CN102585010A

CN102585010A - Immunolatex microsphere for detecting rhbFGF (Recombinant Human Basic Fibroblast Growth Factor) and preparation method thereof

Info

Publication number: CN102585010A
Application number: CN2011100235249A
Authority: CN
Inventors: 董贵俊
Original assignee: Individual
Current assignee: Individual
Priority date: 2011-01-16
Filing date: 2011-01-16
Publication date: 2012-07-18

Abstract

The invention relates to an immunolatex microsphere for detecting an rhbFGF (Recombinant Human Basic Fibroblast Growth Factor) and a preparation method of the immunolatex microsphere. The immnuolatex microsphere is coated by an anti-hbFGF protein polyclonal antibody, and the sizes of the microsphere particles are 50nm. The preparation method of the rhbFGF comprises the steps of redecorating and constructing a full-length human bFGF (hbFGF) gene expression sequence according to preferential codons expressed by procaryotic cells, cutting the gene sequence and inserting the cut gene sequences into GST (Glutathione-S-transferase) gene, fusing in an expression carrier and conducting bacteria expression, utilizing an affinity chromatograph column for purifying so as to obtain GST-hbFGF fusion protein, cutting the fusion protein by thrombin, removing the thrombin by the affinity chromatograph column to obtain hbFGF, fusing adjuvants into antigen so as to immune mice, separating and purifying to obtain hbFGF polyclonal antibody, and coupling the antibody with an activated microsphere by a chemical coupling method to obtain the immunolatex microsphere for detecting hbFGF. The application of the detection of the hbFGF is as follows: an ELISA (Enzyme-Linked Immunosorbent Assay) method is utilized for detecting an hbFGF sample, the lowest detection limit is 50pg/ml of hbFGF protein, and the related coefficient R2 is equal to 0.83. The immunolatex microsphere is used for detecting the rhbFGF has high sensitivity when being used for detecting the rhbFGF, and is simple in operation and strong in repeatability.

Description

Immune latex microsphere of a kind of detection recombination human basic fibroblast growth factor (rhbFGF) and preparation method thereof

Technical field

The present invention relates to the immune latex microsphere of a kind of detection recombination human basic fibroblast growth factor (rhbFGF), and the preparation method of this microballoon.

Background technology

Prostatropin (basic fibroblast growth factor; BFGF) extensively be present in nervous tissue, inoblast, the scavenger cell; As a kind of wide spectrum mitogen of human body, has extremely strong biological activity: 1. short proliferation function, chemotaxis and short cell migration campaign effect are all arranged to deriving from mesoderm and neuroectodermal various cell; 2. can promote the section angiogenesis, improve the nutritional status of microcirculation and tissue, promote inoblast, myoblastic propagation simultaneously, make the granulation ramp; 3. strengthen coming from mesoblastic activity of immune cells, improve the anti-infection ability of section.Research shows that bFGF participates in the processes of wound repair of multiple tissue in vivo, is one of wound healing factor important in the body.The high low energy of the content of bFGF directly reflects the vasculogenesis level in the wound repair after the wound, for detecting and monitor trauma repair and organization healing situation etc. foundation is provided.

Clinically, Prostatropin (bFGF) can be used for treating cranium brain, spinal cord, peripheral nerve injury, poisons, and infects hematencephalon; Cerebral infarction, encephalatrophy, cerebral dysplasia, keratohelcosis, neural heariing loss; Facial paralysis, mucous membrane, skin injury, burn, chronic ulcer, soft tissue injury; Visceral lesion, fracture, some endocrinopathys, the replantation of a severed limb and various postoperative wound healings etc.BFGF can promote wound healing, shortens the course of disease greatly, improves prognosis, but it is few to send out the bFGF content detecting method for damage surface of a wound Central Plains so far, and detection sensitivity, detectability and immunity from interference etc. are in urgent need to be improved.At present, about the RR of bFGF immunoassay technology and related patent U.S. Patent No. technology both at home and abroad report seldom, press for exploitation efficiently, novel immunoassay technology and product accurately and rapidly.

Summary of the invention

The objective of the invention is to: the research and development of further filling up the bFGF immunoassay technology are blank, thereby immune latex microsphere of a kind of detection recombination human basic fibroblast growth factor (rhbFGF) and preparation method thereof is provided.

The objective of the invention is to realize like this: a kind of immune latex microsphere that is used to detect recombination human basic fibroblast growth factor (rhbFGF) of the present invention; It is characterized in that; This immune microsphere is encapsulated by anti-hbFGF protein polyclone antibody; Microsphere particle size 50nm is recombination human basic fibroblast growth factor 50pg/ml through enzyme-linked immunosorbent assay (ELISA) LDL, coefficient R ²=0.83, as shown in Figure 1.

The preparation method of the immune latex microsphere of a kind of detection recombination human basic fibroblast growth factor of the present invention (rhbFGF) is characterized in that, this method comprises following steps:

(1) modifies and makes up total length people bFGF (hbFGF) expressed sequence again according to the preferential codon of procaryotic cell expression;

(2) people bFGF gene fragment is gone in glutathione-S-transferase (GST) the gene fusion expression carrier through the restriction enzyme digestion cleft cutting, make up the bacterium fusion expression vector;

(3) expression vector is carried out bacterial expression after, utilize the affinity chromatography column purification to obtain the GST-hbFGF fusion rotein;

(4) utilize zymoplasm at post cutting GST-hbFGF fusion rotein, eluate obtains people bFGF after affinity chromatographic column is removed zymoplasm;

(5) with the fused antigen (GST-hbFGF fusion rotein) of adjuvant back immune mouse, obtain mouse-anti hbFGF polyclonal antibody through separation and purification;

(6),, obtain detecting the proteic immune latex microsphere of reorganization hbFGF through carboxyl-amino chemical coupling method coupling activation microballoon with mouse-anti hbFGF polyclonal antibody.

Glutathione-S-transferase in the said step 2 (GST) gene fusion expression carrier is pGEX-4T-3, and the fusion expression vector that makes up after accomplishing is pGEX-4T-3-hbFGF.

Affinity chromatographic column in the said step 3 is Glutathione Sepharose 4B.

Affinity chromatographic column in the said step 4 is Benzamidine Sepharose 4B Fast Flow.

Microballoon in the said step 6 is the monodisperse carboxyl polystyrene microsphere, big or small 50nm, and it is carboxyl that group is carried on the surface.The activation of this microballoon is at first to use acvator EDC (carbodiimide) activation microballoon, adds protective material Sulfo-NHS then and makes microballoon form stable active ester midbody, in order to connecting the usefulness of antibody molecule.

The invention has the advantages that: immune latex microsphere of a kind of detection recombination human basic fibroblast growth factor of the present invention (rhbFGF) and preparation method thereof; Adopting the monodisperse carboxyl polystyrene microsphere is substrate material; Obtain with the anti-hbFGF polyclonal antibody of chemical coupling method coupling, it is simple, easy to operate to draw materials; The preparation method is careful rigorous, and repeatability is high; Prepared immune latex microsphere particle is little, and particle diameter is even, is recombination human basic fibroblast growth factor 50pg/ml through enzyme-linked immunosorbent assay (ELISA) LDL, coefficient R ²=0.83.

The object of the invention, characteristic and advantage will combine accompanying drawing explanation through preferred embodiment.

The drawing explanation

Fig. 1 is the carboxylic polystyrene microsphere stereoscan photograph

Fig. 2 is the former sequence of hbFGF gene

Fig. 3 is to be the hbFGF gene order after expressive host is optimized with E.coli

Fig. 4 is that bacterium colony PCR checking hbFGF gene inserts the fragment electrophoresis

Fig. 5 is a plasmid Xba I restriction enzyme digestion and electrophoresis

Fig. 6 is the order-checking of hbFGF expression vector

Fig. 7 is that hbFGF protein SDS-PAGE (A) and Western Blotting (B) detect

Fig. 8 is antiserum titre mensuration figure

Fig. 9 is Bio-Rad

ELIASA pictorial diagram

Figure 10 is that the ELISA method detects the hbFGF pictorial diagram

Figure 11 is that the ELISA method detects hbFGF albumen result

Embodiment

Embodiment 1

Make up total length hbFGF expressed sequence with preparation method of the present invention, the concrete steps of preparation are following:

1, design total length hbFGF gene order, the result is as shown in Figure 2;

2, modify again according to the preferential codon of procaryotic cell expression, adopt full gene synthesis technology to make up total length hbFGF gene, totally 339 pairs of bases are cloned into carrier pGEM-T Easy, and the result is as shown in Figure 3;

3, the exactness of inserting through bacterium colony PCR checking hbFGF gene segment, the result is as shown in Figure 4;

4, will clone hbFGF gene bacterium and implement amplification cultivation, and extract and carry out that enzyme is cut and agarose electrophoretic analysis behind the plasmid, the result is as shown in Figure 5;

5, dna sequencing guarantees that the full sequence 100% of gene is accurate, and the result is as shown in Figure 6;

Embodiment 2

Express and purifying extraction hbFGF albumen with preparation method of the present invention, concrete operation steps is following:

1, the hbFGF gene fragment is gone in the pGEX-4T-3 carrier through the restriction enzyme digestion cleft cutting, make up bacterium fusion expression vector pGEX-4T-3-hbFGF;

2, with fusion expression vector pGEX-4T-3-hbFGF transformed into escherichia coli BL21 (DE3) cell, the cell growth conditions is 200 rev/mins, 37 ℃ of temperature;

3, as thalli growth OD ₅₉₅When value is 0.5-0.8, add the inductor IPTG of 1.0mM, induced 2-3 hour at 25 ℃.In the time of 4 ℃, with 5,000 * g centrifugal 5 minutes, collect thalline;

4, thalline is after PBS buffer solution for cleaning 3 times, ultrasonication.Smudge cells with 15,000 * g centrifugal 1 hour, is collected supernatant in the time of 4 ℃;

5, supernatant is cycled through Glutathione Sepharose 4B affinity chromatographic column, 4 ℃ are spent the night.1 * PBS flushing of 10 times of bed volumes of not conjugated protein usefulness;

6, bonded GST-hbFGF fusion rotein is cut and use the elutriant wash-out with zymoplasm at post, collect eluate;

7, above-mentioned eluate is adsorbed zymoplasm through Benzamidine Sepharose 4B Fast Flow affinity chromatographic column, behind wash-out, obtain hbFGF.

The hbFGF albumen of expressing and purifying extracts is done SDS-PAGE and Western Blotting analysis, and the result is as shown in Figure 7.

Embodiment 3

Prepare anti-hbFGF polyclonal antibody with preparation method of the present invention, concrete preparation process is following:

1, with hbFGF fusion rotein antigen with after freund's adjuvant mixes, fully emulsified.Adopt back multi-point injection method immunity BALB/c mouse.The about 10 μ g of antigen amount of each immunity, immunity three times.Each immune two weeks are surveyed antibody titer after caudal artery is got blood examination.

2, with the antigen coated enzyme plate of suitable concentration, 100 μ l/ holes, 4 ℃ are spent the night;

3, the washing after, add serum sample to be checked, 100 μ l/ holes, 37 ℃ 1 hour; If negative control hole;

4, after the washing, add the antibody reagent of the sheep anti-mouse igg of HRP mark, 100 μ l/ holes, 37 ℃ of lucifuges developed the color 20 minutes; Use 2.5M H ₂SO ₄After the termination reaction, read the A490 value in each hole.

To the hbFGF antibody of preparation, detect through the ELISA method, antibody capable combines with the hbFGF specificity, and its relation conefficient reaches level of signification, tires＞1: 6000, and the result is as shown in Figure 8.

Embodiment 4

After the hbFGF Antibody Preparation was accomplished, through chemical coupling monodisperse carboxyl polystyrene microsphere, preparation was used to detect the proteic immune latex microsphere of hbFGF, and concrete preparation and detection step are following:

1, with the monodisperse carboxyl polystyrene microsphere with acvator EDC (carbodiimide) activation, add protective material Sulfo-NHS then and make microballoon form stable active ester midbody;

2, add anti-hbFGF polyclonal antibody, incubation 1-2 hour;

3, after the washing, collect immune microsphere;

4, through Bio-Rad

ELIASA (as shown in Figure 9); Adopt enzyme-linked immunosorbent assay (ELISA) method, detect hbFGF sample (shown in figure 10).

Through the ELISA method validation, the immune latex microsphere LDL that is used to detect hbFGF is recombination human basic fibroblast growth factor 50pg/ml, coefficient R ²=0.83, the result is shown in figure 11.This shows that the present invention is prepared, and to be used to detect the immune latex microsphere detection sensitivity of hbFGF higher, and simple to operate, repeatability is strong, has shown good prospects for application.

Claims

1. immune latex microsphere that detects recombination human basic fibroblast growth factor (rhbFGF); It is characterized in that; This immune microsphere is encapsulated by anti-hbFGF protein polyclone antibody; Microsphere particle size 50nm is recombination human basic fibroblast growth factor 50pg/ml through enzyme-linked immunosorbent assay (ELISA) LDL, coefficient R ²=0.83.

2. the preparation method of the immune latex microsphere of the described detection recombination human basic fibroblast growth factor of claim 1 (rhbFGF) is characterized in that, this method comprises following steps:

(4) utilize zymoplasm cutting GST-hbFGF fusion rotein, eluate obtains people bFGF after affinity chromatographic column is removed zymoplasm;

3. press the preparation method of the immune latex microsphere of the described detection recombination human basic fibroblast growth factor of claim 2 (rhbFGF); Its characteristic also is; Glutathione-S-transferase in the said step 2 (GST) gene fusion expression carrier is pGEX-4T-3, and the fusion expression vector that makes up after accomplishing is pGEX-4T-3-hbFGF.

4. press the preparation method of the immune latex microsphere of the described detection recombination human basic fibroblast growth factor of claim 2 (rhbFGF), its characteristic is that also the affinity chromatographic column in the said step 3 is Glutathione Sepharose 4B.

5. press the preparation method of the immune latex microsphere of the described detection recombination human basic fibroblast growth factor of claim 2 (rhbFGF), its characteristic is that also the affinity chromatographic column in the said step 4 is Benzamidine Sepharose 4B Fast Flow.

6. press the preparation method of the immune latex microsphere of the described detection recombination human basic fibroblast growth factor of claim 2 (rhbFGF); Its characteristic also is; Microballoon in the said step 6 is the monodisperse carboxyl polystyrene microsphere, big or small 50nm, and it is carboxyl that group is carried on the surface.The activation of this microballoon is at first to use acvator EDC (carbodiimide) activation microballoon, adds protective material Sulfo-NHS then and makes microballoon form stable active ester midbody, in order to connecting the usefulness of antibody molecule.