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Protein nucleic acid compound, as well as preparation method and application thereof

A complex and nucleic acid technology, which is applied in the field of tumor blood vessel targeting drugs, can solve the problems of strong immunogenicity, weak tissue penetration, and poor safety, and achieve a wide range of applications, low immunogenicity, and high safety. Effect

Pending Publication Date: 2020-02-14
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the above two tumor vascular blocking agents use peptides as tumor targeting molecules, which have strong immunogenicity and weak tissue penetration, and cannot reach the target site efficiently, and blocking agents that stay in the normal site may induce thrombosis , has certain toxicity and poor safety

Method used

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  • Protein nucleic acid compound, as well as preparation method and application thereof
  • Protein nucleic acid compound, as well as preparation method and application thereof
  • Protein nucleic acid compound, as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Expression and purification of truncated tissue factor tTF

[0057] (1) Whole-gene synthesis of the gene sequence of truncated tissue factor tTF, designing NdeI and XhoI restriction sites at both ends respectively, and linking the digested gene sequence into pET30a vector to construct the expression vector of truncated tissue factor tTF;

[0058] (2) Transfer the above-mentioned expression vector into BL21 Escherichia coli, put 5 μL of bacterial liquid into 5 mL of LB liquid medium, culture on a shaking table at 37°C and 200 rpm for 16 hours, transfer the cultured bacterial liquid into 500 mL of LB liquid medium, and Cultivate on a shaker at 200 rpm until OD=0.6-0.8, and induce expression with IPTG (0.5mM) for 4 hours;

[0059] (3) Centrifuge the above-mentioned IPTG-induced expression bacterial solution at 6000rpm for 5min, discard the supernatant, collect the bacteria, blow off the precipitate with 25mL 10mM Tris-HCl (pH=8.0), ultrasonically destroy the bact...

Embodiment 2

[0061] Example 2 Connection of truncated tissue factor tTF and nucleic acid aptamer

[0062] The purified truncated tissue factor tTF was vortex mixed with excess Sulfo-SMCC, reacted at room temperature for 1 hour, and the unreacted Sulfo-SMCC was removed by ultrafiltration; Under reduction treatment, the terminal sulfhydryl group is fully activated, and excess TCEP is removed by ultrafiltration; the activated truncated tissue factor tTF is mixed with the activated AS1411 nucleic acid aptamer, reacted at room temperature for 48 hours, and the excess nucleic acid aptamer is removed by ultrafiltration .

[0063] The protein-nucleic acid complex was identified by TBE-PAGE electrophoresis, and the results were as follows figure 2 As shown, the electrophoretic position of the complex tTF-AS1411 is higher than that of the AS1411 nucleic acid aptamer, which is in line with expectations.

Embodiment 3

[0065] Compared with Example 2, SMCC was used as the linker, other conditions were the same as in Example 2, and the result of the prepared protein-nucleic acid complex tTF-AS1411 was similar to that of Example 2.

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PUM

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Abstract

The invention provides a protein nucleic acid compound, as well as a preparation method and application thereof. The compound comprises a coagulation accelerator and a targeting molecule, wherein thecoagulation accelerator comprises a truncated tissue factor (tTF); the targeting molecule comprises a nucleic acid adapter; and the tTF and the nucleic acid adapter are connected through a coupling agent. The compound provided by the invention adopts the nucleic acid adapter as a tumor vessel targeting molecule, the tTF is specifically positioned on the surface of a tumor vessel wall surface, thrombogenesis is induced in a tumor vessel, and the blood supply of a tumor site and a discharge way of metabolic products are cut off, so that the safety is high, the immunogenicity is low, the recognition and the elimination of an immune system can be avoided, the specificity is high, and a tumor cell inhibition effect is achieved.

Description

technical field [0001] The invention belongs to the technical field of tumor blood vessel targeting drugs, and relates to a protein-nucleic acid complex and its preparation method and application, in particular to a protein-nucleic acid aptamer coupling complex and its preparation method and its use in the preparation of tumor blood vessel blocking drugs in the application. Background technique [0002] Tumor blood vessels are channels for tumor cells to obtain nutrients and discharge metabolic waste, and are also one of the main ways for tumor cells to escape and metastasize. Their shape and function are different from the normal vascular system of the body, so they are the key targets of tumor targeted therapy one. Targeted therapy of tumor blood vessels mainly includes three modes: blocking existing tumor blood vessels, destroying existing tumor blood vessels, and inhibiting new angiogenesis. Among them, blocking and destroying tumor blood vessels can cut off the blood ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/115A61K38/36A61K47/54A61P35/00
CPCA61K38/00A61K47/549A61P35/00C07K14/745C07K19/00C12N15/115C12N2310/16
Inventor 聂广军李素萍李博朝
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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