CN104232652B - Come from the recombiant protein rLj-RGD4 of the synthetic gene application in antitumor drug - Google Patents

Abstract

" come from the recombiant protein rLj RGD4 of the synthetic gene application in angiogenesis inhibitors series antineoplastic medicament " and belong to biological technical field, relate to synthetic genelj‑rgd4Gene clone, protein expression, and its recombiant protein rLj potent angiogenesis inhibiting of RGD4 dose dependent and suppression tumor function and as angiogenesis inhibitors application in antitumor drug.

Description

Come from the recombiant protein rLj-RGD4 of synthetic gene in antitumor drug Application

Technical field

" come from the recombiant protein rLj-RGD4 of the synthetic gene application in antitumor drug " and belong to biotechnology Field, relates to recombinate the gene clone of RGD die body albumen Lj-RGD4, protein expression, and rLj-RGD4 is by suppression new life The signal transduction pathway of the integrin of Surface of Vascular Endothelial Cells high expressed and potent angiogenesis inhibiting function and as blood The application in antitumor pharmacy of the pipe new life inhibitor.

Background technology

Vascularization is the basis of growth and metastasis of tumours.When blood vessel volume reaches 1 ~ 2 mm³Time, tumor presents blood vessel Generate phenotype, and supplement blood vessel from surrounding substrate.The advolution of primary solid tumor is highly dependent on the nucleus formation of blood vessel. In preclinical models, the anti-angiogenic drugs with tumor vasculature as targeting has all had shown that obstruction or has postponed swollen Tumor grows, and even promotes tumour regression or dormancy effect.Angiogenesis suppression therapy can make apoptosis of tumor cells speed accelerate, bad Dead and degenerate to initial resting state, so suppression Nasopharyngeal neoplasms and reach to treat the purpose of cancer.The most many blood Pipe formation inhibitor is carrying out the clinical research of I～III phase, such as angiostatin, Endostatin, fumagillin analogs and gold Proteases inhibitive factor and urokinase, IL-12, platelet factor, Bufotanine etc..

RGD(Arg-Gly-Asp) toxin protein is the toxin protein of the bloodsucker secretion containing RGD die body.Due to RGD sequence Row can be specific binding with some integrin of neovascular endothelium cell and tumor cell surface high expressed, so stop its with The combination of extracellular matrix, closes the signal transduction pathway of mediated by integrin, therefore has angiogenesis inhibiting and suppression tumor The propagation of cell, adhere to, infiltrate and shift, and cause vascular endothelial cell and the apoptosis of tumor cell to reach antineoplastic merit Effect.

Up to now, RGD toxin protein is found in five species.Except this laboratory is found in the RGD toxin peptide of Lampetra japonica (Martens). Outward, the RGD die body toxin family being only found that in the poison gland or salivary gland of other four kinds of different plant species, these Four Great families are respectively It is poisonous snake and bloodsucker Hirudo, Ticks class, Lampetra japonica (Martens)., the gadfly ^(1-4).The RGD die body toxin deriving from snake venom also calls to integrate Element, is that to be studied the most also be maximum family, and kind can reach tens of kinds according to Serpentis kind source difference；Derive from Hirudo saliva The RGD die body toxin of liquid gland the most only finds decorsin and ornatin two class；Derive from the malicious containing RGD die body of Ticks class It is called usually as variabilin；The die body toxin containing RGD deriving from the gadfly is referred to as Tablysin-15.

This laboratory is found that three kinds of cDNA sequences that can translate RGD die body in lamprey oral gland EST library, And it has been carried out gene cloning and expression, obtain respectively have the gene recombinant protein rLj-RGD1 of a RGD die body, two The rLj-RGD2 of individual RGD die body, the rLj-RGD3 of three RGD die bodys.Early-stage Study result shows, rLj-RGD3 has potential medicine By value.The powerful RGD toxin protein less in order to obtain molecular weight, we change with rLj-RGD3 gene for prototype Making and the gene order of one 174bp of synthetic it has been carried out gene clone, its recombiant protein has 4 RGD moulds Body, is made up of 58 amino acid residues, is named as rLj-RGD4.RLj-RGD4 is brand-new synthetic gene recombiant protein, The most at home and abroad there is no research and report, the invention belongs to find first.RLj-RGD4 is expected to be applied to angiogenesis inhibitors Series antineoplastic medicament preparation field.

List of references:

1.Chunsik Lee, Molecular Mechanisms of action of Histidine-rich glycoprotein in angiogenesis inhibition, Digital comprehensive summaries of uppsala dissertations from the faculty of medicine 192, ACTA Universitatis Upsaliensis Uppsala (2006).

2.Blank M., Shoenfeld Y., Histidine-rich glycoprotein modulation of immune/autoimmune, vascular, and coagulation systems, Clin Rev Allergy Immunol 34 (2008) 307-312.

3.Vanwildemeersch M., Olsson A.K., Gottfridsson E., Claesson-Welsh L., Lindahl U., Spillmann D., The anti-angiogenic His/Pro-rich fragment of histidine-rich glycoprotein binds to endothelial cell heparan sulfate in a Zn2+-dependent manner, J Biol Chem 281 (2006) 10298-10304.

4.Dongying Ma1, Xueqing Xu2, Su An1 et al. A novel family of RGD- containing disintegrins (Tablysin-15) from the salivary gland of the horseflyTabanus yao targets αIIbβ3 or αVβ3 and inhibits platelet aggregation and angiogenesis. Thrombosis and Haemostasis 105 (2011) 1032-45。

Summary of the invention

The present invention manually designs and synthesizeslj-rgd4Gene, willlj-rgd4Gene is cloned in pET-23b carrier, to it Recombiant protein rLj-RGD4 has carried out efficient abduction delivering in escherichia coli.RLj-RGD4 biologic activity relate to by with Vascular endothelial cell high expressed integrin combines and exercises anti-angiogenic rebirth function, anti-tumor function.

The present invention relates to the gene clone of Japanese lamprey oral gland recombiant protein Lj-RGD4, the table in escherichia coli Reach, and its angiogenesis inhibiting, antineoplastic effect.

lj-rgd4Gene order (open reading frame) 174bp length, its protein is made up of 58 aminoacid, Theoretical molecular Amount is 6.2 kDa, and its cDNA sequence and the protein amino acid sequence derived by it refer to description nucleotide/aminoacid sequence List.

The biologic activity of Lj-RGD4 angiogenesis inhibiting and tumor is as follows:

Human umbilical vein endothelial cells HUVEC induced bFGF by mtt assay detection rLj-RGD4 is bred in dose-dependant Property suppression, rLj-RGD4 suppression HUVEC cell proliferation IC₅₀It is 19.95 μm ol/L.

Body vessel generates experiment and uses chicken allantocherion (CAM) model to carry out.With bFGF (200 ng/embryo ) after newborn 24 h of induction of vascular, act on Embryo Gallus domesticus with the rLj-RGD4 of various dose or the PBS of equivalent.Result shows, bFGF lures The major branch blood vessel of the CAM processed with PBS after leading is clearly flourishing, and capillary network gradually forms；And rLj-RGD4 processed CAM, along with the increasing of dosage, there is obvious decay in CAM major branch blood vessel, quantity significantly reduces, and blood capillary is the most Do not exist.And the CAM angiogenesis that bFGF is induced by rLj-RGD4 is dose-dependent inhibition.

In-vivo tumour Inhibition test result with Hep-2 cell tumor bearing nude mice as animal model shows, with model group Relatively, basic, normal, high (12.5 μ g kg^-1、25.0 μg·kg^-1、50.0 μg·kg^-1) tumor heavily suppresses by dosage rLj-RGD4 Rate is respectively 22.6 %, 35.1 %, 65.4 %, illustrates that rLj-RGD4 is that dose dependent fashion suppresses tumor growth.

Accompanying drawing explanation

Fig. 1: the rLj-RGD4 inhibitory action to the HUVEC cell proliferation of bFGF induction.

Pressing down of six age in days chick chorioallantoic membrane (CAM) angiogenesiss that bFGF is induced by Fig. 2: rLj-RGD4 purifying protein Make and act on instar chicken embryo on the six 24 hours with (shooting of OLYMPUS digital camera) (A) PBS;(B) bFGF of 200 ng acts on Instar chicken embryo 24 hours on the six;(C), after the bFGF of-(D): 200ng induces 24 hours, rLj-RGD4 acts on the angiogenesis of CAM Situation.(C) 20 μ g;(D) 40 μ g;(E) 60 μ g.

Fig. 3: the rLj-RGD4 impact on Hep-2 people's laryngeal carcinoma transplanted tumor tumor weight: successive administration is after 3 weeks, and rLj-RGD4 is agent Amount dependency reduce tumor tumor weight, administration group (dosage is from low to high) tumor tumor be the most respectively 0.80 ± 0.17 g, 0.67 ± 0.14,0.36 ± 0.16, compared with model group, suppression ratio is respectively 22.6 %, 35.1 %, 65.4%(p < 0.01).Note: n=8; * P < 0.05, * * P < 0.01 is compared with model group.

Detailed description of the invention

1、lj-rgd4Gene is connected with pET23b carrier, carries out the screening of positive transformant after being transformed into escherichia coli Identify.

For producing with histidine-tagged fusion protein, selecting the carrier that pET-23b clones as gene, clone is concrete Operate as follows:

(1) recovery of genes of interest and order-checking: the recovery of genes of interest uses TaKaRa PCR Fragment Recovery Kit is carried out；Checking order to reclaiming DNA, this work is completed by Dalian treasured biological engineering company limited.

(2) extraction of plasmid: use precious biological plasmid extraction kit to carry out.

(3) genes of interest DNA fragmentation and the connection of carrier pET23b: owing to designed primer is respectively provided withNde I andHinD III digestion site, and the two restriction enzyme site is also the multiple clone site of pET23b, this makes genes of interest DNA sheet Section is connected to become possibility with carrier pET23b's.

(4) product CaCl will be connected₂Method converts to clone bacterium E.coli In BL21.

(5) Screening and Identification of positive transformant: utilize T₇Universal primer method and double digestion method carry out the sieve of positive transformant Choosing is identified.

2, positive recombinant is carried out the IPTG abduction delivering of final concentration of 1mmol/L.Abduction delivering condition is 30 DEG C of mistakes Night induces.

3, the recombiant protein expressed is carried out histidine affinitive layer purification.

(1) 10000 g is centrifuged 10 min and gathers in the crops thalline, abandons supernatant, and makes residual liquid flow out as far as possible.With every 100 ml's Original fluid adds the ratio re-suspended cell of 1 ice-cold for 4 ml X Binding buffer.

(2) above-mentioned sample is placed in ultrasonic degradation cell on ice, until solution no longer thickness.

(3) 14000 g are centrifuged 20 min to remove cell debris.

(4) supernatant is with the membrane filtration of 0.45 μm.

(5) suck the reservoir of room on His.Bind Column, and open the following mouth of pipe.

(6) with the 1x Binding Buffer of 10 ml, pillar is balanced.

(7) the supernatant loading that will have filtered.

(8) post is washed with the 1x Binding Buffer of 10 ml.

(9) post is washed with the 1x Wash Buffer of 10 ml.

(10) with the 1x Elute Buffer eluting destination protein of 5 ml.

4, mtt assay measures the inhibitory action that Human umbilical vein endothelial cells HUVEC is bred by rLj-RGD4.

Concrete grammar is as follows:

(1) HUVEC cell is inoculated in 96 orifice plates, cultivates in the RPMI-1640 of the final concentration of 3ng/ml of bFGF 24h；

(2) it is separately added into the albumen of gradient concentration in culture fluid, and uses PBS polishing, continue to cultivate 24h；

(3) the MTT solution that concentration is 0.5mg/ml adding culture fluid 10% continues to cultivate 4h；

(4) suck culture fluid, add and the same amount of DMSO of culture fluid；

(5) vibration 10min, makes crystallization fully dissolve；

(6) measuring absorbance value in microplate reader, mensuration wavelength is 490nm；

(7) cell killing rate is calculated:

Killing rate=(average of control wells-test hole average)/control wells average × 100%, 3 experiments are averaged.

5, chicken allantocherion CAM system is utilized to carry out anti-angiogenic rebirth functional examination.

Specific experiment step is as follows:

(1) taking instar chicken embryo on the six, the bromo geramine with 0.1% is sterilized；

(2) window at embryo head 1 cm location at allantocherion body surface projection, and saw out 1 cm²Fenestella；

(3) sterile glass fiber filter paper closes with the bFGF (200ng/disk) of 40 l or equivalent PBS are full, is placed in CAM On, adhesive tape covers；

(4) after hatching 24 h in the water isolation type constant temperature incubator of 37 DEG C of 60% humidity, add various dose rLj-RGD4 or Isopyknic PBS is on the filter paper overlayed on CAM, and adhesive tape covers;

(5) observe with digital camera after continuing to hatch 24 h and take pictures.

6, the foundation of people's Hep-2 cell model of nude mice bearing tumor and rLj-RGD4 are to people's Hep-2 cell transplanted tumor The mensuration of growth effect

(1) Animal Model: when the Hep-2 people's laryngeal cancer cell quantity being in exponential phase cultivated reaches to want After asking, peptic cell is prepared as cell suspension, is inoculated into oxter on the right side of nude mice, every nude mice 0.2mL with 1mL syringe, replicates Model.

(2) rLj-RGD4 impact on people's Hep-2 cell growth of xenografted: take nude mice 40, by concentration be 5 × 10⁵The people's Hep-2 cell suspension inoculation oxter on the right side of nude mice being in exponential phase of individual/mL, every nude inoculation 0.2 mL.When the diameter of tumor reaches 5 ~ 7 mm, tumor bearing nude mice is randomly divided into 5 groups (often groups 8): model (normal saline) Group, positive control drug cisplatin (3mg kg^-1) group, rLj-RGD4 low dosage (12.5 μ g kg^-1) group, dosage in rLj-RGD4 (25.0 μ g kg^-1) group, rLj-RGD4 high dose (50.0 μ g kg^-1) group.Each group nude mice abdominal cavity injection is different by reagent Thing, every day 2 times, is administered capacity 0.2 mL/10g, successive administration 3 weeks.Nude mice takes off after being administered 3 weeks neck put to death, peel off tumor group Knit, survey tumor volume and tumor weight.Formula is utilized to calculate tumor tumor-like hyperplasia.

Tumour inhibiting rate=(model group average tumor weight-administration group average tumor weight) model group average tumor weight^-1·100%。

SEQUENCE LISTING

<110>Liaoning Normal University

<120>the recombiant protein rLj-RGD4 of the synthetic gene application in antitumor drug is come from

<130> Q. Gao, Y. Pang, Y. Wu, F. Ma, Q.W. Li, [Expressed sequence tags

(ESTs) analysis of the oral gland of Lampetra japonica], Yi

Chuan Xue Bao 32 (2005) 1045-1052.

<160> 1

<170> PatentIn version 3.2

<210> 1

<211> 174

<212> DNA

<213> Lampetra japonica

<220>

<221> exon

<222> (1)..(174)

<400> 1

atg gcc att tgt cat aag cag aat tat ccc atg ggt acg gag aca cag 48

Met Ala Ile Cys His Lys Gln Asn Tyr Pro Met Gly Thr Glu Thr Gln

1 5 10 15

gga gac aca cgt gga gac aca cgg gga gac aca cgt gga gac aca cgg 96

Gly Asp Thr Arg Gly Asp Thr Arg Gly Asp Thr Arg Gly Asp Thr Arg

20 25 30

ggg gcc cgc gga gac gca cgg aga cac gga cac aac aaa cat tta cac 144

Gly Ala Arg Gly Asp Ala Arg Arg His Gly His Asn Lys His Leu His

35 40 45

aga atg agt gca gcg gtg agt gaa tgt gtt 174

Arg Met Ser Ala Ala Val Ser Glu Cys Val

50 55

Claims (2)

1. a synthetic genelj-rgd4CDNA sequence and the Protein L j-RGD4 aminoacid sequence derived by it,lj-rgd4Synthetic gene 174bp length, its protein is made up of 58 aminoacid, its cDNA sequence and the egg derived by it White matter aminoacid sequence is:

atg gcc att tgt cat aag cag aat tat ccc atg ggt acg gag aca cag 48

Met Ala Ile Cys His Lys Gln Asn Tyr Pro Met Gly Thr Glu Thr Gln

1 5 10 15

gga gac aca cgt gga gac aca cgg gga gac aca cgt gga gac aca cgg 96

Gly Asp Thr Arg Gly Asp Thr Arg Gly Asp Thr Arg Gly Asp Thr Arg

20 25 30

ggg gcc cgc gga gac gca cgg aga cac gga cac aac aaa cat tta cac 144

Gly Ala Arg Gly Asp Ala Arg Arg His Gly His Asn Lys His Leu His

35 40 45

aga atg agt gca gcg gtg agt gaa tgt gtt 174

Arg Met Ser Ala Ala Val Ser Glu Cys Val

50 55

The most according to claim 1lj-rgd4CDNA sequence connect gene clone's product institute table of any type of carrier The recombiant protein rLj-RGD4 reached application in terms of preparing anti-angiogenic rebirth series antineoplastic medicament.