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Recombinant human serum albumin-collagen binding domain fusion protein of tumor specific targeting matrix and application

A technology of human serum albumin and fusion protein, applied in the direction of serum albumin, albumin peptide, animal/human protein, etc., can solve the problems of production cost disadvantages, difficult production requirements of recombinant fusion protein, etc.

Active Publication Date: 2021-01-15
FORTUNEROCK (CHINA) CO LTD +3
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, the serum albumin in this fusion protein is not of human origin, and there is also a connecting peptide between the C-terminal of CBD and the N-terminal connection of mouse albumin, and the fusion protein also contains a 6His-tag. At the same time, in the literature The disclosed fusion protein is prepared using HEK293 cells. The expression cell system is a non-industrialized cell system, and the output per L is only between the μg-mg level. The recombinant fusion protein obtained in this way is difficult to meet the pharmaceutical grade / kg (kg )-level production requirements, and has a considerable disadvantage in production costs

Method used

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  • Recombinant human serum albumin-collagen binding domain fusion protein of tumor specific targeting matrix and application
  • Recombinant human serum albumin-collagen binding domain fusion protein of tumor specific targeting matrix and application
  • Recombinant human serum albumin-collagen binding domain fusion protein of tumor specific targeting matrix and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1 Human serum albumin (HSA) gene expression and the construction of carrier plasmid

[0061] Using the total RNA extracted from human liver as a template, the HSA gene was synthesized and amplified by reverse transcription polymerase chain polymerization. 5 μg of total RNA synthesized the corresponding DNA strand under the action of reverse transcriptase (purchased from GIBCO / BRL Company), and the oligonucleotide primers were 18 T+1N (N is a random nucleotide). The reaction condition was 45°C for 20 minutes, then the temperature was raised to 55°C, and the temperature was continued for 40 minutes.

[0062] The oligonucleotide primer sequences are: 5'-GAATTCATGAAGTGGGTAACCTTTATTTCC-3' (SEQ ID NO. 5) and 5'-GAATTCTTATAAGCCTAAGGCAGCTTGACTTGC-3' (SEQ ID NO. 6).

[0063] Two EcoRI recognition sites were added to the two ends of the HSA gene and cloned into an expression vector. The PCR reaction conditions were 94°C, 4min, and the DNA encoding HSA was further amp...

Embodiment 2

[0065] Example 2 Expression of long-acting broad-spectrum tumor therapy targeting matrix recombinant fusion protein gene (rhCBD / SA) and construction of vector plasmid

[0066] According to the known literature analysis and the complete amino acid sequence of vWF (von Willebrand factor[Homosapiens]GenBank#AAB59458.1 von Willebrand factor[Homo sapiens]), the human vWF A3domain is Cys1670-Gly1874 (peptide 907-1111 of the human vWF mature peptide amino acid section), which is the collagen binding domain (CBD), and its molecular weight is 20kDa.

[0067] The rhCBD / SA mature peptide was synthesized according to the amino acid sequence shown in Seq ID NO.1: the CBD mature peptide was directly connected to the N-terminus of human serum albumin, and there was no connecting peptide between them.

[0068]Therefore, according to the amino acid sequence of rhCBD / SA mature peptide, the nucleotide sequence of CBD mature peptide was artificially synthesized by DNA sequence synthesis. When ar...

Embodiment 3

[0071] Example 3 Expression of recombinant fusion protein gene (rHSA / CBD) and construction of vector plasmid for long-acting broad-spectrum tumor therapy targeting matrix

[0072] The N-terminus of the collagen-binding domain (CBD) is connected to the C-terminus of human serum albumin, with a rigid linker peptide (GGGSGGGS) in between to ensure that the CBD at the C-terminus of the fused human serum albumin does not interact with the recombinant fusion protein without connecting peptides between proteins figure 2 state. To this end, a novel tumor-specific targeting drug matrix molecular structure as shown in Seq ID NO.3 was artificially synthesized. Refer to Example 2 for the gene synthesis method. Finally, the nucleotide DNA sequence of the obtained rHSA / CBD is shown in SEQ ID NO.4.

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Abstract

The invention provides a recombinant human serum albumin-collagen binding domain fusion protein of a tumor specific targeting matrix and application, and relates to the technical field of biological medicines. According to the invention, gene fusion is formed by utilizing HSA and CBD in different molecular structure forms, expression can be obtained in a constructed recombinant engineering host, and the recombinant fusion protein meeting medicinal requirements is obtained through large-scale fermentation preparation. The recombinant fusion protein has solid tumor specific targeting, can be used as a broad-spectrum matrix carrier starting material, and can be coupled with any tumor treatment drugs, small molecular compounds or cytotoxic proteins to form an innovative drug compound. The recombinant fusion protein drug compound also has long-acting property, can prolong the half-life period of a coupling drug in vivo, and has a clinical curative effect obviously superior to that of a monomeric drug in tumor resistance. The invention also provides an application of the drug compound in preparation of drugs for solid tumor specific targeted therapy and preparation of corresponding drugs.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a recombinant human serum albumin-collagen binding domain fusion protein with tumor-specific targeting matrix and its application. Background technique [0002] Human serum albumin (HSA) is a soluble monomeric protein that constitutes half of the total protein in blood. Albumin is the main component in the blood, its content in the human body is 40 grams per liter of blood, and its half-life is 14-20 days. As a basic carrier, albumin carries molecules such as fatty acids, steroids and hormones, and its stable and inert nature is an important factor in maintaining blood pressure. Serum albumin is a globular non-glycosylated serum protein with a molecular weight of 65 kilodaltons and 585 amino acids. This protein (precursor of albumin) is then processed by the Golgi apparatus to remove the leading polypeptide and then secreted out of the cell. Serum albumin has 35...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/81C12N1/19A61K47/64A61K45/00A61P35/00C12R1/84
CPCC07K14/765C07K14/78C12N15/815A61K47/64A61K47/643A61K47/6435A61K45/00A61P35/00C07K2319/00C07K2319/31C07K2319/70C07K2319/33C07K14/755
Inventor 于在林富岩杨小楠富俞淞乔国强杨波陈颖
Owner FORTUNEROCK (CHINA) CO LTD
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