CN104531742A - Preparation and immunolocalization method of EOLA1 polyclonal antibody - Google Patents

Abstract

The invention discloses a preparation and immunolocalization method of an EOLA1 polyclonal antibody. The preparation and immunolocalization method comprises the following steps: establishing a recombinant plasmid, transforming escherichia coli and carrying out induction expression, extracting interest protein, immunizing rabbits to prepare the polyclonal antibody, and detecting the expression of EOLA1 in a part of malignant tumor tissues of human bodies by using an immunohistochemical method. By adopting the preparation and immunolocalization method, the recombinant plasmid is successfully prepared to express the human EOLA1 protein in escherichia coli, the rabbit anti-EOLA1 polyclonal antibody is prepared through immunizing rabbits, and the immunohistochemical method detection shows that EOLA1 protein is expressed in breast cancer tissue cell cytoplasm.

Description

A kind of preparation of EOLA1 polyclonal antibody and immunolocalization method

Technical field

The present invention relates to a kind of preparation and immunolocalization method of EOLA1 polyclonal antibody, belong to biomedicine technical field.

Background technology

People's endothelium high expression level lipopolysaccharides associated factor 1 (endothelial-overexpressedlipopolysaccharide-associatedfa ctor1, EOLA1) gene is the human gene that this research department is cloned into early stage, because of its be subject to lipopolysaccharides (LPS) stimulate after in endotheliocyte up-regulated, therefore called after endothelial cell protein C receptor (EOLA1), early stage is studied the location of EOLA1 gene, because the protein structure coded by it exists protein kinase C and Tyrosylprotein kinase-phosphorylation site and helix turn helix (HTH) motif, consider that it may be cell transcription factor, participate in intracellular signal transduction, an important prerequisite of research gene function is exactly the expression needing to go to detect with suitable antibody gene, this antibody is not also had to remove gene expression detection in right prior art.

Summary of the invention

The object of the invention is: preparation and immunolocalization method that a kind of EOLA1 polyclonal antibody is provided, the method can successfully prepare EOLA1 polyclonal antibody, confirm that EOLA1 albumen has expression, to overcome the deficiencies in the prior art in breast cancer tissue's cancer cells endochylema.

Technical scheme of the present invention

A kind of preparation of EOLA1 polyclonal antibody and immunolocalization method, the method adopts construction recombination plasmid, transformation of E. coli and abduction delivering, extracting target protein immunizing rabbit prepares polyclonal antibody, and immunohistochemical method detects the expression of EOLA1 in part human body malignant tumor tissue.

Owing to have employed technique scheme, compared with prior art, the present invention adopts the method for prokaryotic expression to prepare EOLA1 full length protein sequence antigen, with the anti-human EOLA1 polyclonal antibody of the rabbit obtaining high-titer after this immunizing rabbit, the expression of EOLA1 in tumor tissues is have detected by immunohistochemical method, the esophagus cancer that result display EOLA1 albumen is detecting, cancer of the stomach, liver cancer, colorectal carcinoma, lung cancer, expression is had in cerebral glioma and mammary cancer mesenchymal cell endochylema, and in essence cancer cells, only have breast cancer cell endochylema to express as seen, mesenchyma stroma of tumors is made up of fibrillar connective tissue and blood vessel, the interstitial of various tumour is substantially identical, do not have a specificity, nutrition and supportive protection effect are play to tumor epithelial cell, EOLA1 albumen great expression is in mesenchyma stroma of tumors cell, prompting EOLA1 albumen plays a significant role in tumor-blood-vessel growth and transfer, vasculogenesis is except having except critical role in tumor development, also take part in retinopathy at the bottom of diabetic eye, the pathophysiological changes such as ischemical reperfusion injury, the activation of vascular endothelial cell plays a major role in vasculogenesis, other cells play auxiliary and supporting function, research shows, after endothelial cell activation, in born of the same parents, MT protein expression increases, activate NF-κ B and AP-1 signal path, promote the expression of Matrix Metalloproteinase-9 (MMP-9), the latter can decompose pericellular matrix, promote migration and the segment dislocation of endotheliocyte, thus cause the acceleration of vasculogenesis, EOLA1 can by affecting the activation of this path with the interaction of MT2A in endotheliocyte, middle Organic Pollutants in Drinking Water acts on liver tissues of rats protein through ADME process, the side chain of primary challenge gal4 amino acid and generate carbonyl, Organic Pollutants in Drinking Water metabolite (hydroxyl radical free radical) directly acts on peptide bond, makes peptide bond rupture and produces carbonyl, be combined with non-protein carbonyl compound, if mda is oxygen containing carbonyl compound, it can be inserted into protein interior and cause protein carbonyl group to rise, and illustrate that tap water organism is in the oxidative damage parameters of rat liver, hepatic tissue PCO is more responsive than hepatic tissue MDA.

Embodiment

Below the present invention is further described in detail, but not as any limitation of the invention.

Embodiments of the invention:

1 materials and methods

1.1 material

Rabbit is purchased from Third Military Medical University's Experimental Animal Center, PET-46EK/LIC plasmid purchased from American Novagen company, human ECV304 strain is that southwestern hospital's burn experiment room is preserved, Trizol extracts reagent purchased from Beijing Ding Guo biotech firm, RT PCR kit is purchased from Dalian Takara company, complete Freund's adjuvant (completeFreund ' sadjuvaant, CFA) and incomplete Freund's adjuvant (incompleteFreund ' sadjuvaant, IFA) purchased from GibcoBRL company, immunohistochemical reagents box is purchased from VECTORLAB company, Protein Extraction Reagent RIPABBuffer available from Sigma, biotin labeling two is anti-purchased from company of Zhong Shan Golden Bridge, ECL luminescence reagent box is purchased from Pierce company, BL21(DE3) plys competent cell is purchased from Suo Laibao bio tech ltd, Shanghai.

1.2 prepare target protein

1.2.1 the human ECV304 about 5 × 10 of extraction-collection cultivation of human ECV304 total serum IgE ⁷individual, through PBS rinsing 2 times, extract reagent description operation according to Trizol, after obtaining total cellular RNA sample, survey A260/A280 ratio Analysis purity, 1% agarose gel electrophoresis analysis.

1.2.2 the Design and synthesis of primer

According to EOLA1 gene open reading frame (ORF) sequences Design upstream primer 5 '--GACGACGACAAGATGAAGTTTGGCTGCC-3 ', downstream primer 5 '-GAGGAGAAGCCCGGTTCACACTTCATG-3 ', transfers to Shanghai Sheng Gong biotechnology company limited to synthesize.

1.2.3 the RT-PCR of cell total rna presses RT-PCR reagent

20 μ l reverse transcription reaction systems prepared by box specification sheets, reverse transcription condition: 50 DEG C, 30min; 99 DEG C, 5min; 4 DEG C, 5min.Acquired results is added 80 μ lPCR reaction systems; PCR condition: 94 DEG C of denaturation 2min, 94 DEG C of sex change 30s, 59 DEG C of annealing 30s, 72 DEG C extend 15min, totally 30 circulations; 72 DEG C extend 10min, and after 1% agarose gel electrophoresis analysis, object fragments gel reclaims purifying.

1.2.4 the structure of recombinant plasmid and transform bacteria

Object fragment is processed into cohesive terminus through T4DNA ligase enzyme and dATP, and with

The glutinous end of PET-46EK/LIC empty plasmid is complementary, and both are built into recombinant plasmid at connection of annealing; The latter is transformed into nonhost with heat shock procedures and expresses bacterium (NovaBlueSinglesTM competence bacterium), coat the upper overnight growth of LB culture plate (Amp50mg/L), the method of application bacterium colony PCR filters out positive single bacterium colony, and bacterium liquid sample serves the order-checking qualification of Hai Shenggong biotechnology company limited.

1.2.5 recombinant plasmid transformed host expresses bacterium

By inoculation correct for order-checking to LB culture plate growth 12h, picking list bacterium colony, 37 DEG C of constant temperature shake bacterium and spend the night, and extract recombinant plasmid by plasmid extraction kit description operation; The latter is transformed into host expresses bacterium BL21(DE3 with heat shock procedures) plysS, PCR screen positive bacterium colony ,-70 DEG C of preservations.

1.2.6 the expression of EOLA1 in intestinal bacteria and target protein analysis

Picking list colony inoculation is to LB nutrient solution, and 37 DEG C are shaken bacterium and cultivate 12h, bacterium liquid is inoculated in fresh LB (containing Amp50mg/L) with 1: 100, and vibration is shaken after bacterium 25h to D600 ≈ 0.6; Be divided into two groups, one group adds isopropyl ss-D thiogalactoside (IPTG) to final concentration is 1mmol/L, induction is shaken bacterium and is cultivated 3h, another group does not add IPTG and shakes bacterium cultivation 3h, get bacterium liquid 1ml respectively, collected by centrifugation thalline, adds 2 times of SDS sample-loading buffers after PBS is resuspended, after ultrasonication, 85 DEG C of heating 3min sex change obtains the total bacterial protein of 10 times of concentrated volumes; Separately get bacterium liquid 10ml respectively, collected by centrifugation thalline, centrifugal after BugBuster liquid 1ml cracking, get and reset and add 2 times of SDS sample-loading buffer mixing, 85 DEG C of heating 3min

Sex change obtains bacterial cytoplasm soluble component; By precipitation 20mmol/LTrisHCl(pH7.5) be resuspended in the 1%SDS of 1ml after repeated washing precipitation, add 2 times of SDS sample-loading buffer mixing, 85 DEG C of heating 3min sex change, obtains the bacterial cytoplasm insoluble component of 10 times of concentrated volumes. above-mentioned sample is carried out SDS-PAGE and west-ern-blot analysis purposes protein expression.

The preparation of 1.3 EOLA1 polyclonal antibodies

1.3.1 fundamental immunity

Rabbit 2, about body weight 3kg.Press 100 μ g polypeptide dose immunization for 1, at the back intracutaneous multi-point injection of rabbit, every 14d injects 1 time, totally 4 times, 1st time use complete Freund's adjuvant immunity, the 2nd, 3,4 time adopt incomplete Freund's adjuvant immunity, another 1 with distilled water subcutaneous injection in contrast.

1.3.2 supplementary immunization

Within after 4 immunity complete the 14th day, again respectively to family's rabbit back intracutaneous multiple spot inoculation, experimental rabbit intradermal injection EOLA1 albumen carries out immunity by 100 μ g polypeptide dosage.1.3.3 anti-EOLA1 polyclonal antibody is collected

After supplementary immunization the 3rd day, put to death by rabbit, collect blood, the centrifugal 10min of 20000 × g, collect serum, 4 DEG C save backup.

1.3.4 saturated ammonium sulphate (SAS) salt precipitation method antibody purification

Get after serum about 1  5ml mixes with equal-volume physiological saline, slowly drip 3mlSAS in stirring is lower, in 4 DEG C of precipitation more than 30min or spend the night, albumen is fully precipitated; At 4 DEG C, the centrifugal 10min of 20000 × g, abandons supernatant liquor; By 18ml physiological saline solution precipitation, ditto drip 12mlSAS, place 1h for 4 DEG C; Repeated centrifugation, precipitates with 13.3ml physiological saline solution; Drip 6.6mlSAS, place 1h for 4 DEG C; The a little salt solution dissolution precipitation of gained precipitation after repeated centrifugation, and load dialysis tubing; With being greater than the PBS of 20 times of volumes in 4 DEG C of dialysis, changing dialyzate for several times ,-20 DEG C of preservations can be put.

1.3.5 enzyme linked immunosorbent assay (ELISA) detects serum titer

EOLA1 albumen coating buffer is diluted to 2mg/L, adds 96 hole enzyme plates, every hole adds 100 μ l, wraps by 24h at 4 DEG C; Abandon coating buffer, add confining liquid every hole 200 μ l, under room temperature, close 2h; Antiserum(antisera) after immune and non-immune rabbit preliminary purification, be diluted to respectively measuring samples (extent of dilution is respectively 1: 100,1: 1000,1: 10000,1: 20000), get 100 μ l respectively and add enzyme plate, 37 DEG C of water-bath 1h; Fill it up with washings and wash 3 times, each 3min;

Add two anti-(1: 2000) every hole 100 μ l of horseradish peroxidase, hatch 2h for 37 DEG C; Fill it up with washings and wash 3 times, each 3min; Add substrate nitrite ion every hole 200 μ l, lucifuge 37 DEG C hatches 10min, and every hole adds 50 μ l stop buffer termination reactions, and OD value (wavelength 490nm) surveyed by enzyme mark spectrophotometer.

1.4 anti-EOLA1 polyclonal antibody qualifications

1.4.1 human umbilical vein endothelial ECV304 cell cultures

Test the cell strain used sum that goes down to posterity and be no more than for 30 ~ 40 generations, cell culture medium is that DMEM adds 10% deactivation newborn calf serum, 100mg/L penicillin and 100mg/L Streptomycin sulphate, 37 DEG C, 5%CO2 incubator cultivate and 2 ~ 3d go down to posterity 1 time; Get 1 bottle of cultured ECV304 cell, 4 times of SDS sample-loading buffer 1ml denaturing treatment 30min, boil 5min; SDS-PAGE electrophoresis, applied sample amount is 20 μ g/ swimming lanes; By protein band from SDS polyacrylamide gel

Be transferred to nitrocellulose filter (NC film).

1.4.2 Western Blot identifies EOLA1 polyclonal antibody

After decolouring, NC film puts into confining liquid (TBST is containing 5% skim-milk), 2 ~ 3h is closed under room temperature, how anti-as primary antibodie (1: 5000) incubated at room 2h with anti-EOLA1, after TBST washs 4 times, immune response band NBT/BCIP develops the color.

1.5 tumor tissues immunolocalizations

Get mammary cancer, esophagus cancer, cancer of the stomach, liver cancer, colorectal carcinoma, lung cancer, cerebral glioma sample that Third Military Medical University's new bridge hospital pathology department is made a definite diagnosis respectively, with 10% neutral formalin fixing organization, conventional ethanol serial dehydration, dimethylbenzene are transparent, specimens paraffin embedding slices, after dewaxing, the protein antibodies of preparation is dripped on tissue sections, after developing a film, the antibody of horseradish peroxidase-labeled is dripped on tissue sections, develop a film again, DAB develops the color, the Morphology and structure of tissues observed cell under light microscopic.

2 results

2.1 ECV304 cell total rnas and RT-PCR product

The total serum IgE ultraviolet spectrophotometer of extracting from human ECV304 is tested, be 1.9412 at A260/A280 ratio, show that sample purity is higher, RT PCR primer electrophoresis can obtain the goal gene segment of about 470bp, in the same size with EOLA1 gene open reading frame.

2.2 expressing protein product analysis qualifications

The recombinant plasmid built is induced being added with in e. coli bl21 (DE3) plys that isopropyl ss-D thiogalactoside (IPTG) cultivates, give expression to obvious target protein, main with inclusion bodies in the insoluble expression of kytoplasm, a small amount of is kytoplasm solubility expression, molecular weight is 20 × 103D about, basically identical with prediction, carry out as primary antibodie the protein band that Western blot can detect specifically expressing with the mouse monoclonal antibody of band His label.

2.3 EOLA1 polyclonal antibody titrations

Titration is carried out to the antibody purification obtained from 2 rabbit, all reach 1: 10000, the concentration of application SDS PAGE gel electrophoresis to antibody purification detects, result shows that antibody concentration all reaches 200mg/L, explanation antibody is successfully prepared, and the serum contrasted prepared by rabbit finds no EOLA1 polyclonal antibody after testing.

2.4 EOLA1 polyclonal antibody specificitys

Extract the albumen of human umbilical vein endothelial ECV304 cytopathy process, the anti-EOLA1 polyclonal antibody of application rabbit carries out Western-blot detection to albumen, the polyclonal antibody of EOLA1 can specific recognition EOLA1 albumen, and its molecular size range conforms to expection.

2.5 Immunohistochemical detection location

Immunohistochemical detection result display EOLA1 albumen is only expressed in mesenchymal cell endochylema at esophagus cancer, cancer of the stomach, liver cancer, colorectal carcinoma, lung cancer, cerebral glioma, achiblastoma cell is without expression, and interstitial and parenchyma endochylema all have expression in breast cancer tissue, and 2 different breast cancer case show same result.

Claims (1)

1. the preparation of an EOLA1 polyclonal antibody and immunolocalization method, it is characterized in that: the method adopts construction recombination plasmid, transformation of E. coli and abduction delivering, extracting target protein immunizing rabbit prepares polyclonal antibody, and immunohistochemical method detects the expression of EOLA1 in part human body malignant tumor tissue.