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Human vascular endothelial cell growth inhibition factor jogged polypeptide, preparation thereof and use in targeted antineoplastic activity

A technology of growth inhibitory factor and chimeric polypeptide, which is applied in the direction of antineoplastic drugs, hybrid peptides, chemical instruments and methods, etc., to achieve great therapeutic value, strong antitumor activity, and rich application forms

Active Publication Date: 2012-07-04
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there is no complete and clear understanding of the three-dimensional structure, mechanism of action and pharmacological properties of VEGI protein. It is necessary to construct a more targeted chimeric recombinant protein and detect its biological activity to study the fusion protein inducing tumor cell apoptosis in vitro , the ability to inhibit the growth of tumor cells, and detect its binding characteristics with tumor cells, identify its tumor targeting, and deeply explore the in vivo targeted tumor inhibitory activity of recombinant proteins constructed with VEGI protein and the application prospects of tumor therapy

Method used

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  • Human vascular endothelial cell growth inhibition factor jogged polypeptide, preparation thereof and use in targeted antineoplastic activity
  • Human vascular endothelial cell growth inhibition factor jogged polypeptide, preparation thereof and use in targeted antineoplastic activity
  • Human vascular endothelial cell growth inhibition factor jogged polypeptide, preparation thereof and use in targeted antineoplastic activity

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1: Construction of chimeric protein RGD-VEGI-192A expression vector

[0044] Amplified from the total RNA of human umbilical vein endothelial cells by RT-PCR technology, according to the VEGI-192A nucleotide sequence shown in SEQID NO: 1, the ACDCRGDCFCG polypeptide and the N-terminal of VEGI-192A were fused and amplified to obtain RGD-VEGI-192A target gene. The upstream primer of PCR is 5'-TTCCATATGGCTTGCGACTGCCGTGGTGACTGCTTCTGCGGTCAACTCACAAAAGGGCCGTCT-3', and the downstream primer is: 5'-CGCGGATCCCTATAGTAAGAAGGCTCCAAAGAAGGTT-3'. Both upstream and downstream primers contain Nde I restriction enzyme cutting sites at the 5' end and BamH I restriction enzyme cutting sites at the 3' end. figure 1 After PCR amplification of the RGD-VEGI-192A target gene, the PCR product is subjected to 1% agarose gel electrophoresis analysis, wherein 1, 2, 3, and 4 are PCR products, and M is a 1Kb gradient DNA molecular weight standard. figure 1 The results show that the size o...

Embodiment 2

[0046] Example 2: Auto-induced expression of RGD-VEGI-192A chimeric protein in Escherichia coli and screening of high-expression clones

[0047] 1. Automatic induction: Use the expression vector pET-30a-RGD-VEGI-192A to transform the competent cell BL21(DE3)pLysS, randomly pick 8 clone colonies into 3mL LB medium, add kanamycin to a final concentration of 50μg / mL, cultured overnight at 37°C. The bacterial cell pellet was collected by centrifugation the next day, and the recombinant expression was analyzed by SDS-PAGE.

[0048] 2.1000× trace metal solution: 0.05M FeCl 3 ;-0.12M HCl; 0.02M CaCl 2 ;0.01M MnCl 2 -4H 2 O; 0.01 M ZnSO 4 -7H 2 O; 0.002M CoCl 2 -6H 2 O; 0.002M CuCl 2 -2H 2 O; 0.002M NiCl 2 -6H 2 O; 0.002M Na 2 MoO 4 -5H 2 O; 0.002M Na 2 SeO 3 -5H 2 O; 0.002MH 3 BO 3 .Add double-distilled water to make up to 100mL.

[0049] 3. Self-inducing expression medium: 1% tryptone (tryptone), 0.5% yeast extract (yeastextract), 25mM Na 2 HPO 4 , 25mM KH 2...

Embodiment 3

[0051] Example 3: Condition optimization of the automatic induction expression system

[0052] 1. Optimization of host bacteria: In order to improve the solubility of the RGD-VEGI-192A chimeric protein, we used two host bacteria, BL21(DE3)pLysS and OrigamiB(DE3), in which the OrigamiB(DE3) host bacteria had mutations in trxB and gor genes, Thereby providing balanced redox potential for the recombinant protein in the host bacteria to facilitate the correct spatial folding of the protein. According to the above image 3As a result, the yield of recombinant protein of RGD-VEGI-192A chimeric protein auto-induced in the two host bacteria is not much different, but in the host bacteria OrigamiB (DE3) auto-induced system in the background protein is slightly smaller, so we finally The host bacterium was selected as OrigamiB(DE3) #1 high-expressing clone colony, the culture time was 16 hours, the culture temperature was 25°C, and the target protein was prepared by high-density cultur...

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PUM

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Abstract

The invention provides chimeric polypeptide of chrotoplast growth inhibitors in human vessels. The chimeric polypeptide is RGD-VEGI-192A chimeric recombinant protein formed by connecting the growth inhibitor VEGI-192A and RGD-4C. The preparation method for the chimeric polypeptide comprises steps that nucleotide sequences shown in the SEQ IDNO:1 construct expression vector and induces the expression in host cells. The chimeric polypeptide is a new angiogenesis antagonist, can be applied to the targeted antitumor and has potential treatment valve in angiogenesis diseases such as tumor and the like. In addition, the chimeric polypeptide enriches the application form of the VEGI-192A in the tumor treatment and provides new idea for the targeted treatment of the tumor and lays foundations forthe development of the RGD-VEGI-192A chimeric polypeptide as a more effective clinical drug.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a chimeric polypeptide of human vascular endothelial cell growth inhibitory factor, its preparation method and its application in targeted anti-tumor activity. Background technique [0002] Malignant tumor is the number one killer that seriously threatens human life and health. Surgery, radiotherapy and chemotherapy are common methods for treating tumors. Radiotherapy and chemotherapy kill tumor cells and normal cells at the same time, and have great side effects. Therefore, finding specific and efficient tumor treatment methods has always been a hot spot in tumor research. Targeted tumor therapy is the use of certain specific carriers to selectively deliver drugs or other active substances that kill tumor cells to the tumor site, and limit the therapeutic effect or drug effect to specific target cells, tissues or organs as much as possible. It is a method that does not affect the f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/63C12P21/02A61K38/19A61P35/00
CPCY02A50/30
Inventor 黎孟枫袁洁李鲁远朱勋吴珏珩何振健陈旭王宁胡洁萍
Owner SUN YAT SEN UNIV
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