Antigen, recombinant expression vector, antibody, preparation method and application

Abstract

The invention relates to the technical field of biological science and detection, in particular to an antigen, a recombinant expression vector, an antibody, a preparation method and application. The amino acid sequence of the antigen is shown as SEQ ID NO.1. The antigen is used for immunizing New Zealand white rabbits; and collecting blood, and collecting antiserum to obtain the insect COXIV polyclonal antibody. The amino acid sequence and the nucleotide sequence of the antigen protein for inducing the insect COXIV polyclonal antibody are obtained, and the polyclonal antibody capable of specifically recognizing COXIV protein can be obtained.

Description

Technical Field

[0001] The present invention relates to the field of biological science and detection technology, and in particular to an antigen, a recombinant expression vector, an antibody, and a preparation method and application thereof. Background Art

[0002] Mitochondrial complex IV, cytochrome C oxidase, abbreviated as COX, is at the end of the system in the cell's mitochondrial respiratory chain and is the site where electrons are catalyzed to combine with O2 to form water. In normal cells, cytochrome C oxidase is often used as an important monitoring point for electron transfer efficiency.

[0003] Reactive oxygen species (ROS) are byproducts of cellular respiration, mainly including superoxide anions (O 2- , hydrogen peroxide H2O2 and hydroxyl radical HO - , which can oxidatively damage macromolecules in the body such as lipids, proteins, nucleic acids, etc., leading to the occurrence of many diseases, including Alzheimer's disease, Parkinson's disease, and diabetes. Intracellular ROS originate from different parts and are catalyzed by multiple enzymes. Studies have shown that about 90% of ROS originate from mitochondria. Cytochrome oxidase subunit IV, abbreviated as COXIV, plays a decisive role in the assembly and activity regulation of COX. It has been confirmed in mammalian cells that changes in COXIV expression can directly affect mitochondrial activity and the generation of ROS. However, the detailed regulatory mechanism is not clear.

[0004] In order to better study the role and mechanism of COXIV in the generation of ROS in insects, it is necessary to develop suitable COXIV antigens to induce the production of specific antibodies. Summary of the invention

[0005] In order to solve the above problems, the present invention provides an antigen, a recombinant expression vector, an antibody and a preparation method and application thereof.

[0006] An insect COXIV antigen, the amino acid sequence of the insect COXIV antigen is shown in SEQ ID NO.1.

[0007] Preferably, the nucleotide sequence of the insect COXIV antigen is shown in SEQ ID NO. 2. In order to effectively promote induced expression, the amino acid sequence of the insect COXIV antigen is optimized to obtain the nucleotide sequence of the insect COXIV antigen.

[0008] A method for preparing the insect COXIV antigen comprises constructing a recombinant expression vector containing the nucleotide sequence shown in SEQ ID NO.2, transforming the recombinant expression vector into Escherichia coli competent cells, and inducing expression to obtain the antigen.

[0009] A recombinant expression vector containing the nucleotide sequence of the insect COXIV antigen.

[0010] Preferably, the nucleotide sequence as shown in SEQ ID NO. 2 is inserted into a basic expression vector and transformed into Escherichia coli.

[0011] Preferably, the basic expression vector is pET-32a.

[0012] Preferably, the Escherichia coli is BL21 (DE3).

[0013] A method for preparing insect COXIV polyclonal antibodies is prepared by immunizing New Zealand white rabbits with the antigen.

[0014] Preferably, the method specifically comprises: immunizing New Zealand white rabbits with antigens of insect COXIV polyclonal antibodies; collecting blood after four immunizations to obtain serum, and purifying the serum to obtain insect COXIV polyclonal antibodies.

[0015] An insect COXIV polyclonal antibody is prepared by the preparation method.

[0016] The insect COXIV polyclonal antibody is used in the preparation of a reagent for detecting COXIV protein.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention obtains for the first time the amino acid sequence and nucleotide sequence of an antigen for inducing insect COXIV polyclonal antibodies.

[0019] The present invention transforms the insect COXIV antigen to obtain the nucleotide sequence of the insect COXIV antigen, uses the nucleotide sequence of the insect COXIV antigen and pET-32a to construct a recombinant expression vector, and effectively promotes the expression of the insect COXIV antigen.

[0020] The invention introduces a recombinant expression vector into BL21 (DE3) cells to induce expression; after protein purification and renaturation, an induced insect COXIV antigen is obtained, thereby obtaining a preparation method of the insect COXIV antigen.

[0021] The present invention obtains polyclonal antibodies after immunizing New Zealand white rabbits with antigen proteins for multiple times, and detects the antibody titer. Based on the specific recognition of the antibody to the antigen, the insect COXIV polyclonal antibody can be used to prepare a reagent for detecting COXIV protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1After sequencing the recombinant plasmid in the present invention, the inserted fragment is translated into an amino acid sequence. Compared with the COXIV antigen protein sequence, there is no mutation in the amino acid sequence, indicating that the recombinant plasmid was successfully constructed;

[0023] Figure 2 In the present invention, SDS-PAGE was used to detect the small-scale induced expression of COXIV antigen;

[0024] Figure 3 For the SDS-PAGE detection, purification and elution in the present invention; the highest elution efficiency is 300mmol / L imidazole elution;

[0025] Figure 4 The antibody serum after immunization is detected by ELISA in the present invention, and the antibody titer can reach 1:128000;

[0026] Figure 5 The Western blot method of the present invention is specific for detecting COXIV polyclonal antibodies in tissues and cells, indicating that it is a specifically recognized COXIV antigen. DETAILED DESCRIPTION

[0027] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0028] Example 1

[0029] Obtaining the Insect COXIV Gene and Constructing Its Expression Vector

[0030] 1. Acquisition of insect COXIV genes

[0031] The laboratory obtained cotton bollworm COXIV by degenerate primer amplification, selected a highly specific COXIV fragment and sent it to a gene synthesis company. In order to effectively promote induced expression, the highly specific COXIV fragment was optimized, and the sequence optimization was completed to obtain a prokaryotic expression fragment. The highly specific COXIV fragment was AN LLLRRALLDAVRVPVCARASSSVGELAKIGNREWVGYGFNGQPNYVDRPDF PMPAIRFRGETPDVKVLREKEKGDWRKLTLEE, recorded as SEQ ID NO.1. The details are as follows:

[0032] According to the distribution of vector restriction sites, Primer5 software was used to design prokaryotic expression-specific primers. The designed prokaryotic expression-specific primers are shown in Table 1, and restriction sites were added to the designed prokaryotic expression-specific primers; the restriction sites were EcoRI "GAATTC" and HindIII "AAGCTT".

[0033] Fresh pupal brain tissue was taken, TRIzol reagent was added and homogenized to extract total RNA. After the total RNA concentration was determined, TaKaRa reverse transcription kit was used to reverse transcribe and synthesize cDNA. Then, using the pupal brain tissue cDNA as a template, COXIV-specific primers containing restriction sites were used to perform PCR amplification to obtain the prokaryotic expression fragment.

[0034] The sequence of the prokaryotic expression fragment is GCTAATTTACTATTGAGGAGAGCATTGCTCGACGCCGTACGTGTTCCGGTTTGCGCGTGCGAGCAGCTCCGTGGGCGAGCTGGCTAAAATCGGCAACCGCGAGTGGGTTGGTTATGGTTTTAA CGGCCAGCCGAATTACGTCGATCGTCCGGACTTCCCGATGCCGGCAATTCGCTTCCGCGTGAAACGCCAGACGTGAAGGTGCTGCGTGAGAAAGAAAAGGGTGATTGGCGTAAACTGACCCTGGAAGAG, denoted as SEQ ID NO.2.

[0035] Table 1 Specific primers for prokaryotic expression of COXIV antigen

[0036]

[0037] The PCR reaction system is as follows: each 25 μL reaction mixture contains 12.5 μL of 2×PCR Mix at a final concentration, 1 μL of prokaryotic primers F / R, 1 μL of cDNA, and is supplemented to 25 μL with sterile water;

[0038] Among them, 2×PCR Mix was purchased from Kangwei Century with the product number #CW3319S.

[0039] The PCR reaction conditions were as follows: pre-denaturation at 98°C for 3 min, denaturation at 98°C for 10 s, annealing at 62°C for 20 s, extension at 72°C for 40 s, and 32 cycles; termination extension at 72°C for 5 min, and storage of the amplified product at 16°C.

[0040] like Figure 1As shown, the amplified product was sequenced, and the expressed amino acids were aligned with the full-length COXIV amino acid sequence. There was no mutation in the amino acid sequence, indicating that the prokaryotic expression fragment was successfully constructed, that is, the recombinant plasmid was successfully constructed.

[0041] The AxyPerp PCR Cleaning Kit was used to purify the amplified product. The specific steps are as follows: (1) Add 3 volumes of Buffer PCR-A to the PCR reaction solution; (2) After mixing, transfer to the preparation tube, place the preparation tube in a 2 mL centrifuge tube, centrifuge at 12,000 × g for 1 min, and discard the filtrate; (3) Place the preparation tube back into the 2 mL centrifuge tube, add 700 μL Buffer W2, centrifuge at 12,000 × g for 1 min, and discard the filtrate; (4) Repeat the above steps once; (5) Finally, place the preparation tube in a clean 1.5 mL centrifuge tube, add 25-50 μL Eluent or deionized water in the middle of the bottom of the preparation tube, and let it stand at room temperature for 1 min. Centrifuge at 12,000 × g for 1 min to elute the DNA; (6) Determine the product concentration using Nanodrop and label to obtain the purified product.

[0042] 2. Construction of expression vector

[0043] According to the restriction sites, double restriction digestion reaction is performed. The purified product is connected to the pET-32a vector, the connected product is transformed into competent cells, and the target plasmid is selected. The specific operation steps are as follows:

[0044] (1) Perform double digestion reaction on the purified product using restriction endonucleases EcoRⅠ and HindIII, incubate in a 37°C water bath for 4 h or overnight, and simultaneously cut the pET-32a vector in another tube; (2) Recover the double digestion product of the purified product and the double digestion product of the pET-32a vector respectively; (3) Perform enzyme ligation reaction according to the volume ratio of double digestion product of the purified product: double digestion product of the pET-32a vector = 4:1, pipet and mix well, then spin down instantly, and ligate at 16°C water bath or use a PCR instrument for more than 2 h to obtain a ligation product. The ligation product was transformed into DH5α competent medium and placed in an ice bath for 30 minutes; after heat shock at 42°C for 90 seconds, it was immediately cooled on ice for 5 minutes; 600 μl LB medium was added to the bacterial solution, and it was revived in a 37°C incubator for 1 hour. 0.2 mL of the revived bacterial solution was taken to coat a solid culture plate containing ampicillin resistance. The culture plate was taken out of a 4°C refrigerator in advance and inverted in an incubator for preheating for 30 minutes; it was cultured at 37°C overnight, and then PCR plaques were picked to obtain positive clones containing the recombinant plasmid, that is, the expression vector pET-32a COXIV was obtained.

[0045] Among them, the pET-32a vector was purchased from Youbao Biotechnology with the catalog number #VT1216.

[0046] Example 2

[0047] Inducible expression of insect COXIV antigen

[0048] 1. Small-scale inducible expression of insect COXIV antigen

[0049] The extracted expression vector pET-32a COXIV was introduced into the transformed BL21 (DE3) competent cells and coated with Amp-resistant solid culture plates. The specific steps are as follows:

[0050] (1) Take 2 μL of expression vector pET-32a COXIV and pET-32a empty vector respectively, transform BL21 (DE3) competent cells, and place on ice for 30 min; (2) Heat shock at 42°C for 90 s, then place on ice again for 5 min; (3) Add 600 μL LB medium to the competent cells and resuscitate at 37°C for 1 h; (4) Pipette 100 μL and spread on ampicillin-resistant LB solid plate and culture at 37°C overnight. The next day, select positive single clones by PCR and sequence them, and add them to 2 mL of Amp-resistant LB solution and start shaking the culture at 37°C and 225 r / min; (5) Shake 5 mL of the culture for more than eight hours to use as the seed solution for large-scale use; (6) Take 10 μL of the seed solution and expand it to 5 mL at a ratio of 1:500, and culture it at 37°C until the absorbance at 600 nm is between 0.8; (7) Add 100 mM IPTG at a ratio of 1:1000 and induce at low temperature for more than 10 hours. IPTG free and empty plasmid transformation groups are set as negative controls; (8) Determine the absorbance at 600 nm, aspirate the bacterial solution according to the value of 160 / OD600, and centrifuge to remove the culture medium; (9) Add an appropriate amount of 5× loading buffer and boil the sample in a metal bath at 100°C for 10 minutes; (10) After shaking, take an appropriate amount of sample for electrophoresis detection and Coomassie Brilliant Blue staining to see if there are positive bands; if the induction is successful, start large-scale induction.

[0051] like Figure 2 As shown, small-scale induced expression of COXIV antigen was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, wherein sodium dodecyl sulfate-polyacrylamide gel electrophoresis is abbreviated as SDS-PAGE.

[0052] 2. Large-scale induced expression and ultrasonic fragmentation of insect COXIV antigen

[0053] The large-scale induced expression of COXIV target protein refers to the small-scale condition settings, but the culture induced expression system is expanded to 1L~2L. The ultrasonic fragmentation process is briefly described as follows:

[0054] After the large-scale cultivation of the seed solution expressing COXIV is completed, the fragmentation treatment is carried out: (1) at room temperature, the rotation speed is 7000g / min, and the bacteria are recovered, not less than 1000mL of bacterial solution, and the supernatant is discarded; (2) 40mL of binding buffer is added to the bacteria and resuspended; (3) After the bacteria are resuspended, they are placed on ice and fragmented for about 0.5h; ultrasonic conditions: on 4s, off 2s, power 50%; (4) centrifuged at 4℃ and 12000 rpm for 20min, the supernatant is collected, and the precipitate is recorded as inclusion body. The inclusion body is resuspended with 30mL of urea binding buffer, and repeated freezing and thawing helps to dissolve; (5) after the precipitate is broken, the rotation speed is 12000 for 20min, and the undissolved precipitate is discarded; (6) 100μL of the inclusion body solution after rupture and the supernatant before rupture are aspirated, 5× Loading buffer is added and the sample is prepared; (7) after protein electrophoresis, R-250 staining is performed, decolorization is performed, and observation is made as to whether the target protein is expressed in the precipitate or the supernatant.

[0055] The staining results showed that the target protein was located in the precipitate, and the inclusion bodies were subsequently lysed and the protein purified.

[0056] Example 3

[0057] Purification of insect COXIV antigen

[0058] Add protein urea binding buffer to the inclusion body. The protein urea binding buffer contains 29.22g NaCl, 7.6g Na3PO4·12H2O and 360.36g urea. The pH is 7.8. Repeated freezing and thawing in a -80℃ refrigerator. After the precipitate breaks, centrifuge at 12000 speed for 20min and discard the undissolved precipitate. Purification is performed as follows:

[0059] (1) Wash the purification column with DDW and add 4 mL of nickel column filler. Slow precipitation can reduce bubbles; (2) Add 20 mL of DDW to the wall of the column at a water flow rate of 10 drops / min and wash once; (3) Add nickel sulfate (2 column volumes) at a flow rate of 0.8 mL / min to ensure the binding efficiency of nickel and filler; (4) Add 20 mL of DDW and set the water flow rate to 4 drops / min to wash away unbound NiSO4; (5) Use 30 mL of Binding buffer to balance the column; (6) Load the broken protein sample and collect the effluent at the same time; (7) Elution setting: 0.05 M, 0.07 M, 0.1 M, 0.3 M and 1 M imidazole buffer to wash and collect each effluent; (8) After washing, take the above effluent and add 5× loading The sample was prepared using buffer, and the purified protein was detected by SDS-PAGE electrophoresis and then stained with R-250. The effluent without obvious impurities was used for the preparation of polyclonal antibodies. (9) The concentration of the purified protein was measured using the Bradford method.

[0060] Figure 3 This is the SDS-PAGE detection, purification and elution in the present invention; when eluted with 0.3M imidazole, the elution amount is the largest and the band is single.

[0061] Example 4

[0062] Preparation and identification of polyclonal antibodies against insect COXIV

[0063] 1. Purchase 3 New Zealand rabbits weighing 2 kg and passing quarantine, and randomly number them 1 to 3 for this immunization experiment. The specific steps of the experiment are as follows:

[0064] (1) Before the first immunization, 2 mL of blood was drawn from the ear vein as a blank control; (2) For the first immunization, 1 μg / μL of purified protein was mixed with complete Freund's adjuvant in a volume ratio of 1:1, and immunization was performed by subcutaneous injection at multiple points in the abdomen; (3) For the second and subsequent immunizations, incomplete Freund's adjuvant was emulsified with protein, and immunization was performed once every half month; (4) Two weeks after the third immunization, 3 mL of blood was collected, and the serum was separated by centrifugation, and the antibody effect was tested by ELISA and WB; (5) If both the antibody titer and specificity meet the requirements, blood was drawn from the artery, left to stand at 4°C overnight, and serum was collected at a speed of 12,000 g and 4°C for 15 min; (6) The Protein A / G ligand compound was fixed to the column carrier; (7) The collected serum was added to the top of the column and allowed to pass through, so that the Protein A / G ligand bound to the target protein; (8) The eluent was used for elution, and the protein was finally washed out at the bottom of the column and collected. The purified polyclonal antibodies were stored at -80°C after aliquoting.

[0065] 2. ELISA to detect the titer of COXIV polyclonal antibodies

[0066] The concentration of the purified COXIV protein solution was determined by the BCA method and coated with carbonate buffer. The specific steps are as follows:

[0067] (1) The purified protein was coated with the coating solution. 50 μL of the coating solution was coated with 2 pmol of protein per well of the ELISA plate. After shaking, the plate was coated overnight at 4°C. (2) The liquid was turned upside down and 200 μL of PBST containing 0.05% Tween-20 was added. The plate was shaken for 5 min and the liquid in the plate was patted dry. This step was repeated 3 times. (3) 200 μL of TBST containing 2% skimmed milk powder was added to each well and blocked at 37°C for 1 h. (4) 200 μL of PBST containing 0.05% Tween-20 was added. The plate was shaken for 5 min and the liquid in the plate was patted dry. The plate was washed 3 times. (5) 200 μL of the diluted primary antibody was added to each well. The dilution ratio was 100:1. 0×, 2000×, 4000×, 8000×, 16000×, 32000×; incubate at 37°C for 1h, and use the pre-immune serum diluted in equal proportion as a negative control; (6) Add 200μL of PBST containing 0.05% Tween-20 each time, shake for 5min, pat the liquid in the wells dry, and wash 4 times; (7) Add 200μL of secondary antibody diluted 1:7500 to each well, incubate at 37°C for 1h, and wash 4 times; (8) Add 100μL of OPD reaction solution, develop color in the dark for 5min, and observe the color change; (9) Use 2M dilute sulfuric acid to terminate the reaction, use an enzyme reader to measure the A490 absorbance, and then take a picture.

[0068] like Figure 4 As shown, the antibody serum titer detected by ELISA after immunization can reach 1:128000.

[0069] 3. Western blot method to detect the specificity of COXIV polyclonal antibodies

[0070] 200 μL NP40 was added to 1.5 mL EP tubes of HzAm1 cells and cotton bollworm tissues, and the mixture was homogenized at 16000 rpm for 20 seconds to extract total protein. After SDS-PAGE separation, the membrane was electrotransferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder solution at room temperature for 1.5 hours, and rabbit anti-COXIV polyclonal antibody diluted at 1:3000 was added, and the membrane was incubated at room temperature for 1 hour or shaken overnight at 4°C. The membrane was washed 4 times with TBS containing 0.1% Tween, each time for 5 minutes. HRP-labeled goat anti-rabbit IgG diluted at 1:5000 was added and incubated at room temperature for 1 hour. The membrane was washed with TBST for 20 minutes × 4 times, and the A and B solutions of the chemiluminescent solution (ECL) kit were mixed in equal volumes, and the PVDF membrane was rinsed for 0.5 minutes to 1 minute, and exposed to light for development.

[0071] like Figure 5 As shown, it indicates that the immunized COXIV polyclonal antibody can specifically recognize the cotton bollworm and HzAm1 cell proteins.

[0072] The present invention successfully cloned the gene encoding COXIV antigen for the first time by designing degenerate primers according to sequence comparison without reference to the cotton bollworm genome, then performed codon optimization, and constructed a corresponding recombinant expression vector; obtained a large amount of antigen protein by IPTG induction; obtained polyclonal antibodies after immunizing New Zealand white rabbits with the antigen protein for multiple times, and detected the antibody titer; based on the specific recognition of the antibody to the antigen, the expression of COXIV in cotton bollworm tissues, HzAm1 cells, fall armyworm Sf9 cells, etc. can be detected, laying a foundation for studying the molecular mechanism of COXIV regulation on mitochondrial activity.

[0073] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0074] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0075] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An insect COXIV antigen, characterized in that: The amino acid sequence of the insect COXIV antigen is shown in SEQ ID NO.

1.

2. The insect COXIV antigen according to claim 1, characterized in that The nucleotide sequence of the insect COXIV antigen is shown in SEQ ID NO.

2.

3. A method for preparing the insect COXIV antigen according to claim 2, characterized in that: A recombinant expression vector containing the nucleotide sequence shown in SEQ ID NO.2 is constructed, the recombinant expression vector is transformed into Escherichia coli competent cells, and induced for expression to obtain the antigen.

4. A recombinant expression vector containing the nucleotide sequence as claimed in claim 2.

5. The recombinant expression vector according to claim 4, characterized in that: The nucleotide sequence shown in SEQ ID NO. 2 is inserted into a basic expression vector and transformed into Escherichia coli.

6. The recombinant expression vector according to claim 5, characterized in that The basic expression vector is pET-32a.

7. The recombinant expression vector according to claim 5, characterized in that The Escherichia coli is BL21 (DE3).

8. A method for preparing insect COXIV polyclonal antibodies, characterized in that: The method is prepared by immunizing New Zealand white rabbits with the antigen described in claim 1.

9. An insect COXIV polyclonal antibody, characterized in that: The invention relates to a novel nanostructured carbon foam. The nanostructured carbon foam is prepared by the preparation method as claimed in claim 8.

10. Use of the insect COXIV polyclonal antibody according to claim 9 in the preparation of a reagent for detecting COXIV protein.

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