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Synthesis method and application of triblock multifunctional fused protein based on mussel attachment protein/zwitter-ion polypeptide

A mussel adhesion protein and fusion protein technology, applied in the biological field, can solve problems such as weakening the signal-to-noise ratio of equipment, reducing detection accuracy, and infectious complications

Inactive Publication Date: 2018-08-14
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, in the immunodiagnosis industry, non-specific protein adsorption on the surface of biochips will weaken the signal-to-noise ratio of the equipment and reduce the accuracy of detection; in the medical field, the harm caused by biofouling is particularly serious, such as artificial heart valves, cardiac pacemakers , hemodialysis systems, vascular stents, implant catheters, artificial bones and other devices in direct contact with tissues and body fluids in the human body, due to the adhesion of bacteria and the adsorption of proteins and cells, it is easy to form biofilms and even cause more serious infections. complication

Method used

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  • Synthesis method and application of triblock multifunctional fused protein based on mussel attachment protein/zwitter-ion polypeptide
  • Synthesis method and application of triblock multifunctional fused protein based on mussel attachment protein/zwitter-ion polypeptide
  • Synthesis method and application of triblock multifunctional fused protein based on mussel attachment protein/zwitter-ion polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1: Construction of the recombinant expression vector of MP-KE-RGD triblock multifunctional fusion protein and the construction of bacterial strain

[0060] The preferred amino acid sequence of the MP-KE-RGD triblock multifunctional fusion protein is SEQ ID NO.14, and the preferred nucleotide sequence is shown in SEQ ID NO.18.

[0061] A NdeI restriction enzyme cutting site was added at the 5' end of the SEQ ID NO.18 gene sequence, and a HindIII restriction enzyme cutting site was added at the 3' end. The gene sequence was artificially synthesized and then amplified by PCR. The total reaction volume was 50 μl, in which 2.5 μl of each primer was added at a concentration of 10 μmol / L, and 1 μl of dNTP at a concentration of 10 mmol / L was added. The DNA polymerase used was Phusion High-Fidelity DNApolymerase, 2 U / μl , add 0.5 μl. The reaction conditions were 98°C for 5s, 55°C for 45s, and 72°C for 30s. After 25 cycles, the product was analyzed by 1.0% agarose gel...

Embodiment 2

[0065] Example 2: High expression of MP-KE-RGD triblock multifunctional fusion protein in Escherichia coli

[0066] The successfully transformed Escherichia coli was streaked on a double-resistant LB solid medium plate containing kanamycin and chloramphenicol, and cultured overnight at 37°C for activation. Pick a single colony in a 5 ml test tube containing 50 μg / ml (final concentration) of kanamycin and chloramphenicol sterilized LB liquid medium, and cultivate overnight at 37° C. with shaking at 200 rpm. Transfer Escherichia coli seed solution to a 500ml shake flask at a ratio of 1:100, which contains 200ml sterile LB liquid medium with a final concentration of 50μg / ml kanamycin and chloramphenicol. Measure the growth curve of Escherichia coli with a UV spectrophotometer, and when the concentration of the bacteria solution is OD600=0.8 2h30min after Escherichia coli transfer (IPTG) induces Escherichia coli to express the fusion protein MP-KE-RGD, and continues culturing for...

Embodiment 3

[0067] Example 3: Separation and purification of MP-KE-RGD triblock multifunctional fusion protein in Escherichia coli

[0068] The Escherichia coli fermentation broth was centrifuged at 4°C and 6000rpm for 10 minutes to discard the culture medium, the Escherichia coli cells were washed with PBS buffer three times, and then centrifuged to collect the E. coli cells under the same conditions. Dilute Escherichia coli cells with PBS solution to 40ml / g cell wet weight, and ultrasonically disrupt the bacterial solution in an ice-water bath for 40min with an output power of 200W, crush for 2s, and rest for 3s. The clarified bacterial liquid obtained by crushing was centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain the supernatant and inclusion bodies respectively, and the E. Acrylamide gel (SDS-PAGE) electrophoresis. The fusion protein MP-KE-RGD exists in the supernatant and inclusion bodies at the same time, so the follow-up experiment uses affinity chromatography to separ...

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Abstract

The invention relates a synthesis method and application of triblock multifunctional fused protein based on mussel attachment protein / zwitter-ion polypeptide. The synthesis method comprises the following steps: cloning a gene of the fused protein to a plasmid vector to obtain a recombinant expression plasmid; after transforming host cells, inducing the host cells by utilizing an induction agent, so as to overexpress the fused protein; after separating and purifying, obtain the fused protein. The mussel attachment protein contains a sequence shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, or SEQ ID NO. 5. The zwitter-ion polypeptide contains a sequence shown as SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 9. Triblock polypeptide contains a sequence shown as SEQ IDNO. 10 (RGD short peptide), SEQ ID NO. 11 (SDE tripeptide), SEQ ID NO. 12 (IKVAV short peptide) or SEQ ID NO. 13 (RADA16-I self-assembled polypeptide). The fused protein is synthesized through microorganisms through a one-step method; the synthesis method has the advantages of moderate and efficient process, low cost and low requirements on equipment and later-period expanded production is facilitated.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to the gene synthesis of a three-block multifunctional fusion protein based on mussel adhesion protein / zwitterionic polypeptide, the construction of a recombinant expression vector containing fusion protein gene fragments, and the use in genetic engineering strains Fermentative expression of fusion protein, separation and purification method of fusion protein, and its application in material modification. Background technique [0002] Biofouling refers to the process of degrading the performance of materials or devices caused by the adhesion, accumulation and reproduction of living substances such as cells, proteins and bacteria on the surface of solid substrates, resulting in the loss of devices. In the fields of medicine, navigation, food, etc., biofouling will not only increase the loss of materials, but also cause many problems such as energy consumption and medical safety. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70
CPCC07K14/43504C07K2319/00C12N15/62C12N15/70C12N2800/22
Inventor 张雷齐海山朱迎男
Owner TIANJIN UNIV
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