CN107050926A - A kind of coating protein G lemon yellow immune affinity column and preparation method thereof - Google Patents

Abstract

The invention provides a tartrazine immunoaffinity column, comprising a carrier, protein G and tartrazine antibody, wherein the protein G is coupled to the carrier through a chemical bond, and the tartrazine antibody is combined with the protein G with a cross-linking Reagent crosslinking. The invention provides a tartrazine immunoaffinity column, comprising a carrier, protein G and tartrazine antibody, wherein the protein G is coupled to the carrier through a chemical bond, and the tartrazine antibody is combined with the protein G with a cross-linking Reagent crosslinking. For the tartrazine affinity column of the present invention, under the condition of a certain capacity of the immunoaffinity column, the amount of antibody used is less, so that the utilization efficiency of the antibody is higher, and the immunoaffinity column prepared in this way is within a batch of , better batch-to-batch repeatability, and lower column-to-column variance.

Description

A kind of tartrazine immunoaffinity column coated with protein G and preparation method thereof

technical field

The invention belongs to the field of food inspection, and in particular relates to a tartrazine immunoaffinity column coated with protein G and a preparation method thereof.

Background technique

Tartrazine is the most widely used artificial synthetic pigment. It is used in food, medicine, cosmetics, medical equipment, tobacco, feed, edible packaging, toys, etc. It is often necessary to quantitatively detect tartrazine in the above products, because if the human body Ingestion of tartrazine in excess can cause harm. Illegal addition of tartrazine in feed also seriously affects people's health and affects the normal development of animal husbandry.

At present, the detection methods of tartrazine stipulated in national standards are physical and chemical methods such as high performance liquid chromatography, thin-layer analysis, enzyme-linked immunosorbent assay (ELISA), and spectrophotometer. Enzyme-linked immunosorbent assay (ELISA) has the advantages of high efficiency, sensitivity, and direct detection after simple processing after sampling. However, this method also has the disadvantage of high false positive rate. HPLC and LC-MS/MS are the most commonly used methods for the quantitative analysis of tartrazine. When using liquid phase or liquid mass method for detection, in order to reduce the interference of impurities and reduce its impact on the detection effect, it is necessary to purify the samples of various matrices first. At present, the commonly used purification treatment method is solid phase extraction, also known as column chromatography. It is currently the most widely used method of purification. Among them, the immunoaffinity column method has fast analysis speed, high sensitivity, high separation efficiency and recovery rate, and is safe and reliable, so it has become the most commonly used method.

The methods currently used to prepare immunoaffinity purification columns include: cyanogen bromide activation direct coupling method, epichlorohydrin method, sodium periodate method, etc. The basic principle of these methods is to chemically activate the carrier matrix and then couple the antibody to the carrier; Protein G (Protein G) is extracted from the cell wall and culture supernatant of many strains of Streptococcus A, C and G A protein that can specifically bind to IgG Fc fragments, this protein can specifically bind to the Fc sites of various animal antibodies, and has high affinity. Although the use of protein G has high affinity, the use of protein G-treated carriers will reduce the elution speed of the affinity chromatography column, thereby reducing the utilization rate of antibodies and the efficiency of detection.

Contents of the invention

In view of this, the first object of the present invention is to provide a tartrazine immunoaffinity column, including carrier, protein G and tartrazine antibody.

Preferably, in the tartrazine immunoaffinity column of the present invention, the protein G includes protein G expressed according to the sequence of GenBankCAA27638.1, and recombinant protein G through sequence optimization.

Preferably, in the tartrazine immunoaffinity column of the present invention, the protein G is a sequence-optimized recombinant protein G based on GenBank CAA27638.1.

Preferably, in the tartrazine immunoaffinity column of the present invention, the protein G is coupled to the carrier through a chemical bond, and the tartrazine antibody is combined with the protein G and then cross-linked with a cross-linking agent.

Preferably, in the tartrazine immunoaffinity column of the present invention, the sequence-optimized recombinant protein G includes reducing the carrier-recombinant protein G steric hindrance, increasing the interaction between the recombinant protein G and tartrazine antibody IgG. Optimization of binding capacity.

Preferably, in the tartrazine immunoaffinity column of the present invention, the optimization site of the recombinant protein G is to truncate 53 amino acids at the N-terminal of GenBank CAA27638.1 sequence.

Preferably, in the tartrazine immunoaffinity column of the present invention, the carrier is agarose gel; the agarose gel includes cyanogen bromide-activated agarose gel, cross-linked agarose gel gel, polyacrylic acid agarose gel, polyacrylamide agarose gel, dextran gel and cellulose; more preferably, the carrier is Sepharose 4B.

Preferably, in the tartrazine immunoaffinity column of the present invention, the anti-tartrazine antibodies include monoclonal IgG antibodies and polyclonal antibodies.

Another object of the present invention is to provide a preparation method of tartrazine immunoaffinity column, comprising the following steps:

1) Carrier activation;

2) The recombinant protein G is coupled with the activated carrier;

3) processing carrier-recombinant protein G, and coupling with tartrazine antibody;

4) Washing the tartrazine antibody-recombinant protein G-carrier, and packing into a column to obtain a tartrazine immunoaffinity column.

Preferably, in the preparation method of tartrazine immunoaffinity column of the present invention, comprise the following steps:

1) Sepharose 4B, add 0.8M NaOH, 30% epichlorohydrin, 2mg/ml sodium borohydride NaBH ₄ , and shake it at 25°C for 8h to activate;

2) Add 30-200 nmol of recombinant protein G per gram of activated Sepharose 4B, and couple in 0.1M NaHCO ₃ , 0.5M NaCl pH 8.8 buffer for 2 hours at room temperature or overnight at 4°C;

3) The coupling product was reacted with 1M Tris·HCl pH 8.0 at room temperature for 2h;

4) Wash the coupled Sepharose-protein G with 20mM PBS pH7.4;

5) Dissolve tartrazine antibody in 20mM PBS pH7.4, add 200nmol～1.2umol antibody per gram of protein G-Sepharose, and react at room temperature for 30 minutes;

6) Wash the tartrazine antibody-white G-Sepharose carrier with 20mM PBS pH7.4, then add triethanolamine and 20mM dimethylpimelate (DMP) at a final concentration of 0.2M, pH8.3, and react at room temperature for 1h ;

7) Terminate the reaction with 20mM ethanolamine at pH7.4, wash the tartrazine antibody-recombinant protein G-Sepharose carrier with 10mMPBS pH7.4 containing 0.01% thimerosal, pack it into a column, and store it at 4°C;

Wherein, the recombinant protein G is a sequence-optimized recombinant protein G based on GenBank CAA27638.1.

As can be seen from the above, the present invention has at least the following advantages:

Compared with prior art, the present invention has following advantage:

1) We cloned the gene sequence of the recombinant protein G (GenBank CAA27638.1), and expressed the recombinant protein G with high affinity to the antibody in Escherichia coli. Utilizing the characteristic of specific binding between protein G and Ig antibody, IgG antibody is composed of two heavy chains and two light chains, and the antibody is divided into a Fab region and an Fc region, wherein the Fab region is an antigen-binding region. Protein G is a special protein on the cell wall of Streptococcus, which has specific binding ability to the Fc region of the antibody. After protein G binds to the antibody, the Fab region of the antibody is released, which does not affect the antigen-binding ability of the antibody. After our gene optimization and recombination of protein G, one molecule of protein G can bind three molecules of IgG antibody, which has a high antibody affinity. And only the antibody can bind to protein G, and the rest of the proteins cannot bind to protein G, so the specificity is very high. The immunoaffinity column prepared on the basis of this protein G has the characteristics of good specificity, large binding capacity of tartrazine and high purification efficiency.

After coupling the genetically recombined protein G to the agarose carrier, the tartrazine antibody coupling was carried out. Compared with the traditional direct coupling, after the genetically recombined protein G was coupled to the carrier as the middle arm, the The effect of steric hindrance increases the binding ability of recombinant protein G and tartrazine antibody IgG, so that impurities can fully flow out, and the purification effect is enhanced, so that the Fab end of the antibody faces outward, and the steric hindrance does not interfere with the antigen binding region , the carrier is first treated with protein G, and then the antibody is coupled, which can not only improve the uniformity between different purification columns, but also improve the utilization rate of the antibody.

The agarose is pretreated with the recombinant protein G of the present invention. Based on the special affinity of Protein G and IgG, after Protein G binds to the Fc fragment of the antibody, the Fab fragment is stretched out, making it easier to grab the antigen. Directly use antibody coupling to agarose, the binding direction and part of the antibody on the agarose are random, but the antibody on the agarose pre-treated with Protein G has a consistent directionality, and the column capacity of the immunoaffinity column Under certain conditions, the amount of antibody used is less, so that the antibody utilization efficiency is higher, and the immunoaffinity column prepared in this way has better repeatability within batches and between batches, and the difference between columns is lower.

Therefore, by designing recombinant protein G to specifically bind to IgG antibody, the antibody is coupled to the carrier via protein G. Moreover, the Fab fragment of the IgG antibody is fully exposed, which greatly improves the capture ability of tartrazine, and the purification efficiency of tartrazine is also effectively improved.

2) The present invention uses the genetically modified protein G by optimizing codons and IgG binding domain genes. The antibody binding ability of protein G is greatly improved, thereby improving the purification efficiency of tartrazine.

3) The method of purifying tartrazine according to the present invention is easy to operate, and tartrazine with higher purity can be obtained in a few steps, which is more convenient for operators to use.

4) The tartrazine obtained by using the present invention has a high purity, and can be directly used for high-performance liquid chromatography detection without further purification treatment in the follow-up, saving the time and expense of the operator.

Description of drawings

Fig. 1 is the structural representation of tartrazine immunoaffinity column in an embodiment of the present invention;

Fig. 2 is a schematic diagram of further coupling with tartrazine antibody after the agarose carrier of an embodiment of the present invention is coupled with recombinant protein G;

Figure 3 is a schematic diagram of the direct coupling of the agarose carrier with the tartrazine antibody;

Fig. 4 is the meaning diagram of the icons in the schematic diagram of the direct coupling of the agarose carrier and the tartrazine antibody;

Fig. 5 is the schematic diagram of the impact of the initial concentration of tartrazine in the solution on the target substance adsorption capacity of the purification column;

Fig. 6 is a schematic diagram of storage stability of tartrazine immunoaffinity column.

detailed description

In one embodiment of the present invention, tartrazine immunoaffinity column and preparation method thereof are provided:

1. Vector activation

Choose agarose carrier Sepharose 4B, and activate it with epichlorohydrin activation method.

Take 2% pre-swelled agarose gel Sepharose 4B, wash it with 20 times the volume of distilled water, wash away the remaining ethanol, and filter out the water with a funnel.

Weigh 5 g of the wet gel after filtering off the water, add 7.5 ml of 0.8M NaOH, 2 ml of 30% epichlorohydrin, 2 mg/ml of sodium borohydride NaBH ₄ , 5 ml, and shake at 25°C The bed was reacted for 8 hours. After the reaction, wash with a large amount of distilled water to remove the mixed epichlorohydrin in the gel.

2. Coupling of recombinant protein G with activation carrier Sepharose 4B

The activated Sepharose 4B was washed 3 times with coupling buffer (0.1M NaHCO ₃ , 0.8M NaCl, pH 8.9). Add 30-200nmol of protein G per gram of activated Sepharose 4B, and couple at room temperature for 2 hours, or overnight at 4°C. The coupled product was reacted with 1M Tris·HCl pH 8.0 at 30°C under nitrogen protection for 2h.

The coupled agarose carrier was washed 3 times with 20 mM, pH 7.4 phosphate buffer PBS. The agarose carrier Sepharose coupled with recombinant protein G is named recombinant protein G-Sepharose.

Among them, the recombinant protein G is the protein G sequence (GenBank CAA27638.1, SEQ ID NO: 1), and the recombinant protein G sequence (SEQ ID NO2) obtained after truncating the N-terminal 53 amino acids is submitted to Jill Biochemical (Shanghai) Co., Ltd. synthesis.

3. The tartrazine antibody is linked to the recombinant protein G on the recombinant protein G-Sepharose carrier

The recombinant protein G has a specific affinity with the antibody. Under certain reaction conditions, the agarose gel coupled with protein G is connected to the antibody, and the antibody is connected to the agarose. Since the binding site between protein G and the antibody is the Fc region of the antibody, the antigen-binding area of the antibody is not affected, which fully guarantees the antigen-binding ability of the antibody.

Dissolve tartrazine antibody in 20mM PBS with pH7.4, add 150nmol～1.0umol antibody per gram of protein G-Sepharose, and react at room temperature for 30 minutes. The bound carrier was washed with PBS. The carrier linked with the antibody is named Antibody-Protein G-Sepharose.

4. Carrier cross-linking

Antibody-Protein G-Sepharose was washed 3 times with 20 mM PBS pH 7.4. Then triethanolamine and 20 mM dimethylpimelate (DMP) were added at a final concentration of 0.2 M, pH 8.3, and reacted at 30° C. for 1 h under nitrogen protection.

The reaction was terminated by adding 20 mM ethanolamine, pH 9.0, and the tartrazine antibody-protein G-Sepharose carrier was washed with 10 mMPBS pH 7.4 containing 0.01% thimerosal.

5. Column packing

Put the cross-linked tartrazine antibody-protein G-Sepharose into a chromatographic column as needed, and store it at 4°C. Tartrazine affinity purification columns with different capacities for the target tartrazine can be prepared according to actual needs.

The technical solution of the present invention will be further described through specific examples below. It should be understood that the following is only an exemplary description of the present invention, and is not intended to limit the protection scope of the claims of the present invention.

The preparation of embodiment 1 tartrazine immunoaffinity purification column

1. Agarose Gel Activation

Take 2% agarose gel Sepharose 4B and wash it with 20 times the volume of distilled water to wash away the remaining ethanol. Use a funnel to filter out the water. Weigh 5g of the wet gel after filtering off the water, add 7.5mL of 0.8M NaOH, 2mL of 30% epichlorohydrin, 5mL of 2mg/mL sodium borohydride NaBH ₄ , and react in a shaking table at 25°C for 8h . After the reaction, wash with 50 mL of distilled water to remove the mixed epichlorohydrin in the gel.

2. Coupling of Recombinant Protein G to Activated Sepharose

One gram of the activated agarose gel was taken and washed three times with coupling buffer (0.1M NaHCO ₃ , 0.8M NaCl, pH 8.9). Add 20 mL of 2 mg/mL protein G, and couple at room temperature for 4 h.

The coupled agarose carrier was washed 3 times with 20 mM, pH 7.4 phosphate buffer PBS. The agarose carrier Sepharose coupled with recombinant protein G is named recombinant protein G-Sepharose.

3. Anti-tartrazine IgG monoclonal antibody combined with protein G-Sepharose

The anti-tartrazine antibody was dissolved in 20 mM PBS, pH 7.4, with a final concentration of 2 mg/mL and a total volume of 10 mL.

Protein G-Sepharose was washed three times with 20 mM PBS buffer, pH 7.4, 30 mL each time. The tartrazine antibody dissolved in PBS was added to the washed protein G-Sepharose, and combined at room temperature for 30 minutes.

The bound carrier was washed three times with 20 mM PBS pH 7.4, 30 mL each time. The carrier linked with the antibody is named Antibody-Protein G-Sepharose.

4. Sepharose Crosslinking of IgG Conjugates

The antibody-Protein G-Sepharose was washed 3 times with 0.1M pH9.0 borate buffer, 30 mL each time.

Add 20 mL of 0.1 M boric acid buffer solution with pH 9.0 to the wet gel solid, and add the cross-linking agent dimethyl pimelimate (DMP) to the buffer solution to a final concentration of 20 mM. Cross-linking at room temperature for 1 h.

Add 20 mL of 100 mM ethanolamine, pH 9.0, to terminate the reaction. And blocked with 20 mL of 50 mM ethanolamine for 10 minutes.

The cross-linked antibody-protein G-Sepharose carrier was washed 3 times with 20mM, pH7.4 PBS, 30mL each time.

5. Column packing

The cross-linked Sepharose was resuspended in 10 mL of 20 mM PBS pH7.4, and loaded into an empty affinity purification cartridge.

Example 2: Purification and Detection of Tartrazine in Corn Gluten Meal Using Tartrazine Immunoaffinity Column

1. Sample processing

1) Weigh 2.0 corn gluten powder samples into a 50mL centrifuge tube;

2) Add 18 mL of acetonitrile aqueous solution (V/V 50:50), oscillate and mix with a vortexer or a homogenizer, extract by ultrasonic oscillation for 2 minutes, and extract by oscillating at 1500 r/min for 3 minutes;

3) Centrifuge at 4000g for 3min at room temperature, carefully take out 5mL of the supernatant into a new centrifuge tube, blow dry with nitrogen at 50°C (take out immediately after drying);

4) Reconstitute with 10mL PBS (0.01M pH7.4) buffer and load all the samples.

2. Equilibrate the lemon yellow immunoaffinity purification column at room temperature for 30 minutes;

3. Take out the immunoaffinity column, connect the injection port with the syringe barrel, and connect the syringe to the air-controlled operating frame;

4. Wash the affinity column with deionized water for 3 times, 10mL each time, adjust the air pump pressure of the stomata operation frame, so that the liquid flows out at a flow rate of 3 drops/second;

5. Put the sample into the purification column and adjust the flow rate to 1-2 drops/second. until all the sample flows out of the purification column.

6. Wash the purification column 3 times with distilled water, 5 mL each time.

7. Add 1 mL of methanol and collect the eluted product.

8. The eluted product was detected by high performance liquid chromatography (HPLC).

1), the preparation of tartrazine standard substance

1.1) Take tartrazine standard substance, dissolve with 0.01M PBS buffer solution of pH7.4, and dilute to 3ppb;

1.2) Take 10 mL of the diluted tartrazine standard substance, and load all samples for detection;

2), equilibrate the lemon yellow immunoaffinity purification column at room temperature for 30 minutes;

3) Take out the immunoaffinity column, connect the injection port to the syringe barrel, and connect the syringe to the air-controlled operating rack;

4) Wash the affinity column with deionized water for 3 times, 10 mL each time, adjust the air pump pressure of the stomata operating frame, so that the liquid flows out at a flow rate of 3 drops/second;

5) Add the sample into the purification column and adjust the flow rate to 1-2 drops/second. Until all the samples flow out of the purification column;

6) Wash the purification column 3 times with distilled water, 5 mL each time;

7), add 1mL methanol, collect the eluted product;

8), the eluted product is detected by high performance liquid chromatography (HPLC).

Liquid phase conditions:

Chromatographic column: Kromasil C18 250mm×4.6mm 5μm

Mobile phase A: Methanol; Mobile phase B: 5mmol/L ammonium acetate

Mobile Phase Gradient Program

Column temperature: 35°C; injection volume: 10μL; detection wavelength: 254nm; flow rate: 1.0mL/min.

Example 3: Determination of Tartrazine Immunoaffinity Column Capacity Comparison of Purification Columns Prepared by Two Methods

Preparation of purification column first treated with protein G

Prepare according to Example 2

Preparation of cleanup column without protein G treatment

Preparing Affinity Columns by Direct Coupling

1. Carrier activation

Select the agarose carrier, Sepharose 4B, and activate it with epichlorohydrin activation method.

Take 2% pre-swelled agarose gel Sepharose 4B, wash it with 20 times the volume of distilled water, wash away the remaining ethanol, and filter out the water with a funnel.

Weigh 5 g of the wet gel after filtering out water, add 7.5 mL of 0.8 M NaOH, 2 mL of 30% epichlorohydrin, 5 mL of 2 mg/mL sodium borohydride NaBH ₄ , and react on a shaking table at 25° C. for 8 h.

After the reaction, wash with a large amount of distilled water to remove the mixed epichlorohydrin in the gel.

2. Anti-tartrazine antibody linked to Sepharose carrier

Under certain reaction conditions, the agarose gel is linked to the antibody, and the antibody is linked to the agarose.

The tartrazine antibody was dissolved in 20mM PBS pH7.4, 200nmol-1.2μmol antibody was added per gram of Sepharose, and reacted at room temperature for 30min.

The bound carrier was washed with PBS. The carrier linked with the antibody is named Antibody-Sepharose.

3. Carrier cross-linking

Antibody-Sepharose was washed 3 times with 20 mM PBS pH 7.4. Then triethanolamine and 20 mM dimethylpimelate (DMP) were added at a final concentration of 0.2 M, pH 8.3, and reacted at room temperature for 1 hour.

The reaction was stopped by adding 20 mM ethanolamine, pH 9.0, and the tartrazine antibody-Sepharose carrier was washed with 10 mM PBS pH 7.4 containing 0.01% thimerosal.

4. Column packing

Put the cross-linked antibody-agarose carrier into a chromatography column as needed, and store it at 4°C. Tartrazine affinity purification columns with different capacities can be prepared as needed.

Column capacity

Load 2 mL of tartrazine PBS solution with a concentration of 100 ng/mL, then wash the column with 5 mL of eluent 100% methanol to elute the target substance, detect by HPLC, and calculate the column capacity according to the following formula.

Take the purification column filled as shown in Figure 1, pass 1mL of tartrazine target substance PBS solution with a certain concentration through the column at a flow rate of 1mL/min, and wash the column with 10mL ultrapure water to wash away the impurities non-specifically adsorbed on the column. Elute with 1 mL of methanol, perform quantitative detection on HPLC, and measure the "capture" capacity of the purification column for the target object.

Figure 2 shows a schematic diagram of the further coupling of the agarose carrier with the tartrazine antibody after being coupled with recombinant protein G, and Figure 3 shows a schematic diagram of the direct coupling of the agarose carrier with the tartrazine antibody, wherein Figure 4 shows the schematic diagram 2. Each diagram in Figure 3 represents agarose, tartrazine antibody and recombinant protein G respectively.

During the purification process, when the volume of the column is constant, as the concentration of the target substance in the solution to be purified increases, the adsorption amount of the "captured" target substance in the purification column will increase, but the adsorption amount will not change. As the concentration of the target increases infinitely, the antibody in the purification column has a certain adsorption capacity—saturation adsorption capacity. In this experiment, the relationship between the amount of antibody adsorbed on the target substance in the purification column and the initial concentration of the target substance in the solution was first investigated. Figure 5 is a curve showing the effect of the initial concentration of the target on the amount of antibody adsorption in the purification column. It can be seen from Fig. 5 that the adsorption amount of the antibody to the target substance in the purification column generally increases with the increase of the concentration of the target substance in the liquid to be purified. However, this curve can also be divided into two regions. Region 1: When the initial concentration of the target substance in the liquid to be purified is lower than 180ng/mL, the adsorption capacity increases rapidly with the increase of the initial concentration of the target substance in the liquid to be purified; Region 2 : When the initial concentration of the adsorbed substance is higher than 180ng/mL, with the increase of the initial concentration of the target substance in the liquid to be purified, the adsorption amount of the target substance by the antibody in the purification column is basically unchanged. Due to the conformation of the protein pre-treated by Protein G, the antibody can effectively bind to the target antigen, resulting in the adsorption amount of the target antigen by the adsorption carrier treated by Protein G under the same amount of purified carrier ( The adsorption capacity is about 260ng/mL) higher than that of the adsorption carrier without Protein G treatment (the adsorption capacity is about 180ng/mL).

Embodiment 4 Tartrazine immunoaffinity column stability

Store the prepared immunoaffinity columns in a refrigerator at 4°C, select different time points such as 0, 20, 40, 60, and 90 days, and equilibrate the immunoaffinity columns and various buffers to room temperature. The operation method is to carry out an addition recovery experiment on it, detect the tartrazine content in the eluent by HPLC, repeat the operation 5 times, and evaluate the stability of the affinity column by calculating the average recovery rate. The results are shown in Figure 6. As the storage time increases, the purification performance of the purification column decreases to a certain extent, from 96% to 80%, which basically meets the requirements of the test.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> Institute of Feed, Chinese Academy of Agricultural Sciences

<120> A tartrazine immunoaffinity column coated with protein G and its preparation method

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 480

<212> PRT

<213> Protein G

<400> 1

Glu Phe Asn Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn Leu Ile Asn

1 5 10 15

Asn Ala Lys Thr Val Glu Gly Val Lys Asp Leu Gln Ala Gln Val Val

20 25 30

Glu Ser Ala Lys Lys Ala Arg Ile Ser Glu Ala Thr Asp Gly Leu Ser

35 40 45

Asp Phe Leu Lys Ser Gln Thr Pro Ala Glu Asp Thr Val Lys Ser Ile

50 55 60

Glu Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr

65 70 75 80

Gly Val Ser Asp Tyr His Lys Asn Leu Ile Asn Asn Ala Lys Thr Val

85 90 95

Glu Gly Val Lys Asp Leu Gln Ala Gln Val Val Glu Ser Ala Lys Lys

100 105 110

Ala Arg Ile Ser Glu Ala Thr Asp Gly Leu Ser Asp Phe Leu Lys Ser

115 120 125

Gln Thr Pro Ala Glu Asp Thr Val Lys Ser Ile Glu Leu Ala Glu Ala

130 135 140

Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr

145 150 155 160

Tyr Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala

165 170 175

Leu Ile Asp Glu Ile Leu Ala Ala Leu Pro Lys Thr Asp Thr Tyr Lys

180 185 190

Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Thr Glu Ala

195 200 205

Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn Asp

210 215 220

Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe

225 230 235 240

Thr Val Thr Glu Lys Pro Glu Val Ile Asp Ala Ser Glu Leu Thr Pro

245 250 255

Ala Val Thr Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly

260 265 270

Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe

275 280 285

Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp

290 295 300

Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Pro Glu Val Ile Asp

305 310 315 320

Ala Ser Glu Leu Thr Pro Ala Val Thr Thr Tyr Lys Leu Val Ile Asn

325 330 335

Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Lys Ala Val Asp Ala Glu

340 345 350

Thr Ala Glu Lys Ala Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp

355 360 365

Gly Val Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu

370 375 380

Met Val Thr Glu Pro Val Gly Asp Ala Pro Thr Glu Pro Glu Lys Pro

385 390 395 400

Glu Ala Ser Ile Pro Leu Val Pro Leu Thr Pro Ala Thr Pro Ile Ala

405 410 415

Lys Asp Asp Ala Lys Lys Asp Asp Thr Lys Lys Glu Asp Ala Lys Lys

420 425 430

Pro Glu Ala Lys Lys Glu Asp Ala Lys Lys Ala Glu Thr Leu Pro Thr

435 440 445

Thr Gly Glu Gly Ser Asn Pro Phe Phe Thr Ala Ala Ala Leu Ala Val

450 455 460

Met Ala Gly Ala Gly Ala Leu Ala Val Ala Ser Lys Arg Lys Glu Asp

465 470 475 480

<210> 2

<211> 427

<212> PRT

<213> recombinant protein G

<400> 2

Gln Thr Pro Ala Glu Asp Thr Val Lys Ser Ile Glu Leu Ala Glu Ala

1 5 10 15

Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr

20 25 30

His Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Asp

35 40 45

Leu Gln Ala Gln Val Val Glu Ser Ala Lys Lys Ala Arg Ile Ser Glu

50 55 60

Ala Thr Asp Gly Leu Ser Asp Phe Leu Lys Ser Gln Thr Pro Ala Glu

65 70 75 80

Asp Thr Val Lys Ser Ile Glu Leu Ala Glu Ala Lys Val Leu Ala Asn

85 90 95

Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn Leu Ile

100 105 110

Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile Asp Glu Ile

115 120 125

Leu Ala Ala Leu Pro Lys Thr Asp Thr Tyr Lys Leu Ile Leu Asn Gly

130 135 140

Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr

145 150 155 160

Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly

165 170 175

Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Lys

180 185 190

Pro Glu Val Ile Asp Ala Ser Glu Leu Thr Pro Ala Val Thr Thr Tyr

195 200 205

Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu

210 215 220

Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn

225 230 235 240

Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr

245 250 255

Phe Thr Val Thr Glu Lys Pro Glu Val Ile Asp Ala Ser Glu Leu Thr

260 265 270

Pro Ala Val Thr Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys

275 280 285

Gly Glu Thr Thr Thr Lys Ala Val Asp Ala Glu Thr Ala Glu Lys Ala

290 295 300

Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Val Trp Thr Tyr

305 310 315 320

Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Met Val Thr Glu Val

325 330 335

Pro Gly Asp Ala Pro Thr Glu Pro Glu Lys Pro Glu Ala Ser Ile Pro

340 345 350

Leu Val Pro Leu Thr Pro Ala Thr Pro Ile Ala Lys Asp Asp Ala Lys

355 360 365

Lys Asp Asp Thr Lys Lys Glu Asp Ala Lys Lys Pro Glu Ala Lys Lys

370 375 380

Glu Asp Ala Lys Lys Ala Glu Thr Leu Pro Thr Thr Gly Glu Gly Ser

385 390 395 400

Asn Pro Phe Phe Thr Ala Ala Ala Leu Ala Val Met Ala Gly Ala Gly

405 410 415

Ala Leu Ala Val Ala Ser Lys Arg Lys Glu Asp

420 425

Claims (10)

1. a kind of lemon yellow immune affinity column, including carrier, Protein G and lemon yellow antibody.

2. lemon yellow immune affinity column according to claim 1, it is characterised in that the Protein G is included according to GenBank Protein G shown in CAA27638.1 sequences, and the recombinant protein G Jing Guo sequence optimisation.

3. lemon yellow immune affinity column according to claim 2, it is characterised in that the Protein G is based on GenBank Recombinant protein Gs of the CAA27638.1 Jing Guo sequence optimisation.

4. the lemon yellow immune affinity column according to claims 1 to 3, it is characterised in that the Protein G passes through chemical bond It is coupled on carrier, lemon yellow antibody uses cross-linking agents after being combined with Protein G.

5. the lemon yellow immune affinity column according to claim 3, it is characterised in that the restructuring egg of described sequence optimisation White G, including reduced carrier-recombinant protein G steric hindrances effect, and add recombinant protein G and lemon yellow IgG antibody The optimization of binding ability.

6. the lemon yellow immune affinity column according to claim 3, it is characterised in that the recombinant protein G optimizes site For amputation GenBank CAA27638.1 sequences 53 amino acid of N-terminal.

7. the lemon yellow immune affinity column according to claim 1, it is characterised in that described carrier is that agarose coagulates Glue；Described Ago-Gel includes the Ago-Gel, the Ago-Gel of crosslinking, polyacrylic acid agarose of cyanogen bromide-activated Gel, polyacrylamide Ago-Gel, the one or more of sephadex and cellulose.

8. the lemon yellow immune affinity column according to claim 1, it is characterised in that the lemon yellow antibody includes Dan Ke Grand IgG antibody and polyclonal antibody.

9. a kind of preparation method of lemon yellow immune affinity column, it is characterised in that comprise the following steps：

1) it is support-activated；

2) recombinant protein G is coupled with the carrier activated；

3) handle carrier-recombinant protein G, and with lemon yellow antibody coupling；

4) washing lemon yellow antibody-recombinant protein G- carriers, dress post is to obtain lemon yellow immune affinity column.

10. preparation method according to claim 9, it is characterised in that comprise the following steps：1) agarose Sepharose 4B, adds 0.8M NaOH, 30% epoxychloropropane, 2mg/ml sodium borohydride NaBH₄, shaking table reaction 8h enters at 25 DEG C Row activation；

2) the Sepharose 4B of every gram of activation add 30~200nmol recombinant protein G, in 0.1MNaHCO₃, 0.5M NaCl In pH8.8 buffer solution, room temperature coupling 2h, or 4 DEG C of couplings are stayed overnight；

3) coupled product reacts at room temperature 2h with 1M TrisHCl pH 8.0；

4) the Sepharose- Protein Gs being coupled, are washed with 20mM pH7.4 PBS；

5) lemon yellow antibody is dissolved in 20mM pH7.4 PBS, every gram of Protein G-Sepharose add 200nmol~ 1.2umol antibody, is reacted at room temperature 30 minutes；

6) lemon yellow antibody-white G-Sepharose carriers are washed with 20mM PBS pH7.4, then adds final concentration 0.2M's The dimethyl pimelate (DMP) of triethanolamine and 20mM, pH8.3 reacts at room temperature 1h；

7) with 20mM pH7.4 monoethanolamine terminating reaction, lemon yellow is washed with the 10mMPBSpH7.4 containing 0.01% thimerosal Antibody-recombinant protein G-Sepharose carriers, fills post, is put in 4 DEG C of preservations；

Wherein, the recombinant protein G is the recombinant protein G based on GenBank CAA27638.1 Jing Guo sequence optimisation.