CN102851296A - Anti-liver cancer protein pp3501, its preparation method and application - Google Patents

Abstract

The invention, belonging to the technical field of medicines or health products, particularly discloses anti-liver cancer protein pp3501, its preparation method and application. The protein can be prepared by genetic engineering. The preparation method comprises the following steps: transforming a recombinant prokaryotic expression plasmid in escherichia coli expression fungus, conducting shake cultivation in a medium containing selective antibiotics at 35-38 DEG C; 1-5h later, inducing with isopropyl-beta-D-sulfo galactose pyranoside having the final concentration of 0.1-3mmol/L for 2-6h; at 4 DEG C, respectively collecting bacteria and a supernatant; condensing the supernatant and then purifying with affinity resin; and treating the protein in the form of inclusion body in escherichia coli with urea, dissolving, and purifying with affinity resin. The invention discloses applications of the protein pp3501 in preparing anti-liver cancer medicines and health products or as a component of anti-liver cancer medicines and health products. The protein disclosed herein can inhibit the growth of a plurality of phenotypic liver cancer cells and enhance sodium butyrate to inhibit the growth of live cancer cells.

Description

A kind of anti-hepatoma protein pp3501 and its preparation method and application

Technical field

The invention belongs to medicine or health product technology field, particularly a kind of anti-hepatoma protein pp3501 and its preparation method and application.

Background technology

Liver cancer is one of disease of serious threat human health, and the research and development of medicines resistant to liver cancer are significant in current medical field.Seeking the medicine of Hepatoma therapy or the method for other treatment liver cancer is to have urgency and far-reaching social effect.

With the restriction differential display polymerase chain reaction analysis of gene sequences to obtain a predicted protein pp3501 with similar gene sequences coding sequence, the amino acid sequence of the protein Met Val Leu Ala AspAla Ala Lys Met Arg Ser Pro Arg Val Arg Met Ala Pro Asn Pro Val ProGly Thr Leu Ile Arg Arg Gly Lys Leu Gly Arg Arg Gln Glu Gly Arg ValLys Thr Glu Ala Glu Thr Gly Gly Gln Gly Val Pro Gly Val Ala Gln SerHis His Gly Leu Gln Glu Val Trp Asp Arg Leu Ser Gln Pro Gln Lys GlyPro Ala Pro His Arg Pro Gly Phe Gln Arg Ala Asn Ile Ser Val Ala GluAla Pro Ser Ala Phe Ala Val Leu Gly Asp Gly Arg Arg His Ile Cys SerTrp Pro Val Ser Arg Ser Val Ala Pro Thr Val Ser Gly His Trp Pro GluGly Asn Arg Lys Ser Trp Arg Gly Arg His Thr His Glu Pro Phe Arg AsnAsn Glu。The encode protein molecule amount is 16kDa, and iso-electric point is 11.6.Theoretical analysis pp3501 albumen contains three α-helixstructures and 6 beta sheet structures.

We test and find that pp3501 suppresses multiple phenotype liver cancer cell growth, and strengthen Sodium propanecarboxylate inhibition liver cancer cell growth.This product can be used for preparing the composition of medicines resistant to liver cancer, and sets up the method that genetically engineered is produced this albumen.Find through retrieval, all do not find the report with the anti-liver cancer of pp3501 at home and abroad.

Summary of the invention

The objective of the invention is increases day by day for the onset of liver cancer rate, and seeking new medicines resistant to liver cancer and target spot is the effective way of the novel medicines resistant to liver cancer of screening or prodrug.A kind of anti-hepatoma protein pp3501 and preparation method thereof is provided, and another object of the present invention provides the new purposes of anti-hepatoma protein pp3501, i.e. application in Hepatoma therapy medicine and healthcare products.This albumen can suppress multiple phenotype liver cancer cell growth, and strengthens Sodium propanecarboxylate inhibition liver cancer cell growth.

For achieving the above object, the technical scheme that the present invention takes is: the preparation method of this anti-hepatoma protein pp3501 produces anti-hepatoma protein pp3501 by genetically engineered, specifically according to the following steps:

(1) with the primer that contains the polyadenylic acid sequence, 35～42 ℃ of reverse transcriptions, then use upstream primer: 5 '-GATCCGAATTCACCATGGTTTTAGCAGATGC-3 ', downstream primer: 5 '-TGTCGACGGGTCTTGCACGTGTCAC-3 ' carries out PCR amplification, polymerase chain reaction parameter: 90 ℃～95 ℃ denaturations 5 minutes, 90 ℃～95 ℃ were heated 10～40 seconds, 53 ℃～60 ℃ were reacted 25～40 seconds, 70 ℃～75 ℃ were reacted 25～40 seconds, 25～40 rear 72 ℃ of extensions 3～10 minutes that circulate, 1～2% agarose gel electrophoresis is identified and is also reclaimed the reverse transcription-polymerase chain reaction product;

(2) respectively with reverse transcription-polymerase chain reaction product and prokaryotic expression carrier behind the property endonuclease digestion purifying processed, after distinguishing purifying again, be built into eucaryon or prokaryotic expression recombinant plasmid with the ligase enzyme connection, the pp3501 that eukaryon expression plasmid carries can be used for the composition of gene therapy, prokaryotic expression recombinant plasmid transformed intestinal bacteria prepare pp3501 albumen with genetic engineering technique;

(3) the recombined pronucleus expression plasmid transformation escherichia coli is expressed bacterium, in containing selective antibiotic substratum, 35 ℃～38 ℃ shaking culture, be that the sec.-propyl-beta-D-thio-galactose pyran-glucoside of 0.1～3mmol/L was induced 2～6 hours with final concentration after 1～5 hour, 4 ℃, collect respectively bacterium and supernatant;

(4) carry out purifying by affine resin after the supernatant concentration, the albumen that exists with the inclusion body form in the intestinal bacteria is used Urea treatment, and dissolving is carried out purifying by affine resin.

The application of anti-hepatoma protein pp3501 of the present invention in preparation Hepatoma therapy medicine and healthcare products is to utilize pp3501 to suppress the growth of multiple phenotype liver cancer cell, strengthens Sodium propanecarboxylate and suppresses liver cancer growth; Adopt gene engineering method to prepare anti-hepatoma protein pp3501, utilize this composition to prepare cancer therapy drug or drug component.

With pp3501 albumen direct effect liver cancer cell, but the direct killing liver cancer cell.Change the pp3501 gene over to liver cancer cell with genophore, can significantly suppress hepatoma cell proliferation.

The brief introduction of anti-liver cancer experimentation:

The not isophenic liver cancer cell of vitro culture adds the target protein of 20 μ g/ml, cultivates 1～5 day (the results are shown in Figure 4).Or set up nude mice lotus knurl model, and adopt intratumor injection, observe target protein anti-liver cancer efficacy (the results are shown in Figure 5).

The function of pp3501 albumen

Suppress multiple phenotype liver cancer cell growth.

The new purposes of pp3501 albumen

The application of pp3501 albumen in preparation medicines resistant to liver cancer and healthcare products.

Concrete type of service

Can be made into injection, use by the injection requirement.

Usage

1. can singlely use.

2. can be used as medicines resistant to liver cancer or one of them component uses.

Working conditions

Working conditions is with reference to the condition of protein and peptide medicine, simultaneously with reference to the antitumor drug scope of application.

Description of drawings

Fig. 1 is clone products agarose gel electrophoresis figure;

Among the figure: 1: molecular weight standard; 2: clone products

Fig. 2 is that sodium dodecyl sulfate-polyacrylamide gel electrophoresis is to the soluble analysis figure of pp3501 fusion rotein;

Among the figure: 1: molecular weight standard; 2: sec.-propyl-beta-D-thio-galactose pyran-glucoside is induced transfection pET-32a-pp3501 bacterium extract supernatant liquor; 3: sec.-propyl-beta-D-thio-galactose pyran-glucoside is induced transfection pET-32a-pp3501 bacterium extract insolubles.

Fig. 3 is the purifying figure that sodium dodecyl sulfate-polyacrylamide gel electrophoresis is analyzed the pp3501 fusion rotein;

Among the figure: 1: sec.-propyl-beta-D-thio-galactose pyran-glucoside is induced transfection pET-32a-pp3501 bacterium extract; 2: the pp3501 fusion rotein behind the purifying

Fig. 4 is that pp3501 suppresses different phenotype liver cancer cell growth figure;

Fig. 5 is results of animal;

* P＜0.05, * * P＜0.01 and control group are relatively;

Compare with the Sodium propanecarboxylate group #P＜0.05.

Embodiment:

1. use special primer

With the primer that contains the polyadenylic acid sequence, 37 ℃ of reverse transcription, then upstream primer: 5 '-GATCCGAATTCACCATGGTTTTAGCAGATGC-3 ', downstream primer: 5 '-TGTCGACGGGTCTTGCACGTGTCAC-3 ' carries out PCR amplification.The polymerase chain reaction parameter: 94 ℃ of denaturations 5 minutes, 94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 30 seconds.30 rear 72 ℃ of extensions 5 minutes that circulate.The reverse transcription-polymerase chain reaction product is identified and reclaimed to 1% agarose gel electrophoresis.

2. the structure of prokaryotic expression recombinant plasmid

Cut reverse transcription-polymerase chain reaction product and pET-32a carrier behind the purifying with property restriction endonuclease EcoR I processed, Sal I enzyme respectively, more respectively behind the purifying, connect with the T4 ligase enzyme and to be built into the prokaryotic expression recombinant plasmid, conversion intestinal bacteria E.coli DH5 α.

3. the prokaryotic expression recombinant plasmid is in colibacillary expression

Prokaryotic expression recombinant plasmid transformed e. coli bl21 (DE3) is expressed bacterium, and picking prokaryotic expression recombinant plasmid is inoculated into the substratum that contains penbritin 100 mcg/ml,, 37 ℃ of shaking culture are spent the night.The bacterium that spends the night of getting spend the night bacterium and the unloaded plasmid of 5 microlitre pET-32a of 5 microlitre prokaryotic expression recombinant plasmids is inoculated in respectively in the substratum that contains penbritin 100 mcg/ml 37 ℃ of shaking culture.Respectively getting the final concentration of drawing after 3 hours is that the sec.-propyl-beta-D-thio-galactose pyran-glucoside of 1mmol/L was induced 5 hours.4 ℃, 12000 rev/mins, centrifugal 1 minute, collect respectively bacterium and supernatant.

4. inclusion body extracts and purifying

Carry out purifying by affine resin after the supernatant concentration.The albumen that exists with the inclusion body form in the intestinal bacteria, with becoming the 6mmol/L Urea treatment, dissolving is carried out purifying by affine resin.Purified product in 10% or 12% sodium lauryl sulphate-polyacrylamide gel in electrophoresis analyze.

5.pp3501 inhibition liver cancer cell growth

With pp3501 albumen direct effect liver cancer cell, but the direct killing liver cancer cell.Change the pp3501 gene over to liver cancer cell with genophore, can significantly suppress hepatoma cell proliferation.Preparation nude mice lotus knurl model inject respectively pp3501 protein (3mg/kg) or Sodium propanecarboxylate (30mg/kg) in the knurl, or both injects simultaneously.Nude mice lotus knurl experiment demonstration, intratumor injection can significantly suppress liver cancer growth, and strengthens the action effect of Sodium propanecarboxylate.

As a result brief introduction

1, people pp3501 gene cloning

The pp3501 recombinant expression plasmid successfully amplifies the encoding sequence of pp3501 gene through the preliminary evaluation of Auele Specific Primer, size is 432 base pairs (seeing Fig. 1).

2, the expression of pp3501 fusion rotein

Through detected through gel electrophoresis, the protein band (seeing Fig. 2) of obvious high expression level has appearred in the intestinal bacteria that the pp3501 recombinant expression plasmid of inducing transforms about molecular weight of albumen standard 34kDa.The molecular weight of albumen of pp3501 genes encoding is 16kDa, and the histidine-tagged molecular weight of Pet-32a vector encoded is 18kDa, and restructuring pp3501 molecular weight of albumen is 34kDa.Through electrophoresis detection, the pp3501 recombinant expression plasmid of inducing transforms in the colibacillary precipitation, the protein band of obvious high expression level occurred at molecular weight of albumen standard 34kDa place, and in supernatant the expression amount of pp3501 fusion rotein lower (seeing Fig. 2).Go out highly purified pp3501 fusion rotein (seeing Fig. 3) with affine resin purification.

3, pp3501 is to the inhibited proliferation of liver cancer cell

The result as shown in Figure 4 and Figure 5.Pp3501 suppresses different phenotype liver cancer cell growths, and each experimental group curve is obviously slow in corresponding control group.Preparation nude mice lotus knurl model is injected respectively pp3501 protein or Sodium propanecarboxylate in the knurl, or both inject simultaneously and all suppress liver cancer growth, and pp3501 strengthens the Sodium propanecarboxylate anti-liver cancer efficacy.

Claims (3)

1. the preparation method of an anti-hepatoma protein pp3501 is to produce anti-hepatoma protein pp3501 by genetically engineered, specifically according to the following steps:

(1) with the primer that contains the polyadenylic acid sequence, 35～42 ℃ of reverse transcriptions, then use upstream primer: 5 '-GATCCGAATTCACCATGGTTTTAGCAGATGC-3 ', downstream primer: 5 '-TGTCGACGGGTCTTGCACGTGTCAC-3 ' carries out PCR amplification, polymerase chain reaction parameter: 90 ℃～95 ℃ denaturations 5 minutes, 90 ℃～95 ℃ were heated 10～40 seconds, 53 ℃～60 ℃ were reacted 25～40 seconds, 70 ℃～75 ℃ were reacted 25～40 seconds, 25～40 rear 72 ℃ of extensions 3～10 minutes that circulate, 1～2% agarose gel electrophoresis is identified and is also reclaimed the reverse transcription-polymerase chain reaction product;

(2) respectively with reverse transcription-polymerase chain reaction product and prokaryotic expression carrier behind the property endonuclease digestion purifying processed, after distinguishing purifying again, be built into eucaryon or prokaryotic expression recombinant plasmid with the ligase enzyme connection, the pp3501 that eukaryon expression plasmid carries can be used for the composition of gene therapy, prokaryotic expression recombinant plasmid transformed intestinal bacteria prepare pp3501 albumen with genetic engineering technique;

(3) the recombined pronucleus expression plasmid transformation escherichia coli is expressed bacterium, in containing selective antibiotic substratum, 35 ℃～38 ℃ shaking culture, be that the sec.-propyl-beta-D-thio-galactose pyran-glucoside of 0.1～3mmol/L was induced 2～6 hours with final concentration after 1～5 hour, 4 ℃, collect respectively bacterium and supernatant;

(4) carry out purifying by affine resin after the supernatant concentration, the albumen that exists with the inclusion body form in the intestinal bacteria is used Urea treatment, and dissolving is carried out purifying by affine resin.

2. an anti-hepatoma protein pp3501 is in the application for preparing cancer therapy drug and healthcare products.

3. the application of anti-hepatoma protein pp3501 in preparation cancer therapy drug component and healthcare products component.

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