CN116903750A - An anti-PIP4K2A splice variant polyclonal antibody and its application in the diagnosis of liver cancer - Google Patents

Abstract

The invention provides an anti-PIP 4K2A splice variant polyclonal antibody and application thereof in liver cancer diagnosis. The preparation method of the anti-PIP 4K2A splice variant polyclonal antibody comprises the following steps: (1) Constructing a recombinant expression vector containing a recombinant PIP4K2A splice variant gene, wherein the nucleotide sequence of the recombinant PIP4K2A splice variant gene is shown as SEQ ID NO. 2; (2) Transforming the recombinant expression vector into escherichia coli competent cells for induction expression to obtain recombinant PIP4K2A splice variant antigens; (3) Immunizing animals with recombinant PIP4K2A splice variant antigen to obtain antiserum, and separating and purifying to obtain the PIP4K2A splice variant polyclonal antibody. The PIP4K2A-S can be used as a diagnosis marker of liver cancer, and has higher guiding significance for judging prognosis of liver cancer and diagnosis of liver cancer.

Description

An anti-PIP4K2A splice variant polyclonal antibody and its use in the diagnosis of liver cancer application

Technical field

The invention relates to the field of bioengineering technology, and in particular to an anti-PIP4K2A splice variant polyclonal antibody and its application in liver cancer diagnosis.

Background technique

Alternative splicing (AS) can edit a single pre-mRNA molecule and produce different mature mRNAs in eukaryotes. These transcript variants can subsequently produce proteins with different structures and biological functions. Therefore, alternative splicing is an important mechanism for post-transcriptional regulation of gene expression and plays a crucial role in the diversity of the transcriptome and encoded proteins. Recent high-throughput sequencing studies have shown that more than 95% of genes undergo alternative splicing and produce at least two alternative pre-mRNA isoforms. Abnormal alternative splicing events may be related to a variety of diseases, especially in the occurrence, development, metastasis and drug resistance of cancer. Alternative splicing events may serve as diagnostic or prognostic biomarkers, as well as for developing therapeutic targets in cancer.

Primary liver cancer ranks fourth in the incidence of malignant tumors in my country and ranks second in mortality, seriously threatening the lives and health of our people. Hepatocellular carcinoma (HCC) (hereinafter referred to as liver cancer) is the most common adult liver cancer, accounting for approximately 75%-85% of primary liver cancers clinically. The liver has rich blood supply, early metastasis and postoperative recurrence are the most important factors for poor prognosis of liver cancer. According to statistics, the 5-year survival rate of liver cancer is only 14.1%. The fundamental reason for the poor prognosis of liver cancer is the insidious onset of liver cancer in its early stages, and the lack of effective early diagnosis methods and early treatment intervention methods.

The full length of the PIP4K2A (phosphatidylinositol-5-phosphate 4-kinase 2α) gene is 179.7Kb, including 10 exons. PIP4K2AcDNA is 3.8kb. The PIP4K2A protein consists of 406 amino acids, has a molecular weight of 53kDa, and has a conserved phosphatidylinositol phosphokinase (PIPK) domain in the C-terminal region. PIP4K2A belongs to PIP kinase type II, which also includes PIP4K2B and PIP4K2C. Originally identified in red blood cells and highly expressed in peripheral blood cells. The main function of this protein family is to recognize and phosphorylate phosphatidylinositol (PtdIns) 5P and synthesize phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). Phosphatidylinositol-4,5-bisphosphate is an important precursor in phosphoinositide signal transduction and can directly regulate the activity of signal transduction proteins and cellular processes. Recent studies have shown that PIP4K2A is involved in the regulation of important biological processes in malignant phenotypes, including cell proliferation, colony formation, and survival. However, there have been no reports on the relationship between PIP4K2A splice variants and liver cancer.

Contents of the invention

In view of the deficiencies in the prior art, the present invention provides a polyclonal antibody against PIP4K2A splice variants and its application in liver cancer diagnosis, which solves the lack of effective early diagnosis methods for liver cancer in the prior art. Issues with early therapeutic intervention.

In one aspect, the present invention provides a method for preparing an anti-PIP4K2A splice variant polyclonal antibody, which includes the following steps:

(1) Construct a recombinant expression vector containing the recombinant PIP4K2A splice variant gene, the nucleotide sequence of the recombinant PIP4K2A splice variant gene is shown in SEQ ID NO. 2;

(2) Transform the recombinant expression vector into Escherichia coli competent cells to induce expression to obtain the recombinant PIP4K2A splice variant antigen;

(3) Immunize animals with the recombinant PIP4K2A splice variant antigen to obtain antiserum, and then isolate and purify the anti-PIP4K2A splice variant polyclonal antibody.

Further, in step (1), the recombinant expression vector is obtained by cloning the fragment of the nucleotide sequence shown in SEQ ID NO. 2 into a prokaryotic expression vector. Preferably, the prokaryotic expression vector is pcDNA3.1- FLAG.

Further, in step (2), the amino acid sequence of the recombinant PIP4K2A splice variant antigen is shown in SEQ ID NO.1.

Further, step (2) also includes the step of purifying the recombinant PIP4K2A splice variant antigen: using FLAG-tag affinity chromatography to purify the PIP4K2A splice variant antigen.

In another aspect, the present invention provides an anti-PIP4K2A splice variant polyclonal antibody, which is prepared by the above-mentioned method for preparing an anti-PIP4K2A splice variant polyclonal antibody.

In yet another aspect, the present invention provides a kit, including the above-mentioned anti-PIP4K2A splice variant polyclonal antibody.

In yet another aspect, the present invention provides the use of the above-mentioned anti-PIP4K2A splice variant polyclonal antibody and the above-mentioned kit in the preparation of products, and the function of the product is at least one of 1)-4):

1) Detection of PIP4K2A splicing variant proteins;

2) Diagnose or assist in the diagnosis of liver cancer;

3) Diagnosis or auxiliary diagnosis of liver cancer metastasis;

4) Determine the prognosis of liver cancer.

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

The present invention identifies the specificity and effectiveness of PIP4K2A-S antibodies through western blot and immunohistochemistry. Experiments show that PIP4K2A-S is an independent risk factor that promotes liver cancer metastasis and is negatively correlated with the survival of liver cancer patients. The experimental results indicate that PIP4K2A-S can be used as a diagnostic marker for liver cancer. Therefore, detecting the expression of splicing variant PIP4K2A-S in tissues of liver cancer patients has high guiding significance for judging the prognosis of liver cancer.

Description of the drawings

Figure 1 is a Westernblot diagram for identifying the specificity of PIP4K2A-S antibodies in Example 1 of the present invention.

Figure 2 is a representative immunohistochemical diagram of PIP4K2A-S in liver cancer tissue chip in Example 2 of the present invention.

Figure 3 is a diagram showing the staining score of PIP4K2A-S in tumor tissues of patients with metastatic liver cancer in Example 2 of the present invention.

Figure 4 is a survival analysis chart of different expression levels of PIP4K2A-S in patients with metastatic liver cancer in Example 2 of the present invention.

Detailed ways

The technical solutions in the present invention will be further described below in conjunction with the accompanying drawings and examples.

Example 1 Preparation of anti-AdPIP4K2A-S antibody

1. Construction of recombinant expression vector

The amino acid sequence of the splice variant PIP4K2A-S protein is shown in SEQ ID NO.3. According to the amino acid sequence shown in SEQ ID NO.3, exon 4 formed by deletion of exon 5 is linked to exon 6. The amino acid sequence at was analyzed for amino acid sequence specificity and conservation. After protein expressibility analysis and protein antigenicity analysis, the selected antigen region (splice variant PIP4K2A-S protein antigen) is: RFGIDDQDFQYIVEC (SEQ ID NO. 1), And further cloned RFGIDDQDFQYIVEC into the modified pcDNA3.1-FLAG vector to obtain pcDNA3.1-PIP4K2A-S-FLAG particles. The details are as follows: PCR amplification target fragment PCR system

PCR reaction conditions

Amplification was performed using touchdown PCR. After purifying the amplified PCR product, perform double enzyme digestion on the PCR product. After the end of the enzyme digestion, the enzyme digestion product is recovered from the gel. At the same time, pcDNA3.1 is also subjected to double enzyme digestion. The digested plasmid and the current fragment were ligated and transformed, and finally the recombinant plasmid was identified through colony PCR screening and enzyme digestion. Among them, the nucleotide sequence encoding the selected antigen region is shown in SEQ ID NO. 2.

And follow the same method to construct PSEB-PIP4K2A-L-3Flag (pcDNA-L-FLAG).

2. Transformation and induced expression of recombinant expression vectors

Transform the constructed pcDNA3.1-PIP4K2A-S-FLAG plasmid into BL21 (DE3) competent cells, and then evenly spread it on an LB plate (containing 50 μg/mL kanamycin sulfate), and then invert it to 37°C for culture box overnight. Select a single colony from the transformed plate and inoculate it into 4 mL of LB medium (containing 50 μg/mL kanamycin sulfate). Cultivate until the OD600 is 0.5-0.8. Add a final concentration of 0.1 mM MIPTG to the test tube culture medium. (isopropylthiogalactopyranoside), and then placed at 37°C to induce expression. The splice variant PIP4K2A-S protein antigen was expressed via an E. coli expression system.

3. Preparation of splicing variant PIP4K2A-S polyclonal antibody

The splice variant PIP4K2A-S protein antigen was affinity purified by FLAG-tag and then immunized in two rabbits. Active serum is run on a FLAG column to remove any antibodies that react with the fusion protein. Flow-through was performed through the second column containing a FLAG column coupled to the PIP4K2A-L or PIP4K2A-S epitope. Antibodies were eluted with 2 mL of 0.2 M Glycine pH 2.6 (200 μL per fraction) into a 1.5 mL tube containing 60 μL of 1 M Tris pH 8.0. Protein peaks were detected using the Bradford method and peak scores were combined. Antibodies were dialyzed against 40% glycerol in PBS overnight at 4°C. Antibodies were collected and stored at -20°C.

4. Identification of polyclonal antibodies

HepG2 cells were transfected with PSEB-3Flag, PSEB-PIP4K2A-L-3Flag (PSEB-L-3Flag), and PSEB-PIP4K2A-S-3Flag plasmids. After 48 hours, the proteins were extracted for Western blot experiments. The results showed that the prepared The PIP4K2A-S antibody can specifically delete the PIP4K2A-S protein after exon 5. The results are shown in Figure 1.

Example 2 Immunohistochemical analysis of human hepatocellular carcinoma

The prepared PIP4K2A-S antibody was used to test 67 cases of human hepatocellular carcinoma specimens. After dewaxing and hydrating the tissue chip, citrate buffer saline and tissue sections were placed in the antigen repair box. After antigen repair, goats were added dropwise. The serum working solution was blocked at room temperature for 1 h. Then add 50ul of the primary antibody dilution prepared in advance, and perform immunohistochemical staining at 4°C overnight. Take it out and rewarm it at room temperature. After washing with PBS, add polymer enhancer A and enzyme-labeled anti-mouse/rabbit polymer (B) for 30 minutes at room temperature. After washing with PBS, DAB solution develops color and protects from light. Observe the color development degree under a microscope and stain with hematoxylin. , after alcohol dehydration, xylene for 10 minutes, air drying in a fume hood, and sealing with neutral resin. The clinicopathological characteristics of HCC patients were analyzed using χ2, and the measured values were used using the 'survminer' R package (version 3.6.3). HCC patients were divided into PIP4K2A-S low expression group (score 89-124.38, 20 cases) and high PIP4K2A-S expression group. Expression group (score 124.38-162, 47 cases). The results are shown in Figure 2.

Patients were stratified according to PIP4K2A-S values. High PIP4K2A-S is closely associated with hepatocellular carcinoma metastasis. The results are shown in Figure 3.

In the tissue chips of patients with liver cancer metastasis, the PIP4K2A-S value and patient survival were analyzed. The results showed that high expression of PIP4K2A-S in the tumor tissue of patients with liver cancer metastasis was negatively correlated with the patient's survival time. The results are shown in Figure 4.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified. Modifications or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention shall be included in the scope of the claims of the present invention.

Claims (7)

1. A method for preparing an anti-PIP4K2A splicing variant polyclonal antibody, which is characterized by: comprising the following steps: (1) Construct a recombinant expression vector containing the recombinant PIP4K2A splice variant gene, the nucleotide sequence of the recombinant PIP4K2A splice variant gene is shown in SEQ ID NO. 2; (2) Transform the recombinant expression vector into Escherichia coli competent cells to induce expression to obtain the recombinant PIP4K2A splice variant antigen; (3) Immunize animals with the recombinant PIP4K2A splice variant antigen to obtain antiserum, and then isolate and purify the anti-PIP4K2A splice variant polyclonal antibody. 2. A method for preparing an anti-PIP4K2A splice variant polyclonal antibody as claimed in claim 1, characterized in that: in step (1), the recombinant expression vector is a nucleoside shown in SEQ ID NO.2 The fragment of the acid sequence is obtained by cloning into a prokaryotic expression vector. Preferably, the prokaryotic expression vector is pcDNA3.1-FLAG. 3. A method for preparing an anti-PIP4K2A splice variant polyclonal antibody according to claim 1, characterized in that: in step (2), the amino acid sequence of the recombinant PIP4K2A splice variant antigen is SEQ ID NO. 1 shown. 4. The preparation method of a kind of anti-PIP4K2A splice variant polyclonal antibody as claimed in claim 1, characterized in that: in step (2), it also includes the step of purifying the recombinant PIP4K2A splice variant antigen: utilizing FLAG- The PIP4K2A splice variant antigen purified by tag affinity chromatography. 5. An anti-PIP4K2A splice variant polyclonal antibody, characterized in that it is prepared by the method for preparing an anti-PIP4K2A splice variant polyclonal antibody according to any one of claims 1-4. 6. A kit, comprising an anti-PIP4K2A splice variant polyclonal antibody according to claim 5. 7. Application of an anti-PIP4K2A splice variant polyclonal antibody according to claim 5 and a test kit according to claim 6 in the preparation of a product, the function of which is at least one of 1)-4) A sort of: 1) Detection of PIP4K2A splicing variant proteins; 2) Diagnose or assist in the diagnosis of liver cancer; 3) Diagnosis or auxiliary diagnosis of liver cancer metastasis; 4) Determine the prognosis of liver cancer.