NaPi2b extracellular domain antigen protein fragment as well as preparation method and application thereof

Abstract

The invention discloses a NaPi2b extracellular domain antigen protein fragment as well as a preparation method and application thereof, according to a pre-designed amino acid sequence shown as SEQ ID NO: 1, escherichia coli codon optimization is performed, then a target gene is synthesized, and the gene sequence is shown as SEQ ID NO: 2; the gene segment is inserted into an expression plasmid between the NdeI and the HindIII; and carrying out plasmid transformation and screening, protein induced expression and protein purification to prepare the antigen protein fragment. The antigen fragment can be used for generating a corresponding NaPi2b antibody through animal immune response, and the antibody can be used for detecting the expression condition of NaPi2b protein in a human tissue sample by an immunohistochemical method, and is also used for an accompanying diagnostic kit of a humanized NaPi2b antibody coupling medicine, so that the aim of assisting in precise medication is fulfilled.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a NaPi2b extracellular region antigen protein fragment and a preparation method and application thereof. Background Art

[0002] Sodium-dependent phosphate transporter type 2b (NaPi-IIb, also known as NaPi2b) is a multi-channel membrane protein composed of 690 amino acids and is a member of the solute carrier SLC34 family, which consists of NaPi-IIa (SLC34A1), NaPi-IIb (SLC34A2) and NaPi-IIc (SLC34A3). Among them, NaPi-IIb protein mediates the entry of inorganic phosphate into epithelial cells through sodium ion co-transport and is responsible for maintaining phosphate balance throughout the body.

[0003] NaPi-IIb is encoded by the gene SLC34A2, whose cDNA was first isolated and cloned from human small intestine and lung cDNA libraries in 1999. Although SLC34A2 is expressed in various human tissues, it is expressed at the highest levels in the lung, small intestine, and kidney. In the lung, SLC34A2 is only expressed on the apical membrane of type II alveolar epithelial cells (ATII cells) and plays a key role in fetal lung development and embryonic development. As multifunctional stem cells, ATII cells may transform into non-small cell lung cancer (NSCLC) after experiencing exogenous or endogenous factors. Recent studies have found that the expression of SLC34A2 is significantly different between surgical specimens of NSCLC and normal tissues, and SLC34A2 is downregulated in the lung adenocarcinoma cell line A549. Upregulating the expression of SLC34A2 can significantly inhibit the cell viability and in vitro invasion of A549. These results suggest that SLC34A2 may play an important role in the development of NSCLC.

[0004] In addition to lung cancer, another disease that SLC34A2 mainly affects is ovarian cancer. It is overexpressed in most epithelial ovarian cancers. In addition, abnormal expression of SLC34A2 is also associated with pulmonary alveolar microlithiasis (PAM), hypophosphatemia, infertility, testicular microlithiasis (TM) and other malignancies (including thyroid cancer, breast cancer, etc.). Summary of the invention

[0005] To address the deficiencies in the prior art, the present invention aims to provide a NaPi2b extracellular region antigen protein fragment and a preparation method and application thereof, wherein the antigen fragment can be used to produce corresponding NaPi2b antibodies through animal immune response, and the antibody can be used to detect the expression of NaPi2b protein in human tissue samples by immunohistochemistry, and at the same time, it can be used in a companion diagnostic kit for humanized NaPi2b antibody-drug conjugates to achieve auxiliary precision medication.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: A NaPi2b extracellular region antigen protein fragment, the amino acid sequence of the antigen fragment is shown in SEQ ID NO: 1: MHHHHHHVEV ATHYLEIITQ LIVESFHFKN GEDAPDLLKV ITKPFTKLIV QLDKKVISQI AMNDEKAKNKSLVKIWCKTF TNKTQINVTV PSTANCTSPS LCWTDGIQNW TMKNVTYKEN IAKCQHIFVN FHLPDL.

[0007] A nucleic acid molecule encoding a NaPi2b extracellular region antigen protein fragment.

[0008] Preferably, the nucleotide sequence of the aforementioned nucleic acid molecule is shown in SEQ ID NO: 2:.

[0009] The method for preparing the extracellular region protein fragment of NaPi2b antigen comprises the following steps: S1. Gene synthesis and plasmid construction: According to the pre-designed amino acid sequence, as shown in SEQ ID NO: 1, the target gene was synthesized after E. coli codon optimization, and the gene sequence was shown in SEQ ID NO: 2; the gene fragment was inserted into the expression plasmid between NdeI and HindIII, and a 6×His tag was used at the end; S2. Plasmid transformation and screening: The synthesized expression plasmid was transformed into Escherichia coli and screened using LB solid medium with Kan resistance; S3, protein induced expression: single colonies were selected from the transformed plates, inoculated into LB medium for culture and protein induced expression; S4, protein purification: collect bacteria, ultrasonically disrupt, and purify recombinant proteins; S5. Analyze and identify the results of induced expression.

[0010] Preferably, in the aforementioned step S1, the expression plasmid is one of pET20b, pET24A-D, pET28A-C, pQE-T7, pQE-30, pQE-60 or pBR322.

[0011] Preferably, in the aforementioned step S2, the Escherichia coli is a BL21 strain.

[0012] Preferably, in the aforementioned step S3, the induction expression conditions are: 0.1-0.8 mM IPTG, 0.2-0.6 M NaCl and LB medium, the temperature is 18° C., and the induction time is 12-24 h.

[0013] Preferably, in the aforementioned step S4, the method for purifying the recombinant protein is nickel column purification.

[0014] Application of the extracellular region protein fragment of NaPi2b antigen in the preparation of a kit for detecting the expression of NaPi2b protein in human tissue samples.

[0015] Application of NaPi2b antigen extracellular region protein fragment in the preparation of companion diagnostic kit for humanized NaPi2b antibody-drug conjugates.

[0016] The present invention is beneficial in that: (1) The present invention designs an extracellular antigen protein fragment for producing NaPi2b antibodies in animal immune response, and uses genetic engineering technology to achieve efficient expression and purification of fusion proteins in an Escherichia coli expression system, significantly improving production efficiency and reducing costs. Compared with traditional protein extraction and purification methods, it is more economical and has strong scalability; (2) The antigen fragment can be used to produce the corresponding NaPi2b antibody through animal immune response. The antibody can be used to detect the expression of NaPi2b protein in human tissue samples by immunohistochemistry. It can also be used in the companion diagnostic kit of humanized NaPi2b antibody-drug conjugates to achieve the purpose of assisting precision medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 To analyze the expression of NaPi2b (ECD) protein in BL21 (DE3); Figure 2 SDS-PAGE analysis of NaPi2b (ECD) protein purification results. DETAILED DESCRIPTION

[0018] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0019] Example 1: A method for preparing a protein fragment of the extracellular region of the NaPi2b antigen, comprising the following steps: S1. Gene synthesis and plasmid construction: The combined polypeptide EEC with the amino acid sequence: MHHHHHHVEV ATHYLEIITQ LIVESFHFKNGEDAPDLLKV ITKPFTKLIV QLDKKVISQI AMNDEKAKNK SLVKIWCKTF TNKTQINVTV PSTANCTSPSLCWTDGIQNW TMKNVTYKEN IAKCQHIFVN FHLPDL was codon-optimized and then gene synthesized, inserted between NdeI and HindIII of the expression plasmid, and a 6×His tag was added to the end; S2. Plasmid transformation and screening: The synthesized expression plasmid was transformed into the Escherichia coli BL21 strain and screened using LB solid medium with Kan resistance; S3, protein induction expression: pick a single colony to induce protein expression in LB liquid culture medium, and culture until OD600 is 0.5-0.8, add inducer to the test tube culture medium, induction expression conditions: 0.1-0.8 mM IPTG, 0.1-0.6 MNaCl, LB medium, 18°C, induction time is 12-24 hours; S4, protein purification: collect the bacteria, disrupt them by ultrasonication, purify them by nickel column purification method, and obtain the recombinant protein; S5. SDS-PAGE analysis was used to identify the results of induced expression. The induced culture medium was centrifuged at 12,000 rpm for 5 min, the supernatant was removed, and PBS was added to resuspend the precipitate. Finally, SDS-PAGE loading buffer was added to heat the sample at 100°C for 10 min, and then centrifuged to obtain the supernatant for electrophoresis. Six groups of experiments were set up, namely: Lane M: SDS-PAGE Protein Marker; Lane 0: Control (without IPTG); Lane 1: Induction at 15°C for 16 h; Lane 2: Induction at 37°C for 16 h; Lane 3: Supernatant after whole bacteria were lysed; Lane 4: Precipitation after whole bacteria were lysed. The whole bacteria were ultrasonically lysed using 20 mM Tris (pH 8.0), 300 mM NaCl, 20 mM Imidazole containing 1% Triton X-100, 1 mM DTT, and 1 mM PMSF. The supernatant and precipitate were taken for SDS-PAGE and WB analysis and detection. The results are shown in the figure. Figure 1 shown.

[0020] according to Figure 1It can be seen that: Lane 0, which serves as a blank control, does not produce clear protein bands without the addition of the inducer IPTG; compared with Lane 1, Lane 2 produces a more obvious band at 15KDa, which indicates that 37°C is more suitable as the experimental temperature for inducing the bacteria to express proteins; comparing Lane 3 with Lane 4, Lane 4 produces a more obvious and accumulated band at 15KDa, indicating that after centrifugation, the protein is located in the precipitate.

[0021] Example 2: A method for purifying a protein fragment of the extracellular region of the NaPi2b antigen, comprising the following steps: The inclusion bodies were washed with 50 mM Tris (pH 8.0), 300 mM NaCl containing 1% Triton X-100, 2 mM EDTA, and 5 mM DTT, and then dissolved with 50 mM Tris (pH 8.0), 300 mM NaCl, 8 M Urea, and 20 mM Imidazole buffer while equilibrating the Ni-IDA column. Finally, the target protein was eluted with equilibration buffer containing different concentrations of imidazole, and each elution fraction was collected for SDS-PAGE analysis and detection. 13 groups of experiments were set up, namely: Lane M: SDS-PAGE Protein Marker; Lane 0: supernatant after inclusion body dissolution and centrifugation; Lane 1: supernatant after inclusion body dissolution and centrifugation; Lane 2: effluent after supernatant incubated with Ni-IDA; Lane 3-6: elution fractions of 50 mM Imidazole; Lane 7-11: elution fractions of 500 mM Imidazole. The results are as follows: Figure 2 shown.

[0022] according to Figure 2 It can be seen that: Lane 0, which serves as a blank control, produces a faint band at 15KDa, indicating that there is a small amount of target protein in the supernatant after centrifugation, and this is also used as a reference for screening concentrations that meet the requirements; the bands at 15KDa from Lanes 1-11 are in a dynamic change from weak to strong and then to weak, reaching the strongest in Lane 7, indicating that the eluate there contains the largest content of target protein. In addition to Lane 7, Lane 6 and Lane 8 also produce bands that are significantly higher than those of other lanes, especially Lane 0, indicating that the eluates represented by these two lanes also contain high levels of target protein and can be used for downstream experiments.

[0023] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A NaPi2b extracellular region antigen protein fragment, characterized in that: The amino acid sequence of the antigen fragment is shown in SEQ ID NO: 1: MHHHHHHVEV ATHYLEIITQ LIVESFHFKN GEDAPDLLKV ITKPFTKLIVQLDKKVISQIAMNDEKAKNK SLVKIWCKTF TNKTQINVTV PSTANCTSPS LCWTDGIQNW TMKNVTYKENIAKCQHIFVN FHLPDL.

2. A nucleic acid molecule encoding the NaPi2b extracellular region antigen protein fragment according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

2.

4. The method for preparing the extracellular region protein fragment of NaPi2b antigen according to claim 3, characterized in that: The following steps are involved: S1. Gene synthesis and plasmid construction: According to the pre-designed amino acid sequence, as shown in SEQ ID NO: 1, the target gene was synthesized after E. coli codon optimization, and the gene sequence was shown in SEQ ID NO: 2; the gene fragment was inserted into the expression plasmid between NdeI and HindIII, and a 6×His tag was used at the end; S2. Plasmid transformation and screening: The synthesized expression plasmid was transformed into Escherichia coli and screened using LB solid medium with Kan resistance; S3, protein induced expression: single colonies were selected from the transformed plates, inoculated into LB medium for culture and protein induced expression; S4, protein purification: collect bacteria, ultrasonically disrupt, and purify recombinant proteins; S5. Analyze and identify the induced expression results.

5. The method for preparing the extracellular region protein fragment of NaPi2b antigen according to claim 4, characterized in that: In step S1, the expression plasmid is one of pET20b, pET24A-D, pET28A-C, pQE-T7, pQE-30, pQE-60 or pBR322.

6. The method for preparing the extracellular region protein fragment of NaPi2b antigen according to claim 4, characterized in that: In step S2, the Escherichia coli is a BL21 strain.

7. The method for preparing the extracellular region protein fragment of NaPi2b antigen according to claim 4, characterized in that: In step S3, the induction expression conditions are: 0.1-0.8 mM IPTG, 0.2-0.6 M NaCl and LB medium, the temperature is 18° C., and the induction time is 12-24 h.

8. The method for preparing the extracellular region protein fragment of NaPi2b antigen according to claim 4, characterized in that: In step S4, the method for purifying the recombinant protein is nickel column purification.

9. Use of the extracellular region protein fragment of the NaPi2b antigen according to claim 1 in preparing a kit for detecting the expression of NaPi2b protein in human tissue samples.

10. Use of the extracellular region protein fragment of the NaPi2b antigen according to claim 1 in a companion diagnostic kit for preparing a humanized NaPi2b antibody-drug conjugate.

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