A recombinant inhibin B antigen and its preparation method and application

Abstract

The present invention discloses a recombinant inhibin B antigen and a preparation method and application thereof, including two subunit antigens, α and β, wherein the coding sequence of the α subunit and the coding sequence of the β subunit are fused with TrxA tags respectively to achieve soluble expression; after a period of expression, the expression of a plasmid encoding SUMO protease is started, and intracellular enzyme cleavage is performed to remove the TrxA tag to obtain the α and β subunit antigens containing only His tags. The α and β subunit antigens of the recombinant inhibin B antigen of the present invention are soluble and do not contain TrxA tags, which can reduce the immunogenicity of non-target antigen peptides during immunization and achieve efficient immunization. The intracellular enzyme cleavage process is simple and convenient for antigen purification.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant inhibin B antigen and a preparation method and application thereof. Background Art

[0002] Inhibin is a member of the transforming growth factor p (TGF-p) superfamily. It is a dimeric glycoprotein hormone composed of two subunits, α and β. The relative molecular mass (Mr) of the α subunit is 20,000, and the relative molecular mass (Mr) of the β subunit is 5,000-15,000, and there are two subtypes, βA and βB. α and β are bound by disulfide bonds to form inhibin A (αβA) and inhibin B (αβB). Studies have found that inhibin A is difficult to detect in the male blood circulation at a low concentration (<2 pg / mL) and has no biological activity, while inhibin B (INH-B) is the main biologically active inhibin in the blood circulation.

[0003] In addition, INH-B exists in a variety of molecular forms in the blood circulation, including mature (Mr: 32000) and incompletely mature (also known as precursor) αβ dimer molecules and free α subunits. Free α subunits lack biological activity. Enzyme-linked immunosorbent assay (ELISA) can also detect α subunit proprotein (Pro-αC) in plasma, which may be a series of proproteins that cross-react with other inhibins, also known as immune-related protein progenitor (Pro-αC-RI). The βB subunit precursor is a peptide chain composed of 392 amino acid (aa) residues, and the mature βB subunit (115aa) is located at the carbon end of this peptide chain. The homology of the nucleotide sequence of the full-length coding region of different mammalian βB subunits is greater than 80%, the homology of the nucleotide sequence of the mature region is greater than 90%, and the homology of the amino acid sequence deduced from the mature region is greater than 96%.

[0004] Inhibin B (INH-B) is secreted by reproductive system cells and is closely related to fertility. It has endocrine, paracrine and autocrine effects in regulating reproductive function. It is involved in the regulation of female follicle development and male spermatogenesis. In assisted reproductive treatment, it is of great significance in ovarian function assessment, follicle monitoring, and prediction of sperm recovery rate in patients with azoospermia.

[0005] Clinical methods for detecting INH-B in serum or seminal plasma include radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (double antibody sandwich ELISA). RIA cannot distinguish INH-A from INH-B and inactive inhibin. ELISA uses a pair of highly specific antibodies that specifically bind only to functional INH-B molecules. Its specificity and sensitivity can fully meet the needs of scientific research and clinical testing. The new βB subunit monoclonal antibody 46A / F makes the detection of INH-B simpler and more sensitive, but it has not yet been put into use. ELISA uses a pair of highly specific antibodies that specifically bind only to functional INH-B molecules. Its detection principle is to capture the antigen with a monoclonal antibody of the βB subunit coated with a micropore, and then bind to the detection antibody (a specific monoclonal antibody of the α subunit coupled to an enzyme) and develop color; although there will be a 0.5% cross-reaction with INH-A, its specificity and sensitivity can fully meet the needs of scientific research and clinical testing.

[0006] However, there is no independent intellectual property right laboratory diagnostic kit for INH-B detection, only products from two foreign companies, DSL and OBI. The imported test kits are expensive and cannot be used for large-scale population research and evaluation. To this end, we explored the development of a double antibody sandwich ELISA inhibin B test kit based on genetic engineering monoclonal antibodies and protein expression platforms, and explored the design of a double antibody sandwich ELISA kit to lay the foundation for rapid, accurate and convenient clinical detection of INH-B. It can also provide a reliable reference value range for Chinese people in the clinic. Summary of the invention

[0007] Technical problems solved: In response to the above technical problems, the present invention provides a recombinant inhibin B antigen and a preparation method and application thereof, which can effectively solve the shortcomings of expensive imported test kits and limited research.

[0008] Technical solution: In the first aspect, the present invention provides a recombinant inhibin B antigen, including two subunit antigens, α and β, wherein the coding sequence of the α subunit and the coding sequence of the β subunit are fused with TrxA tags respectively to achieve soluble expression; after a period of expression, the expression of a plasmid encoding SUMO protease is started, and intracellular enzyme cleavage is performed to remove the TrxA tag to obtain two subunit antigens, α and β, containing only His tags; the amino acid sequence of the α subunit antigen is shown in SEQ ID NO.1, the amino acid sequence of the β subunit antigen is shown in SEQ ID NO.2, the amino acid sequence of the TrxA tag is shown in SEQ ID NO.3, and the amino acid sequence of the SUMO protease is shown in SEQ ID NO.4:

[0009] SEQ ID NO.1:

[0010] STPLMSWPWSPSALRLLQRPPEEPAAHANCHRVALNISFQELGWERWIVYPPSFIFHYCHGGCGLHIPPNLSLPVPGAPPTPAQPYSLLPGAQPCCAALPGTMRPLHVRTTSDGGYSFKYETVPNLLTQHCACI;

[0011] SEQ ID NO.2:

[0012] GLECDGRTNLCCRQQFFIDFRLIGWNDWIIAPTGYYGNYCEGSCPAYLAGVPGSASSFHTAVVNQYRMRGLNPGTVNSCCIPTKLSTMSMLYFDDEYNIVKRDVPNMIVEECGCA;

[0013] SEQ ID NO.3:

[0014] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLA;

[0015] SEQ ID NO.4:

[0016] LVPELNEKDDDQVQKALASRENTQLMNRDNIEITVRDFKTLAPRRWLNDTIIEFFMKYIEKSTPNTVAFNSFFYTNLSERGYQGVRRWMKRKKTQIDKLDKIFTPINLNQ SHWALGIIDLKKKTIGYVDSLSNGPNAMSFAILTDLQKYVMEESKHTIGEDFDLIHLDCPQQPNGYDCGIYVCMNTLYGSADAPLDFDYKDAIRMRRFIAHLILTDALK.

[0017] In a second aspect, the present invention provides a method for preparing the recombinant inhibin B antigen according to the first aspect, comprising the following steps:

[0018] S1. Construction of expression plasmids for α and β subunits of inhibin B

[0019] S1-1, delete the C-terminal 6xHis tag of TrxA;

[0020] S1-2, inserting the recognition sequence of SUMO protease between the enterokinase recognition site (Enterokinase site) and the multiple cloning site (MCS), wherein the amino acid sequence of the recognition sequence of SUMO protease is shown in SEQ ID NO.5,

[0021] SEQ ID NO.5:

[0022] SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG;

[0023] S1-3, inserting the inhibin B α subunit (INHA) and β subunit (INHBB) into the multiple cloning site of the above vector respectively;

[0024] S2. Construction of SUMO protease expression plasmid

[0025] S2-1, replace the resistance tag ampicillin in the pBAD24 vector with chloramphenicol;

[0026] S2-2, inserting the SUMO protease coding sequence into the vector described in step 1;

[0027] S3, inhibin B α and β subunit protein expression and purification

[0028] S3-1, co-transfect α and β subunit plasmids with SUMO protease expression plasmid into BL21 (DE3) competent cells;

[0029] S3-2, pick a single clone for inoculation, and when the culture OD value is 0.6-0.8, add 0.1-1mM IPTG at 16℃ to induce the expression of α or β subunit protein. After 12-16h, add 0.5%-0.001% arabinose to induce the expression of SUMO protease;

[0030] S3-3, collect the cells by centrifugation and resuspend them in PBS. After the cells are ultrasonically disrupted, centrifuge again to collect the supernatant and discard the precipitate;

[0031] S3-4. The supernatant was affinity purified by Ni column, eluted with PBS containing different gradients of imidazole, and the fractions were collected to obtain the target protein.

[0032] In a third aspect, the present invention provides applications of the recombinant inhibin B antigen described in the first aspect, including one or more of the following applications:

[0033] (1) As an immunogen for developing inhibin B antibodies;

[0034] (2) As a quality control product for diagnostic kits;

[0035] (3) As a standard in diagnostic kits.

[0036] The purity and immunogenicity of the antigen directly affect the specificity and quality of the immune response. Antigens should be pure, easy to prepare and stable to ensure that they can effectively stimulate the immune system to produce the required antibodies. The preparation of inhibin B α subunit and β subunit antigens is particularly critical in the process of developing diagnostic antibodies. This lays the foundation for the rapid, accurate and convenient development of INH-B diagnostic reagents in the clinic.

[0037] Beneficial effects: The α and β subunit antigens of the recombinant inhibin B antigen of the present invention are soluble and expressed, have good thermal stability, and do not contain TrxA tags. The advantages of a good antigen in preparing antibodies are mainly reflected in the following aspects:

[0038] 1. Good purity Antigens usually have higher immunogenicity and can more effectively stimulate the immune system to produce antibodies against specific antigens, thereby improving the yield and quality of antibodies; they are usually more effective in diagnostic test applications because they have better binding properties and lower background signals; they bind to antibodies with high specificity, and this property can be used to remove non-specifically bound impurities during antibody purification, simplifying the purification steps and improving purity;

[0039] 2. Removing the TrxA tag helps induce the immune system to produce antibodies against specific antigen epitopes, which means that antibodies can more accurately recognize and bind to target antigens and reduce nonspecific binding; at the same time, it speeds up the screening and optimization process of antibodies because they can more effectively induce immune responses, thereby shortening the time from antigen design to obtaining functional antibodies;

[0040] 3. Good antigen solubility can promote the immune system to produce high-affinity antibodies, which can bind to the target antigen more firmly, which is very important in diagnostic applications; and can help induce the immune system to produce structurally stable antibodies, which are more stable during storage and use, extending their shelf life;

[0041] In summary, good antigens can provide many advantages when preparing antibodies, which help improve the quality and application effect of the final antibody product. In the actual antibody production process, selecting or designing a good antigen is a key step to ensure the successful preparation of high-quality antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of the construction of the expression plasmids for the α and β subunits of inhibin B of the present invention;

[0043] Figure 2Schematic diagram of the construction of the SUMO protease expression plasmid of the present invention;

[0044] Figure 3 1 is the expression result diagram of α subunit obtained by different treatment methods of the present invention, wherein: lane 1 is the whole bacteria before induction, lane 2 is the whole bacteria of single induction (only inducing α subunit expression), lane 3 is the whole bacteria of double induction (first inducing α subunit expression, then inducing SUMO protease expression), lane 4 is the double induction supernatant, lane 5 is the double induction precipitate, lane 6 is the induced supernatant purified by Ni column, M is a protein marker: the molecular weight sizes from top to bottom are 180, 140, 100, 80, 60, 45, 35, 25, 15, 10 (kD);

[0045] Figure 4 1 is the expression result diagram of β-subunit obtained by different treatment methods of the present invention, wherein: lane 1 is the whole bacteria before induction, lane 2 is the whole bacteria of single induction (only inducing β-subunit expression), lane 3 is the whole bacteria of double induction (first inducing β-subunit expression, then inducing SUMO protease expression), lane 4 is the double induction supernatant, lane 5 is the double induction precipitate, lane 6 is the induced supernatant purified by Ni column, M is a protein marker: the molecular weight sizes from top to bottom are 180, 140, 100, 80, 60, 45, 35, 25, 15, 10 (kD);

[0046] Figure 5 It is an electrophoresis diagram of the results of SUMO digestion after the α and β subunits in the comparative example were purified by Ni column, wherein: lane 1 is the target band between 15 and 10 kD after α subunit digestion, lane 2 is a protein marker, lane 3 is the target band between 15 and 10 kD after β subunit digestion, lane 4 is a protein marker; protein marker: the molecular weight sizes from top to bottom are 180, 140, 100, 80, 60, 45, 35, 25, 15, 10 (kD). DETAILED DESCRIPTION

[0047] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0048] Example 1

[0049] The preparation of recombinant inhibin B antigen comprises the following steps:

[0050] S1. Construction of expression plasmids for the α and β subunits of inhibin B, e.g. Figure 1 As shown:

[0051] S1-1, delete the C-terminal 6xHis tag of TrxA;

[0052] S1-2, inserting the recognition sequence of SUMO protease between the enterokinase recognition site (Enterokinase site) and the multiple cloning site (MCS), wherein the amino acid sequence of the recognition sequence of SUMO protease is shown in SEQ ID NO.5;

[0053] S1-3, inserting the inhibin B α subunit (INHA) and β subunit (INHBB) into the multiple cloning site of the above vector respectively;

[0054] S2, SUMO protease expression plasmid construction, such as Figure 2 As shown:

[0055] S2-1, replace the resistance tag ampicillin in the pBAD24 vector with chloramphenicol;

[0056] S2-2, inserting the SUMO protease coding sequence into the vector described in step 1;

[0057] S3, inhibin B α and β subunit protein expression and purification

[0058] S3-1, co-transfect α and β subunit plasmids with SUMO protease expression plasmid into BL21 (DE3) competent cells;

[0059] S3-2, pick a single clone for inoculation, and when the culture OD value is 0.6-0.8, add 0.1-1mM IPTG at 16℃ to induce the expression of α or β subunit protein. After 12-16h, add 0.5%-0.001% arabinose to induce the expression of SUMO protease;

[0060] S3-3, collect the cells by centrifugation and resuspend them in PBS. After the cells are ultrasonically disrupted, centrifuge again to collect the supernatant and discard the precipitate;

[0061] S3-4. The supernatant was affinity purified by Ni column, eluted with PBS containing different gradients of imidazole, and the fractions were collected to obtain the target protein.

[0062] Among them, the expression results of α subunit obtained by different treatment methods are as follows Figure 3 As shown: the whole bacteria before induction was used as the expression background; single induced whole bacteria (only induced α subunit expression): a band near 45KD (theoretical molecular weight 44.3kD); double induced whole bacteria (first induced α subunit expression, then induced SUMO protease expression): α subunit band near 15KD (theoretical molecular weight 16kD) and remaining TrxA residue band near 35kD; double induced supernatant: α subunit band near 15KD (theoretical molecular weight 16kD) and remaining TrxA residue band near 35kD; double induced precipitate: α subunit band near 15KD; induction supernatant purified by Ni column: α subunit band near 15KD.

[0063] The expression results of β subunit obtained by different treatment methods are as follows Figure 4 As shown: the whole bacteria before induction was used as the expression background; single induced whole bacteria (only induced β subunit expression): a band near 45KD (theoretical molecular weight 44.3kD); double induced whole bacteria (first induced β subunit expression, then induced SUMO protease expression): α subunit band near 10kD (theoretical molecular weight 14kD) and remaining TrxA residue band near 35kD; double induced supernatant: β subunit band near 15KD (theoretical molecular weight 14kD) and remaining TrxA residue band near 35kD; double induced precipitate: β subunit band near 10KD; induction supernatant purified by Ni column: β subunit band near 10KD.

[0064] from Figure 3 and Figure 4 Lane 6 shows that the α and β subunit proteins with higher purity were obtained, which can be used as immune antigens for preparing antibodies.

[0065] Comparative Example

[0066] The α and β subunit proteins of inhibin B were expressed and purified. The specific process is as follows:

[0067] 1) The α and β subunits were constructed into the pET32a vector and the SUMO protease recognition sequence was added after TrxA;

[0068] 2) Pick a single clone for inoculation, and when the OD value of the culture reaches 0.6-0.8, add 0.1-1mM IPTG and induce the expression of α or β subunit protein at 16℃ for 16-20h.

[0069] 3) Collect the cells by centrifugation and resuspend them in PBS. After the cells are ultrasonically disrupted, centrifuge again to collect the supernatant and discard the precipitate;

[0070] 4) The supernatant was purified by Ni column affinity purification, eluted with PBS containing different gradients of imidazole, and the fractions were collected to obtain the target protein;

[0071] 5) Dialyze the eluted fraction into PBS to remove imidazole;

[0072] 6) Add the SUMO protease (with His tag) stored in this experiment to the components in step 5 at a final concentration of 20ug / ml and react at 37℃ for 2 hours;

[0073] 7) Purify the enzyme digestion reaction solution through a Ni column, collect the leaked solution and perform electrophoresis. The results are as follows Figure 5 As shown: After purification, the α and β subunit proteins obtained by enzyme digestion have low purity and are not suitable for use as antigens for immunization.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a recombinant inhibin B antigen, characterized in that: The recombinant inhibin B antigen includes two subunit antigens, α and β, and the coding sequence of the α subunit and the coding sequence of the β subunit are fused with TrxA tags respectively to achieve soluble expression; after a period of expression, the expression of the plasmid encoding SUMO protease is started, and intracellular enzyme cleavage is performed to remove the TrxA tag to obtain the α and β subunit antigens containing only His tags; the amino acid sequence of the α subunit antigen is shown in SEQ ID NO.1, the amino acid sequence of the β subunit antigen is shown in SEQ ID NO.2, the amino acid sequence of the TrxA tag is shown in SEQ ID NO.3, and the amino acid sequence of the SUMO protease is shown in SEQ ID NO.4; The preparation method comprises the following steps: S1. Construction of expression plasmids for the α and β subunits of inhibin B. The specific steps are as follows: S1-1, delete the C-terminal 6xHis tag of TrxA; S1-2, inserting the recognition sequence of SUMO protease between the enterokinase recognition site and the multiple cloning site, wherein the amino acid sequence of the recognition sequence of SUMO protease is shown in SEQ ID NO.5; S1-3, inserting the α subunit and β subunit of inhibin B into the vector multiple cloning site respectively; S2. Construction of SUMO protease expression plasmid. The specific steps are as follows: S2-1, replace the resistance tag ampicillin in the pBAD24 vector with chloramphenicol; S2-2, inserting the SUMO protease coding sequence into the vector described in step 1; S3. Expression and purification of the α and β subunit proteins of inhibin B. The specific steps are as follows: S3-1, co-transfect α and β subunit plasmids with SUMO protease expression plasmid into BL21 (DE3) competent cells; S3-2, pick a single clone for inoculation, and when the culture OD value is 0.6-0.8, add 0.1-1mM IPTG at 16℃ to induce the expression of α or β subunit protein. After 12-16h, add 0.5%-0.001% arabinose to induce the expression of SUMO protease; S3-3, collect the cells by centrifugation, resuspend them in PBS, disrupt the cells by ultrasonication, collect the supernatant by centrifugation again, and discard the precipitate; S3-4. The supernatant was affinity purified by Ni column, eluted with PBS containing different gradients of imidazole, and the fractions were collected to obtain the target protein.

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