

[0078] 1) To insert a recombinant expression vector into the yeast genome at a higher copy in yeast cells, it needs to be linearized before being transferred into yeast cells. The enzyme cleavage site analysis function provided in the primer design software Primer 5.0 was used to analyze the cleavage sites of pPIC9k and HFBI-RGD genes, combined with the cleavage site information provided by Ivitrogen in the Pichia pastoris expression system operation manual, and finally The vector was linearized by restriction endonuclease SalI. The reaction system of enzyme cleavage linearization is shown in Table 7. This system was digested in a water bath at 37°C for 2h. Take 5 μL of the product and perform agarose gel electrophoresis to check whether the enzyme digestion is complete, and then the product band is recovered by gel.

[0079] Table 7 Linearization system

[0080]

[008...

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Abstract

The invention discloses a fusion protein HFBI-RGD gene and protein. The nucleotide sequence of the fusion protein HFBI-RGD gene is shown as SEQ ID NO.1. The fusion protein HFBI-RGD provided by the invention has the characteristics of good solubility, short production cycle and high yield. The HFBI-RGD fusion protein not only has the structure and function of HFBI, but also has cell adhesion activity, the fusion protein can locate at a tumor site containing integrin and therefore has good targeting properties. The HFBI-RGD fusion protein still can serve as an emulsifier and enable small oil droplets exist in water stably, and can be used as dispersant to disperse graphene, carbon nanotubes and hydrophobic fluorescent probes to solve the dispersion problem of hydrophobic materials, moreover,as the fusion protein contains short peptide RGD, the fusion protein is endowed with integrin targeting properties, and can be used in tumor diagnosis, drug delivery and other fields.

Description

technical field [0001] The invention belongs to the field of protein genetic engineering, in particular to a fusion protein HFBI-RGD gene and a preparation method thereof. Background technique [0002] Fungal hydrophobins are amphiphilic proteins secreted by filamentous fungi that are rich in hydrophobic amino acids and have a molecular weight of about 10 kDa. They have special physical and chemical properties and play an important role in the contact between hyphae and air. Fungal hydrophobin has two parts, hydrophobic and hydrophilic. Through self-assembly, an amphiphilic membrane of about 10 nm can be formed at the interface. These membranes can reverse the hydrophilic and hydrophobic surfaces, making hydrophilic substances hydrophobic and hydrophobic. Sexual substances are hydrophilic. Fungal hydrophobins are classified into type I and type II according to the solubility of the protein membrane formed by the hydrophobin. Among them, type II hydrophobin contains HFBI. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C07K19/00C12N15/81C12N15/66C12N1/19C12R1/84
CPCC07K14/37C07K2319/00C12N15/66C12N15/815
Inventor 王泽方肖云杰王斌杨海涛
Owner TIANJIN UNIV
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