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Fusion protein HFBI-RGD gene and protein

A fusion protein and gene technology, applied in the field of protein genetic engineering, to achieve short production cycle, good solubility, and solve the effect of dispersion problem

Active Publication Date: 2018-08-21
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] Using the HFBI nucleotide sequence shown in SEQ ID NO.3 in Trichoderma reesei as a template, using F1 as a forward primer, and R1 as a reverse primer, the first round of PCR was carried out to obtain the first PCR product; using the first PCR product as a template, F1 as a forward primer, and R2 as a reverse primer, the second round of PCR was performed to obtain the first sequence containing the fusion protein HFBI-RGD gene, which was digested and T4 DNA ligase ligation, the fusion protein HFBI-RGD gene was constructed on the PET-28a vector, and the PET-28a-HFBI-RGD plasmid was obtained. Specific steps are as follows:

[0052] a) enzyme digestion system

[0053]

[0054] b) conditions

[0055] i. Digest the target gene for 1 hour at 37°C.

[0056] ii. Digest the plasmid for 1 hour at 37°C.

[0057] iii. After digestion, inactivate at 80°C for 5 minutes.

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

Embodiment 2

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

[0072] 1) The vector is selected as pPIC9k, the restriction sites are NotI and EcoRI, and the 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 Example 1 above as a template, PCR can obtain the sequence of the second fusion protein HFBI-RGD gene whose restriction sites are NotI and EcoRI.

[0073] 2) The PCR product and the vector pPIC9k were double-digested with restriction endonucleases NotI and EcoRI, respectively, so that the target gene fragment and the vector had cohesive ends. The specific enzyme digestion system and method are as follows:

[0074]

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

[0076] 4) After conversion and double enzyme digestion verification, the positive results obtained are verified b...

Embodiment 3

[0077] Example 3 Fusion protein HFBI-RGD positive clone screening and expression purification:

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

[0079] Table 7 Linearization system

[0080] ...

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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, and in particular relates to a fusion protein HFBI-RGD gene and a preparation method thereof. Background technique [0002] Fungal hydrophobins are amphoteric proteins with a molecular weight of about 10kDa and rich in hydrophobic amino acids secreted by filamentous fungi. 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 protein film of about 10nm can be formed at the interface. These films can reverse the hydrophilic and hydrophobic surfaces, making hydrophilic substances hydrophobic and hydrophobic. Sexual substances are hydrophilic. According to the solubility of the protein film formed by the hydrophobin, fungal hydrophobins are divided into types I and II. Among them, type II hydrophobins comprise HFBI. The funga...

Claims

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Application Information

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Patent Type & Authority Applications(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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