CN106084057A - The preparation of BACE1 shearing-type high-titer antibody and application - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and relates to a BACE1 shear type high-titer antibody prepared by using a GST expression system and its application. The BACE1 spliced gene was cloned into a prokaryotic expression vector by genetic engineering technology, and after identification by enzyme digestion, the recombinant plasmid was used to transform Escherichia coli, and IPTG induced the production of GST‑BACE1 _146‑189aa and GST‑BACE1 _190‑214aa fusion proteins. New Zealand rabbits were immunized with the fusion protein to prepare antiserum and purified with ProteinA/G. These two anti-BACE1 splicing polyclonal antibodies have good titer and specificity, and can be applied to the detection of different splicing types 52KD and 49KD of BACE1 in cells and the distinction of four splicing types, and can effectively detect BACE1 splicing The differential expression of genotypes in different cells is of great significance for systematically studying the function of BACE1 and its mechanism of action in AD.

Description

Preparation and application of BACE1 shear type high titer antibody

technical field

The invention belongs to the technical field of DNA recombination in bioengineering, and in particular relates to the preparation of a polyclonal antibody of GST-BACE1 fusion protein by using a GST prokaryotic expression system and its application.

Background technique

Alzheimer's disease (AD), commonly known as Alzheimer's disease, is a fatal central nervous degenerative disease mainly characterized by progressive cognitive impairment and memory impairment. One of the main pathological features of AD is the Amyloid plaques formed by β-amyloid protein (Aβ), Aβ is produced by APP (Amyloid Precursor Protein) under the sequential action of β-cleaving enzyme (BACE1) and γ-cleaving enzyme complex.

BACE1 protein, also known as β-site APP lyase 1, is a membrane-bound aspartic acid protease and is the key enzyme for cleaving APP to produce Aβ. It is expressed in various tissues, with the highest expression in the brain and pancreas . The activity of BACE1 in neurons is higher, while the highly expressed BACE1 in the pancreas is an inactive fragment missing exon 3. This expression method of missing some exons is called alternative splicing. According to existing literature reports, there are four long forms of BACE1, A, B, C, and D, which are 55KD, 52KD, 49KD, and 47KD, respectively. So far, there have been no more reports and studies on the functions of these splicing types, the mechanism of action with Aβ, and the relationship with AD. Difficult, so there is an urgent need for technical means that can distinguish these types of shearing.

For the study of the function of a new protein, antibody is one of the most powerful tools. A series of techniques, such as immunohistochemistry, western blotting and immunoprecipitation, are widely used in protein detection and functional research, all based on antigen- Antibody interactions develop. Therefore, the high-titer antibody of BACE1 protein we prepared is used to detect the two splicing types of 49KD and 52KD, which can effectively distinguish these two splicing types. and clues.

Contents of the invention

The purpose of the present invention is to establish a model that can be used for the detection of the 49KD and 52KD splicing types of BACE1 protein, and provide necessary experimental tools for in-depth research on the protein function of the BACE1 splicing type and related diseases from the protein level.

The two antibody fragments used to distinguish the four cut types of BACE1 55KD, 52KD, 49KD and 47KD are: BACE1 _146-189aa (insert1), BACE1 _190-214aa (insert2) [Figure 1]. BACE1 _146-189aa can be used to identify BACE1 splicing types 55KD and 52KD, and BACE1 _190-214aa can be used to identify BACE1 splicing types 55KD and 49KD.

BACE1 splice type 55KD can be recognized by both BACE1 _146-189aa and BACE1 _190—214aa ; BACE1 splice type 52KD can only be recognized by BACE1 _146-189aa ; BACE1 splice type 49KD can only be recognized by BACE1 _190—214aa ; Type 47KD was neither recognized by BACE1 _146-189aa nor BACE1 _190-214aa .

BACE1 splicing type BACE1 _146-189aa (insert1) BACE1 _190—214aa (insert2) BACE1—55KD √ √ BACE1—52KD √ x BACE1—49KD x √ BACE1—47KD x x

Note: "√" in the table means that the fragment is included and can be recognized by the fragment, and "×" means that the fragment is not included and cannot be recognized by the fragment;

According to this principle, we obtained the sequences of these two fragments from Genebank to prepare high-titer antibodies for detecting the two spliced types of BACE1—49KD and BACE1—52KD. Using genetic engineering technology, the DNA fragments corresponding to the 190th to 214th amino acid (insert 2) and 146th to 189th amino acid (insert 1) of the N-terminal of the BACE1 protein were cloned into the prokaryotic expression vector pGEX-4T-1. After digestion and sequence analysis, the recombinant plasmid was used to transform Escherichia coli BL21, and induced by isopropyl-β-D-thiogalactoside (IPTG) to produce fusion of GST-BACE1 _190-214aa and GST-BACE1 _146-189aa protein. Antiserum was prepared by immunizing New Zealand rabbits with the purified fusion protein, and purified by Protein A/G to obtain two polyclonal antibodies, which can detect the 49KD and 52KD splice forms of BACE1 respectively, and were proved by ELISA and Western blot. The titers and specificities of the two antibodies were high.

The GST-BACE1 _190-214aa and GST-BACE1 _146-189aa fusion proteins of the present invention are high-efficiency expression specificity obtained by constructing GST-BACE1 _190-214aa and GST-BACE1 _146-189aa fusion protein prokaryotic expression vectors using special sequences and transforming BL21 GST-BACE1 _190-214aa and GST-BACE1 _146-189aa fusion proteins, the amino acid sequences and corresponding nucleotide sequences of these two segments are respectively:

190--214AA(75bp)

Amino acid sequence:

base sequence:

146--189AA(132bp)

Amino acid sequence:

base sequence:

The preparation of this GST-BACE1 _190-214aa , GST-BACE1 _146-189aa fusion protein and its polyclonal antibody comprises the following steps:

The first step, the construction of the cloning vector

Apply PCR technology from the full length of human BACE1 gene and use pCDNA3.1-Flag-BACE1 as a template to amplify the target fragment and connect it to the PMD18-T vector. After transformation and extraction, the recombinant plasmid is obtained, identified by enzyme digestion and sequenced .

The second step, the construction of prokaryotic expression vector pGEX-4T-1

After the cloned plasmid and plasmid pGEX-4T-1 were double-enzymatically digested, the BACE1 _190-214aa and BACE1 _146-189aa gene fragments were obtained using a recovery kit and connected to the vector. The recombinant expression vector is obtained through transformation, extraction and other steps. Recombinants were identified by enzyme digestion and further confirmed by sequencing.

The third step, induction expression and purification of GST-BACE1 _190-214aa and GST-BACE1 _146-189aa fusion proteins

The recombinant plasmids PGEX-4T-1/BACE1 _190-214aa and PGEX-4T-1/BACE1 _{146-189aa were} transformed into Escherichia coli BL21, induced by IPTG, and the fusion proteins of GST-BACE1 _190-214aa and GST-BACE1 _146-189aa were expressed. Identify by SDS-PAGE, and optimize the expression conditions to induce a large amount of amplification. Bacteria were lysed by sonication, and the obtained protein was purified by Glutathione-Sepharose 4B column, and the purified product was identified by SDS-PAGE.

The fourth step, preparation and purification of rabbit anti-BACE1 _190-214aa and BACE1 _146-189aa antiserum

Male New Zealand white rabbits were immunized with purified BACE1 _190-214aa and BACE1 _146-189aa fusion proteins. For the first immunization, 500 μg of fusion proteins were mixed with an equal volume of complete Freund's adjuvant, fully emulsified, and subcutaneously injected at multiple points on the back. Serum was separated from ear vein blood before immunization as a serum control before immunization. After 2wk, the first booster immunization was carried out. 500 μg of purified GST-BACE1 _190-214aa and GST-BACE1 _146-189aa fusion proteins were mixed with an equal volume of incomplete Freund's adjuvant and injected intramuscularly into the front and rear quadrupeds. Immunization was boosted once every 2wk thereafter. Ear blood was taken 1wk after the last immunization, and the antibody titer was determined by ELISA method. When the antibody titer reached 1:100000, the carotid artery was bled and the serum was collected. Purify the anti-BACE1 serum with ProteinA/G, prepare the protein A sepharose CL-4B affinity column, bind the antibody to the column by affinity chromatography, and elute the antibody after 2 washes to achieve the purpose of purification, and identify it by SDS-PAGE Purify the product to obtain the polyclonal antibody of the invention. A variety of immunological methods were used to test their potency and specificity, and the results showed that these two antibodies could specifically bind to the BACE1 spliced protein.

Using the amino acid gene sequences of BACE1 _190-214aa and BACE1 _146-189aa protein fragments, the prokaryotic expression vector of GST-BACE1 _190-214aa and GST-BACE1 _146-189aa fusion protein was successfully constructed, and the specific GST-BACE1 ₁₉₀ could be highly expressed after transformation of BL21 _-214aa and GST-BACE1 _146-189aa fusion protein. Rabbits were immunized with the fusion protein to obtain high-titer antibodies against GST-BACE1 _190-214aa and GST-BACE1 _146-189aa fusion proteins. The antibody titer and specificity were tested by various immunological methods, and the results showed that the antibody titer was as high as 1:100000 And good specificity.

The antibody can be applied to the detection of BACE1 spliced protein in cells and can effectively detect the differential expression of BACE1 spliced protein in different tissues and cell lines. It is very helpful in terms of correlation, and it is of great significance for systematically studying the function of BACE1 splicing protein and its mechanism of action in Alzheimer's disease. This lays the foundation for determining the relationship between BACE1 protein expression and disease.

Description of drawings:

Figure 1 is a schematic diagram of antibody design

Fig. 2 is the DNA agarose gel electrophoresis figure of the BACE1 gene fragment amplified by PCR;

Fig. 3 is the agarose gel electrophoresis figure of recombinant GST carrier;

Fig. 4 is the SDS-PAGE figure of GST and GST-BACE1 fusion protein;

Figure 5 is the SDS-PAGE diagram of the BACE1 antibody after ProteinA/G purification;

Figure 6 is an indirect ELISA method for determining the titer of the antibody;

Figure 7 is the Western blot analysis of antiserum specificity.

Figure 8 is the overexpression of mutant plasmids of different shear types in cells, antibody Westernblot analysis

detailed description:

Embodiment 1: the preparation of anti-GST-BACE1 _190-214aa and GST-BACE1 _146-189aa shear type serum

1. Construction of PCR-PMD18-T/BACE1 _190-214aa and BACE1 _146-189a recombinant plasmids

The full length of the BACE1 gene was obtained from Genebank, and the sequence number of the gene is NM 001207048. Using pCDNA3.1-Flag-BACE1 as template, 190-214aa upstream primer is GGA TCC ATG CCT GAC GAC TCC CTG GAG CCT TTCTTT GAC TCT CTG GTAAAG (including Bamh1 restriction site); downstream primer is 5'-CTC GAG CTA CTG CAG GGAGGA GAG GTT GGGAAC GTG GGT CTG CTT TAC CAG (with XhoI restriction site). 146-189aa The upstream primer is 5'-GGATCC ATG GTA AGC ATC CCC CAT GGC (containing the Bamh1 restriction site); the downstream primer is 5'-CTC GAGCTA CCT GGC AAT CTC AGC ATA (containing the XhoI restriction site). The DNA sequences of 75bp and 132bp corresponding to 25 and 44 amino acids on the BACE1 gene were successfully amplified by PCR [Figure 2]. The amplified fragment was ligated with the PMD18-T vector, and the ligated product was transferred into competent Escherichia coli DH5α, the clones were selected on the Amp ⁺ agar plate, and the recombinant plasmid was extracted by alkaline lysis method, and BamH I and Xhol I Double digestion identification.

2. Construction of prokaryotic expression vector pGEX-4T-1

The pMD18-T plasmid containing the BACE1 gene fragment was double digested with BamHI and Xho1, and the BACE1 _190-214aa and BACE1 _146-189a gene fragments were obtained using the recovery kit, and the plasmid pGEX-4T-1 was treated with the same enzyme. Then, the recovered BACE1 _190-214aa and BACE1 _146-189a gene fragments were ligated with the digested vector pGEX-4T-1 under the action of T4 DNA ligase at 16°C overnight. Recombinants were identified by enzyme digestion, and the results showed that both fragments of the BACE1 gene were correct [Figure 3].

3. Induced expression and purification of GST-BACE1 _190-214aa and GST-BACE1 _146-189a fusion proteins

The recombinant plasmid PGEX-4T-1/BACE1 was transformed into Escherichia coli BL21 in shear type, and a single colony was picked and inserted into LB/Ampr medium, and cultured overnight at 37°C with shaking. The next day, the culture was transferred to LB medium containing Amp ⁺ at a ratio of 1:50, and cultured on a shaker at 37°C until mid-logarithmic growth. When the A600 of the culture medium is 0.5-0.6, add IPTG to a final concentration of 0.08mmol/L, and the one without IPTG is used as a negative control, and culture is continued at 25°C for 4-5h. The cells were collected by centrifugation, and the expressions of GST-BACE1 _190-214aa and GST-BACE1 _146-189a fusion proteins were identified by SDS-PAGE [Figure 4], and the expression conditions were optimized for massive amplification induction. Centrifuge at 5000r/min at 4°C for 5min to collect the bacteria, and suspend 1L of the bacteria solution with 60mL of ice-cold NETN. Lyse the bacteria with ultrasound, then centrifuge at 9600rpm at 4°C for 15min, take the supernatant, pass it through a Glutathione-Sepharose 4B column, and first elute with an equal volume of buffer 1 (containing 20mM glutathione, 50mM TrisCl, pH=8.0) Elution, collect the eluate, wash twice with an equal volume of elution buffer 2 (containing 100mM glutathione), collect the eluate, and identify the purified product by SDS-PAGE [Figure 5].

4. Preparation of rabbit anti-BACE1 _190-214aa and BACE1 _146-189a antiserum

Male New Zealand white rabbits were immunized with purified GST-BACE1 _190-214aa and GST-BACE1 _146-189a fusion proteins. For the initial immunization, 500 μg fusion protein was mixed with an equal volume of complete Freund's adjuvant and then injected subcutaneously at multiple points on the back . Serum was separated from ear vein blood before immunization as a serum control before immunization. After 2wk, the first booster immunization was carried out. 500 μg of purified GST-BACE1 _190-214aa and GST-BACE1 _146-189a fusion proteins were mixed with an equal volume of incomplete Freund's adjuvant and injected intramuscularly into the front and rear quadrupeds. Immunization was boosted once every 2wk thereafter. Ear blood was taken 1wk after the last immunization, and the antibody titer was determined by ELISA method. When the antibody titer reached 1:100000, the carotid artery was bled and the serum was collected.

5. Anti-BACE1 _190-214aa and BACE1 _146-189a serum purified by ProteinA/G

Pack the ProteinA/G sepharose CL-4B filler slowly, add diluted antiserum after equilibrating the column, control the flow rate to ensure the combination of antiserum and filler, that is, affinity chromatography makes the antibody bind to the column, after 2 washes , add pH2.7 elution buffer solution to elute the antibody, collect the eluate and measure the 280nm optical density of each collection tube, mix the collection tubes containing the antibody, and identify by SDS-PAGE and Coomassie brilliant blue staining. The staining results showed that the purification effect was obvious, and the purified samples had clear light and heavy chain bands.

Example 2: Analysis and application of GST-BACE1 _190-214aa and GST-BACE1 _146-189a antibodies

1. Determination of GST-BACE1 _190-214aa and GST-BACE1 _146-189a antibody titers

New Zealand white rabbit serum before immunization with GST-BACE1 _190-214aa and GST-BACE1 _146-189a fusion protein was used as a control, and the purified GST-BACE1 _190-214aa and GST-BACE1 _146-189a antibodies were first diluted 10 times and then doubled After serial dilution, the titer of the antibody was determined by indirect ELISA. The results showed that the rabbit serum before immunization did not detect antibodies against fusion proteins GST-BACE1 _190-214aa and GST-BACE1 _146-189a , and the titers of GST-BACE1 _190-214aa and GST-BACE1 _146-189a antibodies were as high as 1: More than 100,000 [Figure 6].

2. GST-BACE1 _190-214aa and GST-BACE1 _146-189a antibodies are applied to the detection of BACE1 spliced protein in cells

Two high titer sera of rabbit anti-mouse GST-BACE1190-214aa and GST-BACE1146-189a prepared by using soluble GST-BACE1190-214aa and GST-BACE1146-189a fusion proteins as immunogens were used for Western blot. Specific protein bands appeared, and the results suggested that anti-GST-BACE1190-214aa and GST-BACE1146-189a sera were highly specific [Figure 7].

The cultured prostate HK293T was collected, and the mutant plasmids overexpressing BACE1 shearing types 55KD, 52KD, 49KD and 47KD were collected. After lysis and ultrasound, the BCA protein quantification kit was used to quantify the protein. Electrotransfer to nitrocellulose membrane. Block with 5% skimmed milk powder for 1 hour, add rabbit anti-BACE1 antibody (2 hours at room temperature, wash 3 times with PBS) and goat anti-rabbit IgG2HRP (react at room temperature for 1 hour, wash 3 times with PBS) in sequence, and finally add substrate DAB for color development, and take pictures [Figure 8]. The results showed that the BACE1190-214aa and BACE1146-189a antibodies we prepared had antigen-antibody reactions with the BACE1 spliced protein expressed in the 293T cell line, and under the instructions of Marker, our antibody 1 could specifically recognize BACE1 —49KD, Antibody 2 can specifically recognize BACE1—52KD.

190--214AA(75bp)

146--189AA(132bp)

190--214AA(75bp)

Amino acid sequence:

146--189AA(132bp)

Amino acid sequence:

Claims (5)

1. two kinds of antibody of invention protection are provided commonly for the differentiation of tetra-kinds of shearing-types 55KD, 52KD, 49KD and 47KD of BACE1, and Both antibody is the qualification of 52KD and 49KD in BACE1 shearing-type；

It is respectively as follows: for distinguishing 2 antibody fragments of BACE1 tetra-kinds of shearing-types 55KD, 52KD, 49KD and 47KD BACE1_146-189aa(insert1), BACE1_190—214aa(insert2)；

BACE1 shearing-type BACE1_146-189aa(insert1) BACE1_190—214aa(insert2) BACE1—55KD √ √ BACE1—52KD √ × BACE1—49KD × √ BACE1—47KD × ×

Note: in table, " √ " represents and comprise this fragment and can be by this antibody recognition, and "×" represents and do not comprises this fragment and can not be by this Antibody recognition；

Antibody BACE1_146-189aaCan be used for identifying BACE1 shearing-type 55KD and 52KD, antibody BACE1_190—214aaCan be used for identifying BACE1 shearing-type 55KD and 49KD；

BACE1 shearing-type 55KD can be simultaneously by antibody BACE1_146-189aaWith antibody BACE1_190—214aaIdentify；BACE1 shearing-type 52KD is only by antibody BACE1_146-189aaIdentify；BACE1 shearing-type 49KD is only by antibody BACE1_190—214aaIdentify；BACE1 Shearing-type 47KD can not be by antibody BACE1_146-189aaIdentify, again can not be by antibody BACE1_190—214aaIdentify；

Therefore both antibody may be used for the differentiation of four kinds of shearing-types 55KD, 52KD, 49KD and 47KD.

2. invention protection is used for identifying BACE1 shearing-type 55KD, 52KD antibody BACE1_146-189aa(insert1) aminoacid sequence Row and corresponding nucleotide sequence；

It is characterized in that utilizing GST expression system to prepare using the 146th to 189 aminoacid of BACE1 albumen n end as distinguished sequence GST-BACE1_146-189aaFusion protein.

146--189 aminoacid sequence:

146--189AA (132bp) base sequence:

3. invention protection is used for identifying BACE1 shearing-type 55KD, 49KD antibody BACE1_190—214aa(insert2) aminoacid sequence Row and corresponding nucleotide sequence；

It is characterized in that utilizing GST expression system to prepare using the 190th to 214 aminoacid of BACE1 albumen n end as distinguished sequence GST-BACE1_190—214aaFusion protein.

190--214AA aminoacid sequence:

190--214AA (75bp) base sequence:

4. this preparation method for antibody is protected in invention；

Prokaryotic expression GST-BACE1 as described in claim 2,3_190-214aaAnd GST-BACE1_146-189aaMany grams of fusion protein The preparation method of grand antibody, it is characterised in that utilize GST expression system to obtain GST-BACE1_190-214aaAnd GST- BACE1_146-189aaFusion protein, then obtain anti-GST-BACE1 through immunity rabbit_190-214aaAnd GST-BACE1_146-189aaAntiserum.

5. invention protection is sheared according to the BACE1 in the diseases such as alzheimer's disease of two kinds of antibody described in right 1,2,3,4 Differentiate, detect, treat and apply.