CN103044549A - Preparation method of apis cerana antimicrobial peptide AccRoyalisin polyclonal antibody and use thereof - Google Patents

Abstract

The invention discloses a preparation method and application of a polyclonal antibody of the antibacterial peptide AccRoyalisin of Apis mellifera. The antimicrobial peptide AccRoyalisin of Chinese bee royal jelly recombinantly expressed in Escherichia coli was used as the antigen to immunize the polyclonal antibody prepared by white rabbits, and the antibody titer was more than 40000 times; the polyclonal antibody was used as the primary antibody, and the recombinantly expressed GST-AccRoyalisin was used as the standard The protein antigen was serially diluted, and the absorbance of different concentrations of GST-AccRoyalisin protein samples was detected by Elisa method with a microplate reader, and a standard curve was established between the absorbance and MRJP1 protein concentration. The hemolymph and royal jelly freeze-dried powder of different bee species or different bee colonies were diluted as antigens, and the absorbance of the samples was detected by Elisa method, which was substituted into the established standard curve to calculate the content of Royalisin in the samples. The present invention provides a new application for the Royalisin antibody, and provides a fast qualitative method for the evaluation of the disease resistance of bees and bee colonies in the field of beekeeping, the selection of disease-resistant bee colonies, and the evaluation of the antiseptic and antibacterial properties of royal jelly A new method for quantitative detection.

Description

Preparation method and application of a polyclonal antibody against antimicrobial peptide AccRoyalisin of Apis mellifera

technical field

The invention relates to the fields of genetic engineering and immunology, in particular, the invention relates to a polyclonal antibody prepared by using the antimicrobial peptide AccRoyalisin gene fusion expression product GST-AccRoyalisin of Apis cerana cerana royal jelly. More specifically, the present invention relates to the preparation method of AccRoyalisin antibody, and using the antibody Elisa method to detect the Royalisin content in the hemolymph of Apis mellifera and Apis mellifera, as the basis for judging the disease resistance of bee species or bee colonies; and using the antibody Elisa The content of Royalisin in royal jelly was detected by this method, which was used as the basis for evaluating the antiseptic performance of royal jelly.

Background technique

Royal jelly is a biologically active substance secreted by the royal jelly glands on the head of bees. It has many functions such as promoting human growth and development, anti-fatigue, improving immunity, anti-tumor, anti-inflammatory and antibacterial. It has a long history as a nutritional health product for human beings. Royalisin is an antimicrobial peptide in royal jelly. It is a low-molecular-weight peptide produced by the head or chest of bees when they are infected by microorganisms or other exogenous harmful substances. Japanese scholar Fujiwara et al. (Fujiwara, S. et al. J. Biol. Chem., 1990, 265: 11333-11337) initially obtained Royalisin from royal jelly by acid treatment. Its molecular weight is 5523 Da, and its primary The structure consists of 51 amino acid residues, which are almost identical to the sequence of defensin in bee hemolymph, only the 50th amino acid residue is changed, and the arginine in defensin replaces the tyrosine in royalisin in royal jelly. Royalisin has no specificity, and the induced product not only has antimicrobial effects on specific microorganisms, but also has antibacterial effects on other microorganisms. Bilikova and Bachanová et al. (Bilikova, K., et al. Apidologie, 2001, 32 : 275-283; Bachanová, K., et al. Apidologie, 2002, 33 : 259-269.) on Royalisin isolated from natural royal jelly Corresponding antibacterial tests were carried out, and it was confirmed that the polypeptide had a strong antibacterial effect on the Gram-positive bacterium Paenibacillus larvae larvae, and it also had an antibacterial effect at a concentration as low as 1 μM; Gram-positive bacteria Bacillus subtilis (Bucillus. subtilis) and lutea Sarcina lutea (Sarcina lutea), including Botrytis cinerea also have a good antibacterial effect, but have no effect on Gram-negative bacteria.

Among the 9 members of the genus Apis reported in the world, only Apis mellifera (A. mellifera mellifera, the most widely distributed Italian bee) and A. Artificial domestication and large-scale artificial breeding, and used for honey production.Compared with Western honeybees, Oriental honeybee ( A.cerana ), including China's unique subspecies Chinese honeybee ( A.cerana cerana, referred to as the honeybee) The number of bee colonies is shrinking in the place of origin. However, domestic and foreign studies have shown that because Chinese honeybees have long-term adaptation to China's geographical and climate environment, they have disease resistance, cold resistance and other stress resistance properties that Western honeybees do not have. The biological characteristics are also very different from the former, and it has very important value in bee breeding and ecological diversity protection. Because antimicrobial peptides play an irreplaceable role in the bee's own defense system. Bee mites and the outbreaks in recent years Bee collapse disease is very harmful to Western honeybees, and the prevention and control of bee diseases has been highly valued by various countries. Many bee experts have carried out a lot of research on the selection of disease-resistant varieties as the most important control method. In recent years, domestic scholars (Xu, P., et al . PLoS ONE. 2009, 4: 1-9.) Studies have shown that the disease resistance of oriental honeybees ( Apis cerana ), including Apis cerana subspecies, is better than that of western bees including Italian honeybees, rather than secreting antibacterial Strong peptide ability and many types are closely related.

At present, the antimicrobial peptides discovered in the world are divided into four categories: (a) cecropins, (b) defensins or sapecins, (c) attacin-like proteins, (d) proline-rich peptides. Defensins are the most numerous class of insect antimicrobial peptides, which include three subclasses: classic defensins, β-defensins and insect defensins. Royalisin belongs to the subclass of insect defensins. The insect defensins subclass generally consists of 36-51 amino acid residues, three pairs of disulfide bonds, a stable structure composed of an α-sheet, two β-sheets and an N-terminal loop, all of which have anti-gram-positive bacteria Activity (Cho, W. L., et al Insect Biochem. Molec. Biol. 1996, 26: 395-402).

The amino acid composition of Royalisin between Zhongfeng and Italian bee has more than 95% homology. At present, Royalisin, in addition to reports on the Royalisin gene sequence and amino acid sequence of Apis mellifera, and the antibacterial test of Royalisin isolated from natural royal jelly, there is no use of Royalisin antibodies to detect Royalisin in bee species and bee colony hemolymph at home and abroad. content, and Royalisin content in royal jelly, as the basis for evaluating the disease resistance of bee species and bee colonies, and the antiseptic performance of royal jelly.

Contents of the invention

The purpose of the present invention is to address the deficiencies of the prior art, and provide a method and application for preparing a polyclonal antibody from a recombinant expression product of the antibacterial peptide AccRoyalisin gene of Apis mellifera royal jelly.

The purpose of the present invention is achieved through the following technical solutions:

A preparation method of royal jelly antibacterial peptide AccRoyalisin antibody, the steps are as follows:

(1) Using the bee head cDNA containing the AccRoyalisin gene as a template, its precursor fragment was amplified by PCR, and cloned into the expression vector pGEX-4T-2 containing the GST-encoded gene. The recombinant pGEX-4T-2/AccRoyalisin obtained by screening was transformed into E. coli BL21 and expressed by fusion with glutathione; the target protein GST-AccRoyalisin was purified by affinity separation method;

(2) Using purified GST-AccRoyalisin as an antigen, white rabbits were immunized, and the serum was collected to obtain GST-AccRoyalisin polyclonal antibody.

The GST-AccRoyalisin polyclonal antibody prepared by the method.

The described GST-AccRoyalisin polyclonal antibody detects the Elisa method of antimicrobial peptide Royalisin content in bee hemolymph,

(1) Use the GST-AccRoyalisin polyclonal antibody as the primary antibody, and use the recombinant expressed GST-AccRoyalisin as the standard antigen protein for doubling dilution, and use a microplate reader to detect the absorbance of different concentrations of GST-AccRoyalisin protein samples by the Elisa method, and establish the absorbance Standard curve with AccRoyalisin protein concentration;

(2) Using the hemolymph of Apis cerana and Apis mellifera as antigens and GST-AccRoyalisin polyclonal antibody as the primary antibody, the absorbance of the obtained samples was detected by Elisa method, and substituted into the established standard curve to calculate the content of Royalisin in the samples.

The described Elisa method was applied to determine the difference in the anti-bacteria ability of Apis mellifera and Apis mellifera.

The GST-AccRoyalisin polyclonal antibody is used to detect the Elisa method of antimicrobial peptide Royalisin content in royal jelly,

(1) Using the GST-AccRoyalisin polyclonal antibody as the primary antibody and the recombinantly expressed GST-AccRoyalisin as the standard antigen protein for doubling dilution, use a microplate reader to detect the absorbance of different concentrations of GST-AccRoyalisin protein samples by Elisa method, and establish Standard curve of absorbance and AccRoyalisin protein concentration;

(2) The protein extract of royal jelly freeze-dried powder was used as the antigen, the GST-AccRoyalisin polyclonal antibody was used as the primary antibody, and the absorbance of the sample was detected by Elisa method, which was substituted into the established standard curve to calculate the content of Royalisin in the sample.

The described Elisa method is applied to the determination of the antiseptic properties of royal jelly.

The beneficial effects of the present invention are:

(1) In the present invention, for the first time, a polyclonal antibody with higher titer was prepared by gene recombination and fusion expression product of antibacterial peptide AccRoyalisin of Apis mellifera royal jelly.

(2) The establishment of Royalisin antimicrobial peptides as a new indicator to identify the authenticity and quality of royal jelly can fill in the deficiency of the current national standard for royal jelly. Because the current national standard for royal jelly (GB 9697-2008) uses royal jelly acid 10-HDA as the unique active ingredient of royal jelly, it is also a key indicator for measuring the quality and authenticity of royal jelly in international trade. However, Antinelli et al. (Antinelli, et al. 2003, Food Chem, 80: 85-89) showed that the content of 10-HDA was only reduced by 0.1% and 0.2% and 0.4%, very stable, only a small amount of degradation even at high temperatures. Therefore, it is not suitable as an indicator of the freshness and quality of royal jelly.

(3) The present invention provides a simple and easy, high-accuracy Elisa rapid determination method for the determination of Royalisin content in royal jelly. As a quantitative analysis method of antigen and antibody, the Elisa method can greatly amplify the reaction effect due to the high catalytic efficiency of the enzyme, so that the determination method can achieve high sensitivity. At the same time, the method is simple, convenient, rapid and low in cost, and it is very feasible to use this method to detect the quality of royal jelly.

Description of drawings

Figure 1 is the SDS-PAGE electrophoresis of GST-AccRoyalisin, the fusion expression product of AccRoyalisin gene in Escherichia coli. Among them, lanes 1-3 are the supernatant of the product induced by the positive clone (soluble protein); lane M is the protein standard; lanes 7-9 are the precipitate of the product induced by the positive clone (inclusion body protein); lane 10: contains the empty expression vector The bacterial solution induces product precipitation (inclusion body protein). GST-AccRoyalisin protein is marked with an arrow.

Figure 2 is the SDS-PAGE electrophoresis of the recombinant fusion expression product GST-AccRoyalisin through affinity separation. Wherein, lane M is the protein standard, lane M is the protein standard, lanes 3-7 are the elution peaks obtained from purification, and lane 9 is the elution peak.

Figure 3 shows the absorbance at 450 nm measured by Elisa method using recombinant GST-AccRoyalisin antibody as primary antibody and purified recombinant GST-AccRoyalisin as standard antigen after serial dilution. Taking the absorbance value OD ₄₅₀ as the ordinate, and the concentration of GST-AccRoyalisin standard solution as the abscissa, the standard curve drawn is: y=0.017x+0.067, R ² =0.998.

Figure 4 is a comparison of the results of determining the Royalisin content in the hemolymph of the two bees using the GST-AccRoyalisin antibody as the primary antibody and using the hemolymph of the Chinese bee and the Italian worker bee as antigens by using the Elisa method. The vertical axis is the Royalisin content per μl of bee hemolymph, and the horizontal axis is the time (hours) after the bees were inoculated with E. coli.

Detailed ways

The invention discloses that the antibacterial peptide AccRoyalisin of royal jelly of Chinese bee ( Apis cerana cerana ) recombinantly expressed by Escherichia coli is used as an antigen to immunize white rabbits to prepare a polyclonal antibody, and then the polyclonal antibody is used as a primary antibody, and the recombinantly expressed GST-AccRoyalisin is used as a standard The protein was diluted as the antigen, and the absorbance of different concentrations of GST-AccRoyalisin protein samples was detected by Elisa method with a microplate reader, and a standard curve between absorbance and GST-AccRoyalisin protein concentration was established. The hemolymph and royal jelly freeze-dried powder of different bee species or bee colonies were diluted and used as antigens, and the absorbance of samples was detected by Elisa method, which was substituted into the established standard curve to calculate the content of Royalisin in the samples.

1. The preparation method, steps and usage of royal jelly antibacterial peptide AccRoyalisin antibody are as follows:

(1) Using the bee head cDNA library plasmid containing the AccRoyalisin gene (GenBank accession number: EF660337) as a template, its precursor fragment was amplified by PCR and cloned into the expression vector pGEX-4T- In 2, the recombinant pGEX-4T-2/ Accroyalisin obtained by screening was transformed into Escherichia coli BL21 and fused with glutathione (GST) for expression. Both royal jelly antimicrobial peptide AccRoyalisin and AccRoyalisin precursor and Apis mellifera antimicrobial peptide AmRoyalisin precursor are composed of 95 amino acid residues, with a homology of 90%-92%; both mature peptides are composed of 51 amino acid residues , only 3-4 amino acid residues are different, and the homology is above 90%.

The recombinant bacteria were cultured and induced to express. The total bacterial protein was electrophoresed by SDS-PAGE. Compared with the BL21 bacteria containing pGEX-4T-2 empty vector, the pGEX-4T-2/ Acc-royalisin engineering bacteria had a specific band of about 36 ku in size (Figure 1 ). The theoretical molecular mass of Acc-royalisin is 5.52 kDa, plus the GST protein with a mass of 26 kDa fused with it expressed in pGEX-4T-2 vector, the expected mass of GST-A ccRoyalisin fusion protein is 31.2 kDa, therefore, the specific The molecular weight of the target protein was consistent with that of the expected protein. The recombinant expression product was further subjected to ultrasonic crushing and centrifugation, and the obtained supernatant and precipitate were analyzed by SDS-PAGE electrophoresis. There are two forms of inclusion body protein and inclusion body protein, among which the soluble protein which has biological activity and can be directly separated by affinity purification method accounts for the majority. Transfer the GST-AccRoyalisin fusion protein on the SDS-PAGE electrophoresis gel to the nitrocellulose membrane, and use GST antibody for Western blot analysis, showing specific blot.

(2) Inoculate the screened AccRoyalisin-containing recombinant bacteria into LB liquid medium, and induce expression with IPTG at 37°C for 12 hours. Transfer 500 mL of bacterial liquid to a centrifuge tube, centrifuge for 10 min (5000 rpm, 4 °C), remove the supernatant, and place the pellet on ice. Use 50 μL of ice-bathed PBS for every 1 mL of the original culture solution to completely suspend the pellet. Cells were briefly disrupted by sonication on ice until the sample was not viscous. The sonicated bacterial solution was centrifuged at 10,000 rpm, 4°C for 10 min. Carefully transfer the supernatant to a clean, ice-cooled vial. The cell pellet was then completely suspended in ice-bathed PBS (50 μL ice-bathed PBS was used for every 1 mL of original culture medium).

Separation of target protein by affinity chromatography prepacked column with GST antibody connected to AKTA explorer 100. The absorption peak of UVA at ₂₈₀ nm was used as a detection tool for protein. Samples must be centrifuged or 0.45 μm filter before passing through the column. Samples are too viscous and can be diluted with Binding Buffer.

Water and buffer solution must use high-purity water during operation, and buffer solution must pass through a 0.45 μm filter, and then ultrasonically degassed. Inject the binding buffer into the syringe or pump tubing, and connect the column to the syringe to prevent air from entering the column. Remove the small plug at the outlet at the bottom of the column. Equilibrate the column with 5 bed volumes of binding buffer. Inject with a syringe or a pump, the recommended injection flow rate: 0.2-1 mL/min. Wash the column with 5-10 times column volume binding buffer or until there is no UV absorption peak in the effluent, and the flow rate is 1-2 mL/min. Elute with 5-10 column volumes of elution buffer at a flow rate of 1-2 mL/min. When the elution peak appears, collect the eluate.

Take the elution peak of affinity separation and analyze it by SDS-PAGE electrophoresis. The result (see Figure 2) shows that the elution peak obtained from this purification system is the molecular weight A single band protein with a size of about 32 kDa is consistent with the size of the target protein, indicating that a single target protein was obtained after purification. The elution peak contains multiple protein bands, belonging to mixed proteins, which is consistent with the peak shape diagram. There are also target protein bands in the elution peak, indicating that part of the target protein fails to bind to the column in time and directly flows out in the form of effluent.

(3) Take the GST-AccRoyalisin purified and eluted by AKTA column, put it into a dialysis bag, dialyze overnight, transfer the dialysis desalted solution into a 1.5 mL eppendorf tube, freeze-dry, dissolve the purified GST-AccRoyalisin with 0.9% normal saline protein as an antigen.

2. Preparation of polyclonal antibody GST-AccRoyalisin

Two healthy male rabbits aged 14-16 weeks were selected, and each rabbit was injected with about 0.5 mL of purified fusion protein antigen by subcutaneous injection. Before immunization, blood samples were drawn from rabbit ears as negative controls. The fusion protein antigen was dissolved in 700 μL of normal saline, emulsified with 700 μL of Freund’s complete adjuvant and appropriate amount of penicillin and streptomycin, and injected subcutaneously at multiple points for immunization. Three weeks later, a booster immunization was performed with the same antigen, adjuvant and penicillin emulsion. After 5 weeks, the fusion protein antigen was dissolved in 1 mL of normal saline for intramuscular injection; after 6 weeks, the immunization was boosted again with 1 mL of normal saline containing the fusion protein antigen. At the same time, blood samples were drawn from the rabbit ears, and the titer of multiple antiserum was determined by indirect ELISA; after 7 weeks, the blood was collected from the carotid artery and allowed to coagulate at room temperature. Put the coagulated blood in an incubator at 37°C for half an hour, then put it in a refrigerator at 4°C overnight to shrink the blood clot and precipitate the antiserum; suck out the antiserum, centrifuge at 3,000 rpm for 10 min, aspirate the supernatant and store at -80°C spare.

3. Determination of Royalisin content in bee worker bee hemolymph by Elisa method

(3.1) Coating: use the recombinant GST-AccRoyalisin described in claim 1 as the antigen, and use carbonate buffer (CBS, Na ₂ CO ₃ 1.59g, NaHCO ₃ 2.93g, ddH ₂ O 950 ml, adjust the pH to 9.6, add ddH ₂ O to 1000 ml, store at 4°C) add 10 μg/ml AccRoyalisin standard protein solution to a 96-well plate by doubling gradient dilution method, add 100 μl/well to each well, 4 ℃ coated overnight;

(3.2) Blocking: Discard the coating solution in the wells of the enzyme-linked plate the next day, use PBST (PBS+0.05% Tween-20) as the washing solution, wash 5-10 times in each step, and pat dry. Then add 100 μL of PBST solution containing 5% BSA or skimmed milk powder to block at 37 °C for 1.5 h;

(3.3) Discard the blocking solution in the wells of the enzyme-linked plate and wash with PBST. Add primary antibody, use phosphate buffer solution (PBS, KH ₂ PO ₄ 0.27g _, Na ₂ HPO ₄ 1.42g, NaCl 8g, KCl 0.2g, ddH ₂ O 800mL, adjust pH to 7.4 with concentrated hydrochloric acid, volume to 1L), dilute 1000 times, 100uL/well, stand at 37°C for 1-2 h;

(3.4) Washing: Rinse 10 times with tap water and pat dry;

(3.5) Discard the primary antibody in the small well of the enzyme-linked plate, and wash it with PBST. Use HRP-labeled goat anti-rabbit IgG as the secondary antibody, dilute 5000 times, 100 μl/well, and stand at 37°C for 1 hour;

(3.6) Washing: Rinse 10 times with tap water and pat dry;

(3.7) solution, 50 μl/well, measure the absorbance value at a wavelength of 450 nm on a microplate reader immediately after adding (450 nm is the maximum absorption wavelength of the chromogen);

(3.8) Table 1 was obtained by antibody detection. Taking the average OD ₄₅₀ absorbance value (n=3) as the ordinate, and the concentration of GST-AccRoyalisin standard protein solution as the abscissa, the standard curve (Figure 3) was obtained: y = 0.017x + 0.067, R ² =0.998.

Table 1 The absorbance obtained by measuring the gradient dilution standard polypeptide with GST-AccRoyalisin antibody

(3.9) Collection of bees and infection treatment

Randomly pick a beehive of Apis chinensis and Apis mellifera from the apiary, respectively comb, and randomly take about 100 worker bees with jars. Divided into three groups:

不处理组：各取中蜂、意蜂工蜂30只。

No treatment group: 30 worker bees from Zhongfeng and Italian bees were taken respectively.

② Take 60 worker bees of Chinese bees and Italian bees respectively. After being anesthetized with CO ₂ , each worker bee is injected with 2 ul of Escherichia coli at 50 cfu/g in the abdomen, and then placed in insect cages and fed with 10% honey water. Recapture after 24 hours of isolation.

各取中蜂、意蜂工蜂60只，用CO₂麻醉后，每只工蜂在腹部注射50 cfu/mL 的大肠杆菌2 ul染菌，然后置养虫笼内，用10%的蜂蜜水饲养，隔离48小时候回捕。

60 worker bees of Chinese bees and Italian bees were taken respectively. After being anesthetized with CO ₂ , each worker bee was injected with 2 ul of Escherichia coli at 50 cfu/mL in the abdomen, and then placed in insect cages and fed with 10% honey water. Recapture after 48 hours of isolation.

(3.10) Extraction of bee hemolymph

A total of 15 Chinese bees and Italian bees were randomly collected from the above three groups of bees, and 5 bees were used as a sample, and 3 replicates were set for each. Cut the back of the bee’s neck with scissors, let the hemolymph flow out automatically, absorb it with a capillary tube, add the collected hemolymph to the centrifuge tube, and add a small amount of phenylthiourea at the same time to prevent the activation of phenol-based oxidase in the plasma and blacken the hemolymph , centrifuged to remove blood cells. Take the supernatant for later use.

(3.11) Pipette 10 uL of hemolymph from Apis chinensis and Apis mellifera respectively, add 990 uL of PBS, shake well, make a solution with a concentration of 10 μg/ml, and add 100 uL/well to each well. According to the above Elisa method, the absorbance value (OD ₄₅₀ ) of the hemolymph of Zhongfeng and Italian bee was measured respectively, and the results are shown in Table 3. Each absorbance value is substituted into the Y item of the standard curve y=0.017x+0.067 in embodiment 4, and conversion obtains the antimicrobial peptide content (X) in the hemolymph.

Table 2 The results of the determination of antimicrobial peptides in the hemolymph of Chinese bee and Italian bee by GST-AccRoyalisin antibody

the Time after inoculation (h) the I II III average value the 0 Absorbance 0.1883 0.1861 0.1865 the the the Content (μg/μL) 7.1353 7.0059 7.0294 7.06±0.07 the the the the the the the Chinese bee twenty four Absorbance 0.2043 0.2122 0.2107 the the the Content (μg/μL) 8.0765 8.5412 8.4529 8.36±0.25 the the the the the the the the 48 Absorbance 0.2437 0.2462 0.2461 the the the Content (μg/μL) 10.3941 10.541 10.5353 10.49±0.08 the the the the the the the the 0 Absorbance 0.1606 0.1619 0.1659 the the the Content (μg/μL) 5.5059 5.5824 5.8176 5.64±0.16 the the the the the the the Italian bee twenty four Absorbance 0.1717 0.1702 0.1799 the the the Content (μg/μL) 6.1588 6.0706 6.6412 6.29±0.32 the the the the the the the the 48 Absorbance 0.2091 0.2029 0.2017 the the the Content (μg/μL) 8.3588 7.9941 7.9235 8.09±0.23

The results in Table 2 were used for t-test and plotted for analysis (Figure 4). The results showed that the antimicrobial peptide content in the hemolymph of the Chinese bee was significantly higher than that of the Italian bee ( p <0.01). It shows that the resistance immunity of Chinese bees to pathogenic bacteria is higher than that of Italian bees. At the same time, after 24 hours and 48 hours after immunization, the content of antimicrobial peptides in the hemolymph of Zhongfeng and Italian bee were significantly increased, showing an obvious immune response. And the immune response of Chinese bees is stronger than that of Italian bees. This is a good verification of the previous domestic scholars (Xu, P., et al. PLoS ONE. 2009, 4:1-9.) report that the antimicrobial peptide ability of Chinese bee is higher than that of Italian bee. Therefore, the GST-AccRoyalisin antibody is suitable for evaluating the disease resistance of bee species and bee colonies.

4. Determination of Royalisin content in royal jelly by Elisa indirect method

Weigh 1.5 mg of royal jelly freeze-dried powder, dissolve in 1 ml of PBS, shake well, make a solution with a concentration of 1.5 mg/ml, centrifuge at high speed, take the supernatant as the antigen, dilute 10 times, add the sample to a 96-well plate, Add 10 uL/well to each well, according to the above Elisa method, use GST-AccRoyalisin antibody as the primary antibody, and measure the absorbance value (OD ₄₅₀ ). The results are shown in Table 3. Substituting each absorbance value into the Y item of the standard curve y = 0.017x + 0.067, the average content (X) of antimicrobial peptides in the royal jelly freeze-dried powder is converted to 0.49±0.23 μg/μL, that is, 100×0.49/15=3.3%.

Table 3 Determination results of Royalisin in royal jelly (n=3)

the 1 2 3 average OD value 0.0789 0.0758 0.0713 the Antimicrobial peptide content (ug/15ug) 0.70 0.52 0.25 0.49±0.23 Antimicrobial peptide content (%) 4.67 3.45 1.69 3.27±1.50

The present invention will be further described below in conjunction with specific embodiments and accompanying drawings. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.

Example 1 Recombinant expression of AccRoyalisin gene and separation and purification of fusion expression product

1. Recombinant expression and identification of AccRoyalisin gene

Using the bee head cDNA library plasmid containing the antibacterial peptide AccRoyalisin gene of Apis cerana royal jelly (GenBank acceptance number: EF660337) as a template, its precursor fragment was amplified by PCR and cloned into the expression vector pGEX-4T-2, The recombinant pGEX-4T-2/ Accroyalisin obtained by screening was transformed into Escherichia coli BL21 and fused with glutathione (GST) for expression. Both royal jelly antimicrobial peptide AccRoyalisin and AccRoyalisin precursor and Apis mellifera antimicrobial peptide AmRoyalisin precursor are composed of 95 amino acid residues, with a homology of 90%-92%; both mature peptides are composed of 51 amino acid residues , only 3-4 amino acid residues are different, and the homology is above 90%.

The recombinant bacteria were cultured and induced to express. The total bacterial protein was electrophoresed by SDS-PAGE. Compared with the BL21 bacteria containing pGEX-4T-2 empty vector, the pGEX-4T-2/ Acc-royalisin engineering bacteria had a specific band of about 36 ku in size (Figure 1 ). The theoretical molecular mass of Acc-royalisin is 5.52 kDa, plus the GST protein with a mass of 26 kDa fused with it expressed in pGEX-4T-2 vector, the expected mass of GST-A ccRoyalisin fusion protein is 31.2 kDa, therefore, the specific The molecular weight of the target protein was consistent with that of the expected protein. The recombinant expression product was further subjected to ultrasonic crushing and centrifugation, and the obtained supernatant and precipitate were analyzed by SDS-PAGE electrophoresis. There are two forms of inclusion body protein and inclusion body protein, among which the soluble protein which has biological activity and can be directly separated by affinity purification method accounts for the majority. Transfer the GST-AccRoyalisin fusion protein on the SDS-PAGE electrophoresis gel to the nitrocellulose membrane, and use GST antibody for Western blot analysis, showing specific blot.

2. Affinity separation of recombinant fusion expression product GST-AccRoyalisin

The screened AccRoyalisin-containing recombinant bacteria were inoculated into LB liquid medium, and the expression was induced by IPTG at 37°C for 12 hours. Transfer 500 mL of bacterial liquid to a centrifuge tube, centrifuge for 10 min (5000 rpm, 4 °C), remove the supernatant, and place the pellet on ice. Use 50 μL of ice-bathed PBS for every 1 mL of the original culture solution to completely suspend the pellet. Cells were briefly disrupted by sonication on ice until the sample was not viscous. The sonicated bacterial solution was centrifuged at 10,000 rpm, 4°C for 10 min. Carefully transfer the supernatant to a clean, ice-cooled vial. The cell pellet was then completely suspended in ice-bathed PBS (50 μL ice-bathed PBS was used for every 1 mL of original culture medium).

Separation of target protein by affinity chromatography prepacked column with GST antibody connected to AKTA explorer 100. The principle is: the target protein has a GST tag and can bind to the GST affinity column in an equilibrium buffer environment, and the protein without the GST tag will be eluted in the form of an elution peak. During elution, the collected eluent is the salt solution of the purified single target protein. The absorption peak of UVA at ₂₈₀ nm was used as a detection tool for protein. Samples must be centrifuged or 0.45 μm filter before passing through the column. Samples are too viscous and can be diluted with Binding Buffer.

Water and buffer solution must use high-purity water during operation, and buffer solution must pass through a 0.45 μm filter, and then ultrasonically degassed. Inject the binding buffer into the syringe or pump tubing, and connect the column to the syringe to prevent air from entering the column. Remove the small plug at the outlet at the bottom of the column. Equilibrate the column with 5 bed volumes of binding buffer. Inject with a syringe or a pump, the recommended injection flow rate: 0.2-1 mL/min. Wash the column with 5-10 times column volume binding buffer or until there is no UV absorption peak in the effluent, and the flow rate is 1-2 mL/min. Elute with 5-10 column volumes of elution buffer at a flow rate of 1-2 mL/min. When the elution peak appears, collect the eluate.

Take the elution peak of affinity separation and analyze it by SDS-PAGE electrophoresis. The result (see Figure 2) shows that the elution peak obtained from this purification system is the molecular weight A single band protein with a size of about 32 kDa is consistent with the size of the target protein, indicating that a single target protein was obtained after purification. The elution peak contains multiple protein bands, belonging to mixed proteins, which is consistent with the peak shape diagram. There are also target protein bands in the elution peak, indicating that part of the target protein fails to bind to the column in time and directly flows out in the form of effluent.

Example 2 Preparation of GST-AccRoyalisin polyclonal antibody

Take the GST-AccRoyalisin purified and eluted by AKTA column, put it into a dialysis bag, dialyze overnight, transfer the dialysis desalted solution into a 1.5 mL eppendorf tube, freeze-dry, dissolve the purified protein with 0.9% normal saline, and use as Antigen back up.

Two healthy male rabbits aged 14-16 weeks were selected, and each rabbit was injected with about 0.5 mL of purified fusion protein antigen by subcutaneous injection. Before immunization, blood samples were drawn from rabbit ears as negative controls. The fusion protein antigen was dissolved in 700 μL of normal saline, emulsified with 700 μL of Freund’s complete adjuvant and appropriate amount of penicillin and streptomycin, and injected subcutaneously at multiple points for immunization. Three weeks later, a booster immunization was performed with the same antigen, adjuvant and penicillin emulsion. After 5 weeks, the fusion protein antigen was dissolved in 1 mL of normal saline for intramuscular injection; after 6 weeks, the immunization was boosted again with 1 mL of normal saline containing the fusion protein antigen. At the same time, blood samples were drawn from the rabbit ears, and the titer of multiple antiserum was determined by indirect ELISA; after 7 weeks, the blood was collected from the carotid artery and allowed to coagulate at room temperature. Put the coagulated blood in an incubator at 37°C for half an hour, then put it in a refrigerator at 4°C overnight to shrink the blood clot and precipitate the antiserum; suck out the antiserum, centrifuge at 3,000 rpm for 10 min, aspirate the supernatant and store at -80°C spare.

Example 3 Elisa method to detect GST-AccRoyalisin polyclonal antibody titer

Using the recombinant GST-AccRoyalisin isolated in Example 1 as the antigen, and the GST-AccRoyalisin antibody prepared in Example 2 as the primary antibody, follow the steps below:

(1) Coating: use the recombinant GST-AccRoyalisin described in Example 1 as the antigen, adjust the pH value with carbonate buffer (CBS, Na ₂ CO ₃ 1.59 g, NaHCO ₃ 2.93 g, ddH ₂ O 950 ml to 9.6, add ddH ₂ O to 1000 ml, store at 4°C), dilute to 1 μg/ml, add 100 μl/well into a 96-well plate, and store at 4°C overnight;

(2) Blocking: Discard the coating solution in the small wells of the enzyme-linked plate the next day, use PBST (PBS+0.05% Tween-20) as the washing solution, wash 5-10 times in each step, and pat dry. Then add 100 μL of PBST solution containing 5% BSA or skimmed milk powder to block for 1.5 h at 37 °C.

(3) Discard the blocking solution in the small wells of the enzyme-linked plate, and wash with PBST. Add primary antibody, use phosphate buffer solution (PBS, KH ₂ PO ₄ 0.27g _, Na ₂ HPO ₄ 1.42g, NaCl 8g, KCl 0.2g, ddH ₂ O 800mL, adjust pH to 7.4 with concentrated hydrochloric acid, Volume to 1L) was diluted 1000 times, 100uL/well, and stood at 37°C for 1-2 h. The negative control was diluted in the same ratio as the positive antiserum. A blank control group without primary antibody was set up.

(4) Washing: Rinse 10 times with tap water and pat dry.

(5) Discard the primary antibody in the wells of the enzyme-linked plate and wash it with PBST. Use HRP-labeled goat anti-rabbit IgG as the secondary antibody, dilute it 5000 times, 100 μl/well, and let stand at 37°C for 1 h.

(6) Washing: Rinse 10 times with tap water and pat dry;

(7) Use TMB as the chromogenic substrate, 100 μl/well, place at 37°C for 15 minutes; use 2 M H ₂ SO ₄ as the stop solution, 50 μl/well, measure the absorbance at a wavelength of 450 nm on a microplate reader immediately after adding value. 450 nm is the maximum absorption wavelength of the chromogen.

(8) Calculate the average value P of the OD value of the sample, and the OD value N of the negative control. If P≥2N, it is regarded as a positive reaction, otherwise it is a negative reaction.

According to the measurement results in Table 4, it shows that the polyclonal potency of the prepared GST-AccRoyalisin is higher than 40000 times.

Table 4 The potency of GST-AccRoyalisin polyclonal antibody against standard peptides determined by Elisa (n=6)

Antibody dilution factor 1 2 3 average value 5000 2.7566 2.9229 2.8987 0.8389±0.0069** 10000 2.3228 3.1097 2.9991 0.6189±0.0076** 15000 3.0539 3.0255 3.1185 0.4528±0.0008** 20000 2.8319 3.1513 2.8339 0.3568±0.0017** 40000 1.2354 1.4088 1.5779 0.2444±0.0059** control 0.9020 0.8233 0.9516 0.1811±0.0058

p< 0.00001, **: means there is a very significant difference compared with the control p<0.01

Embodiment 4 The establishment of AccRoyalisin standard curve

Dissolve the GST-AccRoyalisin standard protein obtained by affinity separation in CBS to make a 10 μg/ml solution. The GST-AccRoyalisin polyclonal antibody was diluted at a ratio of 1:1000 as the primary antibody; HRP-labeled goat anti-rabbit IgG was used as the enzyme-labeled secondary antibody, which was added to PBS at a ratio of 1:5000 to form a secondary antibody solution.

Add 10 μg/ml AccRoyalisin standard protein solution to a 96-well plate by doubling gradient dilution method, add 100 μl/well to each well, and coat overnight at 4°C. The liquid in the wells was discarded, washed 5 times with PBST buffer, and skimmed milk blocking solution was added at 200 μl/well, and blocked at 37°C for 1.5 h. Discard the skimmed milk blocking solution, wash with PBST buffer 5 times, add primary antibody and secondary antibody solution at 100 μl/well, and react at 37°C for 1 h. The primary antibody solution was discarded, washed 5 times with PBST buffer, and the secondary antibody solution was added in an amount of 100 μl/well, and reacted at 37°C for 0.5 h. Discard the secondary antibody solution, wash 5 times with PBST buffer, add chromogenic solution TMB at 100 μl/well, react at 37°C for 15 min, add TMB stop solution 2M H ₂ SO ₄ at 50 μl/well. Immediately place it on a microplate reader and measure the absorbance at 450 nm.

(1) Table 1 was obtained by antibody detection. Taking the average OD ₄₅₀ absorbance value (n=3) as the ordinate, and the concentration of GST-AccRoyalisin standard protein solution as the abscissa, the standard curve (Figure 3) was obtained: y = 0.017x + 0.067, R ² =0.998.

Table 1 The absorbance obtained by measuring the gradient dilution standard polypeptide with GST-AccRoyalisin antibody

Example 5 Elisa determination of Royalisin content in bee worker bee hemolymph

1. Sampling, Immunization Processing, and Hemolymph Extraction of Honey Bees

(1) Collection of bees and infection treatment

Randomly pick a beehive of Apis chinensis and Apis mellifera from the apiary, respectively comb, and randomly take about 100 worker bees with jars. Divided into three groups:

不处理组：各取中蜂、意蜂工蜂30只。

No treatment group: 30 worker bees from Zhongfeng and Italian bees were taken respectively.

② Take 60 worker bees of Chinese bees and Italian bees respectively. After being anesthetized with CO ₂ , each worker bee is injected with 2 ul of Escherichia coli at 50 cfu/g in the abdomen, and then placed in insect cages and fed with 10% honey water. Recapture after 24 hours of isolation.

各取中蜂、意蜂工蜂60只，用CO₂麻醉后，每只工蜂在腹部注射50 cfu/mL 的大肠杆菌2 ul染菌，然后置养虫笼内，用10%的蜂蜜水饲养，隔离48小时候回捕。

60 worker bees of Chinese bees and Italian bees were taken respectively. After being anesthetized with CO ₂ , each worker bee was injected with 2 ul of Escherichia coli at 50 cfu/mL in the abdomen, and then placed in insect cages and fed with 10% honey water. Recapture after 48 hours of isolation.

(2) Extraction of bee hemolymph

A total of 15 Chinese bees and Italian bees were randomly collected from the above three groups of bees, and 5 bees were used as a sample, and 3 replicates were set for each. Cut the back of the bee’s neck with scissors, let the hemolymph flow out automatically, absorb it with a capillary tube, add the collected hemolymph into the centrifuge tube, and add a small amount of phenylthiourea at the same time to prevent the activation of phenol-based oxidase in the plasma and blacken the hemolymph , centrifuged to remove blood cells. Take the supernatant for later use.

2. Determination of Antimicrobial Peptide Content in Serum of Chinese Bee and Italian Bee with GST-AccRoyalisin Polyclonal Antibody

Pipette 10 uL of the hemolymph of the Chinese bee and Italian bee, add 990 uL of PBS, shake well, make a solution with a concentration of 10 μg/ml, and add 100 uL/well to each well. According to the above Elisa method, the absorbance value (OD ₄₅₀ ) of the hemolymph of Zhongfeng and Italian bee was measured respectively, and the results are shown in Table 2. Each absorbance value is substituted into the Y item of the standard curve y=0.017x+0.067 in embodiment 4, and conversion obtains the antimicrobial peptide content (X) in the hemolymph.

Table 2 The results of the determination of antimicrobial peptides in the hemolymph of Chinese bee and Italian bee by GST-AccRoyalisin antibody

the Time after inoculation (h) the I II III average value the 0 Absorbance 0.1883 0.1861 0.1865 the the the Content (μg/μL) 7.1353 7.0059 7.0294 7.06±0.07 the the the the the the the Chinese bee twenty four Absorbance 0.2043 0.2122 0.2107 the the the Content (μg/μL) 8.0765 8.5412 8.4529 8.36±0.25 the the the the the the the the 48 Absorbance 0.2437 0.2462 0.2461 the the the Content (μg/μL) 10.3941 10.541 10.5353 10.49±0.08 the the the the the the the the 0 Absorbance 0.1606 0.1619 0.1659 the the the Content (μg/μL) 5.5059 5.5824 5.8176 5.64±0.16 the the the the the the the Italian bee twenty four Absorbance 0.1717 0.1702 0.1799 the the the Content (μg/μL) 6.1588 6.0706 6.6412 6.29±0.32 the the the the the the the the 48 Absorbance 0.2091 0.2029 0.2017 the the the Content (μg/μL) 8.3588 7.9941 7.9235 8.09±0.23

 The results in Table 2 were used for t-test and plotted for analysis (Figure 4). The results showed that the antimicrobial peptide content in the hemolymph of the Chinese bee was significantly higher than that of the Italian bee ( p <0.01). It shows that the resistance immunity of Chinese bees to pathogenic bacteria is higher than that of Italian bees. At the same time, after 24 hours and 48 hours after inoculation, the content of antimicrobial peptides in the hemolymph of Zhongfeng and Italian bee were significantly increased, showing an obvious immune response. And the immune response of Chinese bees is stronger than that of Italian bees. This is a good verification of the previous domestic scholars (Xu, P., et al. PLoS ONE. 2009, 4:1-9.) report that the antimicrobial peptide ability of Chinese bee is higher than that of Italian bee. Therefore, the GST-AccRoyalisin antibody is suitable for evaluating the disease resistance of bee species and bee colonies.

Example 5 Elisa indirect quantitative determination of the content of Royalisin in royal jelly

Weigh 1.5 mg of royal jelly freeze-dried powder, dissolve in 1 ml of PBS, shake well, make a solution with a concentration of 1.5 mg/ml, centrifuge at high speed, take the supernatant as the antigen, dilute 10 times, add the sample to a 96-well plate, Add 10 uL/well to each well, according to the above Elisa method, use GST-AccRoyalisin antibody as the primary antibody, and measure the absorbance value (OD ₄₅₀ ). The results are shown in Table 3. Substituting each absorbance value into the Y item of the standard curve y=0.017x+0.067 in Example 4, the average content (X) of antimicrobial peptides in the royal jelly freeze-dried powder obtained by conversion is 0.49 ± 0.23 μg/μL, i.e. 100 × 0.49/15 =3.3%.

       Table 3 Determination results of Royalisin in royal jelly (n=3)

the 1 2 3 average OD value 0.0789 0.0758 0.0713 the Antimicrobial peptide content (ug/15ug) 0.70 0.52 0.25 0.49±0.23 Antimicrobial peptide content (%) 4.67 3.45 1.69 3.27±1.50

Claims (6)

1. a preparation method of royal jelly antimicrobial peptide AccRoyalisin antibody, characterized in that, the steps are as follows: (1) Using the bee head cDNA containing the AccRoyalisin gene as a template, its precursor fragment was amplified by PCR, and cloned into the expression vector pGEX-4T-2 containing the GST-encoded gene. The recombinant pGEX-4T-2/AccRoyalisin obtained by screening was transformed into E. coli BL21 and expressed by fusion with glutathione; the target protein GST-AccRoyalisin was purified by affinity separation method; (2) Using purified GST-AccRoyalisin as an antigen, white rabbits were immunized, and the serum was collected to obtain GST-AccRoyalisin polyclonal antibody. 2. A GST-AccRoyalisin polyclonal antibody prepared by the method as claimed in claim 1. 3. a GST-AccRoyalisin polyclonal antibody as claimed in claim 2 detects the Elisa method of antimicrobial peptide Royalisin content in honeybee hemolymph, it is characterized in that, (1) Use the GST-AccRoyalisin polyclonal antibody as the primary antibody, and use the recombinant expressed GST-AccRoyalisin as the standard antigen protein for doubling dilution, and use a microplate reader to detect the absorbance of different concentrations of GST-AccRoyalisin protein samples by the Elisa method, and establish the absorbance Standard curve with AccRoyalisin protein concentration; (2) Using the hemolymph of Apis cerana and Apis mellifera as antigens and GST-AccRoyalisin polyclonal antibody as the primary antibody, the absorbance of the obtained samples was detected by Elisa method, and substituted into the established standard curve to calculate the content of Royalisin in the samples. 4. a kind of Elisa method as claimed in claim 3 is applied to the difference of measuring Apis mellifera and Italian honeybee antibacterial ability. 5. a GST-AccRoyalisin polyclonal antibody as claimed in claim 2 is used to detect the Elisa method of antimicrobial peptide Royalisin content in royal jelly, it is characterized in that, (1) Using the GST-AccRoyalisin polyclonal antibody as the primary antibody and the recombinantly expressed GST-AccRoyalisin as the standard antigen protein for doubling dilution, use a microplate reader to detect the absorbance of different concentrations of GST-AccRoyalisin protein samples by Elisa method, and establish Standard curve of absorbance and AccRoyalisin protein concentration; (2) The protein extract of royal jelly freeze-dried powder was used as the antigen, the GST-AccRoyalisin polyclonal antibody was used as the primary antibody, and the absorbance of the sample was detected by Elisa method, which was substituted into the established standard curve to calculate the content of Royalisin in the sample. 6. a kind of Elisa method as claimed in claim 5 is applied to measuring royal jelly antiseptic property.

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