CN100491399C - Hybrid protein of p53 protein epitope and filobactivirus gene 8 protein and application thereof - Google Patents

Abstract

This invention belongs to the DNA recombination field in bioengineering. Wherein, with the recombination technique, inserting the gene fragment of B cell epitope composed by 37-46 peptide fragments on N-end of P53 protein into the carrier integrated with main coat protein gene of filamentous phage to form the new recombinant plasmid; in host, secreting the epitope gene product out of the cell to assemble in coat protein and form heterozygous protein that can be used as a antigen to detect antibody in clinical tumor patient serum (such as, gastric cancer, liver cancer, breast cancer and lung cancer).

Description

The hybrid protein and the application thereof of proteic epi-position of p53 and filobactivirus gene 8 protein

Technical field

The invention belongs to the DNA recombinant technology field in the biotechnology.

Background technology

Tumour is the second in the world number that is only second to cardiovascular disorder " killer " disease.By to the end of the year 1999, global tumour patient sum has exceeded 4,000 ten thousand people.China has 1,300,000 people to die from tumour every year.At present, also lack the medicine that to diagnose effectively, to prevent and to treat the carrying out of tumour.

P53 is a kind of cancer suppressor gene, and its albumen is to keeping the normal cell fission and the important regulatory role of grow, but in case undergo mutation, the prolongation of mutain transformation period loses cancer suppressing action, accumulates in cell then stimulating immune system generation corresponding antibodies.The gene fragment of the B cell epitope that N end 20-25 peptide section, 37-46 peptide section constitute is inserted in the carrier that is integrated with the main coat protein gene of filobactivirus, is built into new recombinant vectors.This recombinant vectors is changed over to the host bacterium, and in the intestinal bacteria, under the condition that helper phage exists, tumour epitope gene product is secreted into the extracellular, and is assembled in the bacteriophage coat protein, forms the hybrid protein with corresponding epi-position.(Rui-juan Gao, Hui-zheng Bao, QiongYang, Qi Cong, Jin-na Song, Li Wang*.The presence of serum anti-p53 antibodiesfrom patients with invasive ductal carcinoma of breast:correlation to otherclinical and biological parameters.Breast Cancer Research and Treatment.2005; 93 (2): 111-115; Wang Li. the displaying of polypeptide on filamentous phage coat protein. Northeast China Normal University's journal, 1998 the 1st phases: 46-48).

The advantage of utilizing phage display allogenic polypeptide technology is to have realized the external conversion of genotype and phenotype effectively, make the investigator can on gene molecule clone's basis, realize the external control of protein conformation fully effectively, have good biological in external acquisition and learn active expression product.This novel genetic engineering technique has aspect the preparation biological products: easy, quick, sensitive, low cost and other advantages (Wang Li*.Foreign peptides displayed on the major coatprotein of filamentous bacteriophage.Chinese Science Bulletin, 1998.Vol.43 No.12:1242-1246).

Summary of the invention

The objective of the invention is to establish a kind of biological products that the preparation clinical tumor detects that can be used in.

Hybrid protein of the present invention is the filobactivirus gene 8 heterozygosis coat protein that contain the epi-position of human p53N end 37-46 peptide section formation.

The nucleotide sequence of P53-37-46 hybrid protein gene:

atg?aaa?aag?tct?tta?gtc?ctc?aaa?gcc?tcc?gta?gcc?gtt?gct?acc?ctc 48

gtt?ccg?atg?ctg?tct?ttc?gcc?gcg?gag?ggt?tct?caa?gct?atg?gat?gat 96

tta?atg?tta?tct?cca?tcg?aac?gat?cct?gca?aaa?gcg?gcc?ttt?gac?tcc 144

ctg?caa?gcc?tca?gcg?acc?gaa?tat?atc?ggt?tat?gcg?tgg?gcg?atg?gtt 192

gtt?gtc?att?gtc?ggc?gca?act?atc?ggt?atc?aag?ctg?ttt?aag?aaa?ttc 240

acc?tcg?aaa?gca?agc 255

The aminoacid sequence of P53-37-46 hybrid protein is:

Met?Lys?Lys?Ser?Leu?Val?Leu?Lys?Ala?Ser?Val?Ala?ValAla?Thr

1 5 10 15

Leu?Val?Pro?Met?Leu?Ser?Phe?Ala?Ala?Glu?Gly?Ser?Gln?Ala?Met

20 25 30

Asp?Asp?Leu?Met?Leu?Ser?Pro?Ser?Asn?Asp?Pro?Ala?Lys?Ala?Ala

35 40 45

Phe?Asp?Ser?Leu?Gln?Ala?Ser?Ala?Thr?Glu?Tyr?Ile?Gly?Tyr?Ala

50 55 60

Trp?Ala?Met?Val?Val?Val?Ile?Val?Gly?Ala?Thr?Ile?Gly?Ile?Lys

65 70 75

Leu?Phe?Lys?Lys?Phe?Thr?Ser?Lys?Ala?Ser

80 85

The preparation of hybrid protein may further comprise the steps:

The first step, construction of expression vector and recombinant vectors.

Handle the pfd88 plasmid with SacII and two kinds of restriction enzymes of BstB I, the oligonucleotide fragment with synthetic is inserted into the plasmid vector of handling through enzyme then, obtains recombinant vectors pfd8p53-37.

Second goes on foot, and synthesizes the gene order of p53 albumen n end 37-46 peptide segment table position, and adds restriction enzyme site at the sequence two ends.Dna synthesizer with commercial company carries out synthetic.The sequence of epitope gene is: (be 5 '-3 ' direction)

Positive-sense strand:

GGAGGGTTCTCAAGCTATGGATGATTTAATGTTATCTCCAT

Antisense strand:

CGATGGAGATAACATTAAATCATCCATAGCTTGAGAACCCTCCGC。

In the 3rd step, change recombinant vectors over to host cell.Host cell is the large intestine bacterial strain TG1 bacterial strain that has F-factor.The transformant that obtains is new engineering strain pfd8p53-37.

The 4th step, the preparation hybrid protein.The culturing engineering bacterial strain, steps such as process is centrifugal, precipitation can obtain the hybrid protein of this invention.

Immunodetection:

The antibody that hybrid protein can be used as among a kind of antigen and the kinds of tumors patients serum produces immunne response: the hybrid protein with the present invention's preparation is an antigen, adopt immunologic detection method (as ELISA), can detect clinical tumor (such as, cancer of the stomach, colon knurl, liver cancer, mammary cancer and lung cancer etc.) associated antibodies among the patients serum.Illustrate that this hybrid protein can be used for preparing the detection of drugs of clinical tumor.

Embodiment

1. epitope gene is synthetic:

Utilize the dna synthesizer of commercial biotech firm to carry out the epitope gene fragment

GGAGGGTTCTCAAGCTATGGATGATTTAATGTTATCTCCAT，

Synthesizing CGATGGAAGTAACATTAAATCATCCATAGCTTGAGAACCCTCCGC).

2. the structure of recombinant vectors

1) gets pfd88 plasmid 5 μ g, add the SacII enzyme of 1-2 μ L, add damping fluid and sterilized water then to cumulative volume 200 μ l.37 ℃ are incubated 24 hours.After enzyme is cut, with ordinary method extracting plasmid DNA.After the extracting,, and be dissolved in the 50 μ L TE liquid with amine acetate and the long-pending ethanol sedimentation DNA of diploid.

2) handle first kind of plasmid DNA that enzyme is cut with second kind of restriction enzyme BstBI, method is the same.

3) behind the double digestion, carry out agarose gel electrophoresis, measure carrier concn.

4) carrier mixes by mole concentration 1:3 with the exogenous dna fragment of synthetic.10 μ L mixed solutions add 0.5 μ LT ₄Dna ligase and 1 μ L damping fluid.Under 15-20 ℃ of condition, overnight incubation obtains recombinant vectors (pfd8p53-37).

3. preparation competent cell

The TG1 cell cultures on the LB substratum, was cultivated 16-20 hour for 37 ℃.Select single colony inoculation in 2ml LB liquid culture medium, 37 ℃ of overnight incubation.Nutrient solution changes in the fresh LB nutrient solution of 100ml, cultivates 5 hours for 37 ℃.Centrifugal 15 minutes of 5000rpm.0.1mol/L CaCl with the 8ml precooling ₂Suspend and precipitate, obtain competent cell.

4. recombinant vectors transformed host cell

Get the recombinant vectors dna solution 1.25 μ L that concentration is about 150 μ g/mL; The competent cell that adds 100 μ l placed trash ice 15 minutes; Add 600 μ l LB nutrient solutions, place 37 ℃ of constant temperatures to cultivate 1 hour; Centrifugal 5 minutes of 10000rpm; Abandon supernatant, remainder is applied to the LB media surface that contains penbritin, overnight incubation in 37 ℃ of thermostat containers.Be selected in single bacterium colony of cultivating planar growth, carry out enlarged culturing and cryopreservation (70 ℃).

5. the preparation of hybrid protein

It is streak culture on the LB flat board to get frozen transformant, spends the night; The menu colony inoculation in the 3mlLB nutrient solution that contains penbritin, 37 ℃ of overnight incubation; Change nutrient solution in 80-100ml LB nutrient solution continuation cultivation, and add IPTG (final concentration is 0.5-1mM/mL), cultivated 30 minutes for 37 ℃; Centrifugal (10 000rpm) 10 minutes; Precipitation suspends again with nutrient solution, cultivates 2-4 hour for 37 ℃; Centrifugal (8000rmp) 20 minutes abandons supernatant, suspends with TE liquid and precipitates; Centrifugal (5000rmp) 20 minutes gets supernatant, and adds 20% polyoxyethylene glycol and 2.5mol/L sodium-chlor.Placed 4-5 hour centrifugal (8000rmp) 10 minutes in the cold environment; Abandon supernatant, suspend with TE liquid and precipitate, obtain the filobactivirus heterozygosis coat protein that has allogenic polypeptide to show.

6. the detection of Zhi Bei hybrid protein

1) adopt SDS-polyacrylamide gel electrophoresis to carry out proteic purity detecting

The testing protein sample was handled in boiling water 3-5 minute.Carry out electrophoresis according to a conventional method.Behind the electrophoresis, offset plate is dyeed with silver nitrate method staining.If hybrid protein is expressed successfully, two bands appear on the offset plate.The top band is the hybrid protein band, and the below is a wild-type filamentous phage coat protein band.Because silver nitrate method staining is relatively sensitiveer,, illustrate that the albumen of preparation has reached pure standard as there not being other assorted taking out of now on the offset plate.

2) adopting ultraviolet spectrophotometry to be prepared proteic concentration detects

Get the sample of 5-10 μ L, dilute 100 times after, be determined at the light absorption value of 270 nanometers.With light absorption value divided by 3.84 concentration (μ g/ μ L) that just obtain protein solution.

7. immunodetection experiment

Take the ELISA method, as envelope antigen, person's to be checked serum is detected with het.Antigenic bag is 60 μ g/mL by concentration, an anti-patients serum for 200 times of dilutions, and two anti-ly are the goat anti-human igg.Dyeing process adopts the TMB method.Detecting wavelength is 450/620.The result that 367 tumour patient serum are detected shows: colorectal carcinoma 28.6% (8/28), the esophageal carcinoma 27.3% (6/22), cancer of the stomach 26.9% (7/26), lung cancer 23.5% (23/98), nasopharyngeal carcinoma 23.1% (3/13), mammary cancer 1 8.8% (27/144), ovarian cancer 18.2% (2/11), brain tumor 16.7% (2/12), liver cancer 15.4% (2/13).The specific degree 94.2% that detects, sensitivity is 21.8%.

SEQUENCE?LISTING

＜110〉Northeast Normal University

＜120〉hybrid protein and the application thereof of proteic epi-position of p53 (SQAMDDLMLS) and filobactivirus gene 8 protein

<130>none

<140>---------

<141>2005-10-23

<160>2

<170>PatentIn?version?3.1

<210>1

<211>255

<212>DNA

<213>Artificial?sequence

<220>

＜223〉recombination sequence of p53 albumen epi-position (SQAMDDLMLS) gene fragment and filobactivirus gene VIII

<220>

<221>CDS

<222>(1)..(255)

<223>

<220>

<221>misc_recomb

<222>(79)..(108)

<223>

<300>

＜302〉hybrid protein and the application thereof of proteic epi-position of p53 (SQAMDDLMLS) and filobactivirus gene 8 protein

<308>none

<309>2007-04-25

<310>none

<311>2005-10-25

<312>2007-04-25

<313>(1)..(255)

<400>1

<210>2

<211>85

<212>PRT

<213>Artificial?sequence

<220>

＜223〉recombination sequence of p53 albumen epi-position (SQAMDDLMLS) gene fragment and filobactivirus gene VIII

<400>2

Claims (2)

1, the hybrid protein of proteic epi-position SQAMDDLMLS of a kind of P53 and filobactivirus gene 8 protein, the aminoacid sequence that it is characterized in that this hybrid protein are shown in the sequence 2 in the sequence table.

2, be used to prepare the purposes of the clinical detection medicine that detects tumour by the described hybrid protein of claim 1.