Anti-pip4k2a splice variant polyclonal antibody and application thereof in diagnosis of liver cancer

Abstract

The application provides an anti-PIP4K2A splice variant polyclonal antibody and application thereof in liver cancer diagnosis. A preparation method of the anti-PIP4K2A splice variant polyclonal antibody comprises the following steps: (1) constructing a recombinant expression vector containing a PIP4K2A splice variant antigen coding gene, wherein the amino acid sequence of the PIP4K2A splice variant antigen is RFGIDDQDFQYIVEC; (2) transforming the recombinant expression vector into an E. coli competent cell to induce expression, and obtaining a recombinant PIP4K2A splice variant antigen; (3) immunizing animals with the recombinant PIP4K2A splice variant antigen to obtain an antiserum, and then isolating and purifying the antiserum to obtain the anti-PIP4K2A splice variant polyclonal antibody. The PIP4K2A-S can be used as a diagnostic marker of liver cancer, and has a high guiding significance for judging the prognosis of liver cancer and liver cancer diagnosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, and particularly relates to an anti-PIP4K2A splice variant polyclonal antibody and application thereof in liver cancer diagnosis. BACKGROUND

[0002] Alternative splicing (AS) can edit a single pre-mRNA molecule and produce different mature mRNAs in eukaryotes, and these transcription variants can subsequently produce proteins with different structures and biological functions. Therefore, alternative splicing is an important mechanism of post-transcriptional regulation of gene expression, and plays a crucial role in the diversity of 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 subtypes. Abnormal alternative splicing events can be associated with a variety of diseases, especially in the occurrence, development, metastasis and drug resistance of cancer. Alternative splicing events can be used as diagnostic or prognostic biomarkers, and for developing therapeutic targets for cancer.

[0003] Primary liver cancer accounts for the fourth place of the incidence of malignant tumors in China, and the mortality rate ranks the second place, which seriously threatens the life and health of the people in China. Hepatocellular carcinoma (HCC) (hereinafter referred to as liver cancer) is the most common adult liver cancer, which accounts for about 75%-85% of primary liver cancer in clinic. The liver has rich blood supply, and 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%, and the fundamental reason for poor prognosis of liver cancer is that liver cancer is occult in early stage, and there is lack of effective early diagnosis method and early treatment intervention means.

[0004] PIP4K2A (phosphatidylinositol-5-phosphate 4-kinase 2 alpha) gene full length 179.7Kb, including 10 exons. PIP4K2A cDNA is 3.8kb. PIP4K2A protein is composed of 406 amino acids, with a molecular weight of 53kDa, and has a conserved phosphatidylinositol phosphate kinase (PIPK) domain in the C-terminal region. PIP4K2A belongs to PIP kinase type II, also including PIP4K2B and PIP4K2C. It was first identified in red blood cells and is highly expressed in peripheral blood cells. The main function of this protein family is to recognize and phosphorylate phosphatidylinositol (PtdIns) 5P to synthesize phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). Phosphatidylinositol-4,5-bisphosphate is an important precursor in phosphoinositide signal transduction, which can directly regulate the activity of signal transduction proteins and cell processes. Recent studies have shown that PIP4K2A is involved in the regulation of important biological processes of malignant phenotype, including cell proliferation, clonal formation and survival. However, there is no report on the relationship between PIP4K2A splice variants and liver cancer. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides an anti-PIP4K2A splice variant polyclonal antibody and its application in the diagnosis of liver cancer, which solves the problem of lack of effective early diagnosis method and early treatment intervention means for liver cancer in the prior art.

[0006] In one aspect of the present application, a preparation method of an anti-PIP4K2A splice variant polyclonal antibody is provided, comprising the following steps:

[0007] (1) Constructing a recombinant expression vector containing a PIP4K2A splice variant antigen coding gene, wherein the amino acid sequence of the PIP4K2A splice variant antigen is RFGIDDQDFQYIVEC;

[0008] (2) Transforming the recombinant expression vector into an E. coli competent cell to induce expression, and obtaining a recombinant PIP4K2A splice variant antigen;

[0009] (3) Immunizing animals with the recombinant PIP4K2A splice variant antigen to obtain antisera, and then isolating and purifying to obtain an anti-PIP4K2A splice variant polyclonal antibody.

[0010] Further, in step (1), the recombinant expression vector is obtained by cloning the PIP4K2A splice variant antigen coding gene into a prokaryotic expression vector, preferably, the prokaryotic expression vector is pcDNA3.1-FLAG.

[0011] Further, step (2) further comprises a step of purifying the recombinant PIP4K2A splice variant antigen: the PIP4K2A splice variant antigen is purified by FLAG-tag affinity chromatography.

[0012] In another aspect of the present application, an anti-PIP4K2A splice variant polyclonal antibody is provided, which is prepared by the method for preparing the anti-PIP4K2A splice variant polyclonal antibody.

[0013] In yet another aspect of the present application, a kit is provided, which comprises the anti-PIP4K2A splice variant polyclonal antibody.

[0014] In still another aspect of the present application, the anti-PIP4K2A splice variant polyclonal antibody and the kit are used in the preparation of a product, and the product has at least one of the following functions:

[0015] 1) detecting PIP4K2A splice variant protein;

[0016] 2) diagnosing or assisting in the diagnosis of liver cancer;

[0017] 3) diagnosing or assisting in the diagnosis of liver cancer metastasis;

[0018] 4) judging the prognosis of liver cancer.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] The present application identifies the specificity and effectiveness of the PIP4K2A-S antibody by western blot and immunohistochemistry, and experiments show that PIP4K2A-S is an independent risk factor for promoting liver cancer metastasis and is negatively correlated with the survival of liver cancer patients. The experimental results show that PIP4K2A-S can be used as a diagnostic marker for liver cancer, and therefore, detecting the expression of the splice variant PIP4K2A-S in the tissues of liver cancer patients has a high guiding significance for judging the prognosis of liver cancer. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 A Western blot diagram for identifying the specificity of the PIP4K2A-S antibody in Example 1 of the present application.

[0022] Figure 2 A representative immunohistochemical diagram of PIP4K2A-S in a liver cancer tissue chip in Example 2 of the present application.

[0023] Figure 3 A staining score diagram of PIP4K2A-S in the tumor tissue of a metastatic liver cancer patient in Example 2 of the present application.

[0024] Figure 4 Figure 2 is a survival analysis chart of different expression levels of PIP4K2A-S in patients with metastatic liver cancer in Embodiment 2 of the present application. DETAILED DESCRIPTION

[0025] The technical solutions in the present application are further described below in combination with the drawings and embodiments.

[0026] Embodiment 1 Preparation of anti-AdPIP4K2A-S antibody

[0027] 1. Construction of recombinant expression vector

[0028] The amino acid sequence of the splice variant PIP4K2A-S protein is shown in SEQ ID NO. 1. According to the amino acid sequence shown in SEQ ID NO. 1, the amino acid sequence at the junction between the 4th exon and the 6th exon after deletion of the 5th exon is analyzed for amino acid sequence specificity and conservation, protein expressibility, and protein antigenicity, and the antigen region (splice variant PIP4K2A-S protein antigen) is selected as RFGIDDQDFQYIVEC. The nucleotide sequence encoding RFGIDDQDFQYIVEC is further cloned into a modified pcDNA3.1-FLAG vector to obtain a pcDNA3.1-PIP4K2A-S-FLAG plasmid. The specific process is as follows: PCR amplification of the target fragment

[0029]

[0030]

[0031] Touchdown PCR is used for amplification. After purification of the amplified PCR product, the PCR product is double-digested, and the digested product is recovered by gel. The plasmid is also double-digested. The digested plasmid and the current fragment are ligated and transformed, and finally screened by colony PCR and recombinant plasmid digestion. The nucleotide sequence encoding the splice variant PIP4K2A-S protein is shown in SEQ ID NO. 2.

[0032] PSEB-PIP4K2A-L-3Flag (pcDNA-L-FLAG) is constructed by the same method.

[0033] 2. Transformation of recombinant expression vector and induction of expression

[0034] The constructed pcDNA3.1-PIP4K2A-S-FLAG plasmid was transformed into BL21(DE3) competent cells, which were then uniformly spread on LB plates containing 50 μg / mL kanamycin sulfate, and then inverted in a 37 °C incubator overnight. Single colonies were selected from the transformed plates and inoculated into 4 mL of LB medium containing 50 μg / mL kanamycin sulfate. When the culture reached an OD600 of 0.5-0.8, 0.1 mM IPTG (isopropyl thiogalactoside) was added to the test tube culture solution, and then the culture was induced at 37 °C. The splice variant PIP4K2A-S protein antigen was expressed by the E. coli expression system.

[0035] 3. Preparation of polyclonal antibody of splice variant PIP4K2A-S

[0036] The splice variant PIP4K2A-S protein antigen was immunized in two rabbits after FLAG-tag affinity purification. The active serum was run on a FLAG column to remove any antibodies that reacted with the fusion protein. Flow-through was performed by a second column containing a FLAG column coupled to PIP4K2A-L or PIP4K2A-S epitopes. The 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. The protein peak was detected by the Bradford method, and the peak fractions were combined. The antibodies were dialyzed against PBS with 40% glycerol overnight at 4 °C. The antibodies were collected and stored at -20 °C.

[0037] 4. Identification of polyclonal antibody

[0038] PSEB-3Flag, PSEB-PIP4K2A-L-3Flag (PSEB-L-3Flag), PSEB-PIP4K2A-S-3Flag plasmids were transfected into HepG2 cells, and after 48 hours, the proteins were extracted for Western blotting. The results showed that the prepared PIP4K2A-S antibody could specifically delete the PIP4K2A-S protein after the 5th exon. The results are shown in Figure 1 .

[0039] Example 2 Immunohistochemical analysis of human hepatocellular carcinoma

[0040] The prepared PIP4K2A-S antibody was used to perform immunohistochemical staining on 67 human hepatocellular carcinoma samples. After dewaxing and hydration of the tissue chip, the citrate buffer and tissue sections were placed in an antigen retrieval box for antigen retrieval. Then, 50 ul of pre-prepared primary antibody diluent was added, and the mixture was incubated at 4°C overnight. After removal, the mixture was incubated at room temperature, washed with PBS, and then added with polymer enhancer A and enzyme-labeled anti-mouse / rabbit polymer (B). After incubation at room temperature for 30 min, the mixture was washed with PBS, and then DAB solution was added for color development in the dark. The color development degree was observed under a microscope, followed by hematoxylin staining, alcohol dehydration, xylene treatment for 10 min, air drying in a fume hood, and neutral resin mounting. The clinical and pathological characteristics of HCC patients were analyzed using χ2analysis. The measured values were used to divide the HCC patients into a PIP4K2A-S low expression group (score 89-124.38, 20 cases) and a high expression group (score 124.38-162, 47 cases) using the'survminer' R package (version 3.6.3). The results are shown in Table 1. Figure 2

[0041] The patients were stratified according to the PIP4K2A-S values. High PIP4K2A-S was closely related to hepatocellular carcinoma metastasis. The results are shown in Table 2. Figure 3

[0042] The PIP4K2A-S values were analyzed in relation to patient survival in the tissue chip of liver cancer metastasis patients. The results showed that high expression of PIP4K2A-S in the tumor tissue of liver cancer metastasis patients was negatively correlated with patient survival time. The results are shown in Table 3. Figure 4

[0043] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application, and all such modifications or replacements should be included in the scope of the claims of the present application.​​​

Claims

1. A method for preparing anti- PIP4K2A splice variant polyclonal antibodies, characterized by: The method comprises the following steps: (1) constructing a recombinant expression vector containing a PIP4K2A splice variant antigen coding gene, wherein the amino acid sequence of the PIP4K2A splice variant antigen is RFGIDDQDFQYIVEC; (2) transforming the recombinant expression vector into an E. coli competent cell to induce expression, and obtaining a recombinant PIP4K2A splice variant antigen; (3) immunizing an animal with the recombinant PIP4K2A splice variant antigen to obtain an antiserum, and then isolating and purifying the antiserum to obtain an anti-PIP4K2A splice variant polyclonal antibody.

2. The method for preparing a polyclonal antibody against PIP4K2A splicing variant as described in claim 1, characterized in that: In step (1), the recombinant expression vector is obtained by cloning the PIP4K2A splice variant antigen coding gene into a prokaryotic expression vector, and the prokaryotic expression vector is pcDNA3.1-FLAG.

3. The method for preparing a polyclonal antibody against a PIP4K2A splice variant as described in claim 1, characterized in that: In step (2), the recombinant PIP4K2A splice variant antigen is further purified by using FLAG-tag affinity chromatography.

4. An anti-PIP4K2A splice variant polyclonal antibody, characterized in that: The anti-PIP4K2A splice variant polyclonal antibody is prepared by the method of any one of claims 1-3.

5. A kit comprising the anti-PIP4K2A splice variant polyclonal antibody of claim 4.

6. Use of the anti-PIP4K2A splice variant polyclonal antibody of claim 4 or the kit of claim 5 in the preparation of a product, wherein the product has at least one of the following functions: 1) detecting PIP4K2A splice variant protein; 2) diagnosing or assisting in the diagnosis of liver cancer; 3) diagnosing or assisting in the diagnosis of liver cancer metastasis.

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